Renal Fibrosis (Contributions to Nephrology)

Renal Fibrosis Contributions to Nephrology Vol. 139 Series Editor Claudio Ronco Vicenza Renal Fibrosis Volume E...

Author: Mohammed S. Razzaque | Takashi Taguchi

31 downloads 1050 Views 2MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

Renal Fibrosis

Contributions to Nephrology Vol. 139

Series Editor

Claudio Ronco

Vicenza

Renal Fibrosis

Volume Editors

Mohammed S. Razzaque Boston, Mass. Takashi Taguchi Nagasaki

39 figures, 2 in color, and 11 tables, 2003

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Contributions to Nephrology (Founded 1975 by Geoffrey M. Berlyne)

Mohammed S. Razzaque

Takashi Taguchi

Department of Oral Medicine, Infection and Immunity Harvard School of Dental Medicine 188 Longwood Avenue Boston, MA 02115 (USA) E-Mail [email protected] and Department of Pathology Nagasaki University Graduate School of Biomedical Sciences 1-12-4, Sakamoto, Nagasaki 852-8523 (Japan) E-Mail [email protected]

Department of Pathology Nagasaki University Graduate School of Biomedical Sciences 1-12-4, Sakamoto, Nagasaki 852-8523 (Japan) E-Mail [email protected]

Library of Congress Cataloging-in-Publication Data Renal fibrosis / volume editors, Mohammed S. Razzaque, Takashi Taguchi. p. ; cm. – (Contributions to nephrology, ISSN 0302–5144 ; v. 139) Includes bibliographical references and index. ISBN 3–8005–7568–8 (hard cover : alk. paper) 1. Kidneys–Fibrosis–Molecular aspects. I. Razzaque, Mohammed S. II. Taguchi, Takashi. III. Series. [DNLM: 1. Kidney Diseases–physiopathology. 2. Fibrosis. WJ 300 R39159 2003] RC903.9.R473 2003 616.6⬘107–dc21 2003044735

Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2003 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0302–5144 ISBN 3–8055–7568–8

Contents

VII Preface Razzaque, M.S. (Boston, Mass./Nagasaki); Taguchi, T. (Nagasaki) IX Foreword Olsen, B.R. (Boston, Mass.) 1 Factors That Influence and Contribute to the Regulation of Fibrosis Razzaque, M.S. (Boston, Mass./Nagasaki); Taguchi, T. (Nagasaki) 12 Disturbance of Syncytial Cell Function in Glomerular Mesangial Cells Involved in the Progressive Glomerular Diseases Oite, T.; Yao, J.; Morioka, T. (Niigata) 20 Proteinuria and Tubulointerstitial Injury:The Causative Factors for the Progression of Renal Diseases Matsuo, S.; Morita, Y.; Maruyama, S.; Manchang, L.; Yuzawa, Y. (Nagoya) 32 Molecular Mechanism of Cell Injury Maeshima, Y.; Makino, H. (Okayama) 44 Role of TGF-␤ in the Progression of Renal Fibrosis Tamaki, K.; Okuda, S. (Fukuoka) 66 Chemokines in Renal Fibrosis Yokoyama, H.; Wada, T.; Furuichi, K. (Kanazawa) 90 Pathological Significance of Renal Expression of NF-␬B Inan, M.S. (Boston, Mass.); Razzaque, M.S. (Boston, Mass./Nagasaki); Taguchi, T. (Nagasaki)

V

102 Oxidant Stress and Regulation of Chemokines in the Development of Renal Interstitial Fibrosis Cochrane, A.L.; Ricardo, S.D. (Clayton, Vic.) 120 Role of Myofibroblasts in Progressive Renal Diseases Moriyama, T.; Imai, E. (Osaka) 141 Regulation of Renal Extracellular Matrix Metabolism Miyazaki, M.; Nishino, T.; Abe, K.; Furusu, A.; Koji, T.; Kohno, S. (Nagasaki) 156 Implication of Apoptosis in Progression of Renal Diseases Kashihara, N. (Kurashiki); Sugiyama, H.; Makino, H. (Okayama) 173 Factors Regulating the Progression of Hypertensive Nephrosclerosis Razzaque, M.S. (Boston, Mass./Nagasaki); Azouz, A. (Boston, Mass.); Shinagawa, T. (Kawatana); Taguchi, T. (Nagasaki) 187 Pathogenesis and Molecular Mechanisms of Chronic Allograft Nephropathy Ahsan, N. (New Brunswick, N.J.); Cheung, J.Y. (Danville, Pa.) 205 Author Index 206 Subject Index

Contents

VI

Preface

This book, with chapters contributed by leading experts in their field, contains the information required for a basic orientation on the molecular mechanisms of renal fibrosis. It is especially formatted for scientists and clinicians who need a quick update of fibrogenesis in their research and practice. Recent developments in our understanding of the cellular and molecular events of fibrosis have either offered new therapeutic choices or have led to the discovery of gene-based therapeutic options. This book provides a synopsis on the rapid progress encountered in the last couple of years in renal cell biology, which has enhanced our understanding of fibrogenesis in general. Given the complexity of renal diseases, providing information on all aspects of fibrogenesis is clearly an overwhelming task. However, we selected topics that we believe will provide necessary information on the molecular basis of renal fibrosis. We organized the subject matter with the intent that it may be useful to general health professionals, while also being of interest to clinicians and researchers familiar with the field. Our editorial approach was to make the book easy to read. We encouraged our authors to provide diagrams and tables, which in most cases summarize complex biological processes in simple terms. We hope our readers will find this book a clear, useful and informative tool for understanding some of the basic molecular mechanisms of fibrotic renal diseases. Our sincere hope is that amongst the readers, a few will be inspired to take up the challenges and reap the rewards for themselves to further enhance the knowledge and understanding of the pathomechanisms of fibrotic diseases, eventually leading to better patient care.

VII

We are fortunate to have a group of basic and clinical investigators who have devoted their time and effort to bring this book up to date. We would like to take this opportunity to express our thanks and gratitude to each of the contributors for kindly sharing their knowledge and expertise. We are grateful to Karger Publishers, Switzerland, for their support in bringing this project to completion. A special thanks goes to Peter Roth and his staff. Last but not least, we acknowledge the kind support of our families (Rafi, Yuki, Kanako, Ai, and Kazuko) for their encouragement and patience during our many hours of writing and editing. We hope that this book will be important reading for all those involved in the basic and clinical research of matrix biology. Mohammed S. Razzaque, Boston, Mass. Takashi Taguchi, Nagasaki

Preface

VIII

Foreword

Postnatal injury to skin or internal organs in humans most often initiates a repair process that results in the formation of a collagen-rich scar or a fibrotic mass instead of a regenerated and fully functional organ. In internal organs, such as the lung, liver, or kidney, the consequences of fibrosis can be devastating. Understanding and controlling tissue repair processes with the goal of preventing fibrosis has been a high priority in clinical research for a long time. This volume on Renal Fibrosis, with chapters contributed by a number of well-known clinical investigators, therefore addresses a significant set of problems. The book provides a strong reminder that progress in this field is intimately connected with advances in research on fundamental molecular and cellular mechanisms in the areas of immunology, inflammation, and extracellular matrix biology. Exciting progress in each of these areas has led to the identification of a large number of genes, gene products, and biochemical pathways that are likely to be involved in the cellular responses that lead to fibrosis, allowing a rich and molecularly detailed context for discussing fibrotic repair. A large number of cytokines, intracellular signaling molecules, transcription factors, and extracellular matrix components are important participants and modulators at different stages of the progression of fibrosis, and up-to-date reviews of many of these molecules can be found in this book. The reviews do an excellent job in defining where current work on renal fibrosis stands and in which directions the work ought to proceed. Studies in the suggested directions will continue to define markers of disease progression and molecular targets for drugs to treat fibrotic conditions.

IX

Additional insights will undoubtedly also come from research that is not directly addressed in this book. For example, as we learn more about regenerative wound repair, it may become possible to define the critical step(s) during wound healing when one imperfect process (scarring/fibrosis) wins out over the more perfect process of regeneration. It will therefore be important for investigators of fibrosis to keep an eye on research into regenerative mechanisms in model organisms. It could well be that critical insights into the pathogenesis of fibrosis, leading to effective clinical strategies of prevention, will ultimately come from studies of tissue/organ regeneration in organisms (such as the zebrafish) where regenerative repair is a potent alternative to fibrotic repair of wounds. Bjorn Reino Olsen, Boston, Mass.

Foreword

X

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 1–11

Factors That Influence and Contribute to the Regulation of Fibrosis Mohammed S. Razzaquea,b, Takashi Taguchia a Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, and bDepartment of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Mass., USA

Introduction

Scar formation is an important component of post-injury tissue repair process and is tightly regulated at cellular and molecular levels. Injury of any tissue, whether caused by trauma, microbes or a foreign body, initiates an inflammatory response which leads to the coordinated interplay of a variety of physiologic processes that results in a healed wound. Wound healing is a complex process of cellular and molecular events, which comprises an ordered sequence of events that include cell migration and proliferation, synthesis of extracellular matrix (ECM), angiogenesis and matrix remodeling, to restore the structure and function of the damaged and/or injured tissue. These events are partly regulated by cytokines and other mediators of cellular functions. In many instances, tissue repair or the healing process results in scar formation. Tissue repair is initiated in the extracellular space and surrounding cells (often by fibroblasts) in the connective tissue. Accumulation of excessive amounts of collagens and other ECM components in the skin and internal organs results in fibrosis [1–5]. Fibrosis is the end result of all the above listed pathophysiological events occurring in a living tissue, organ or an appendage. In most instances, when inflammation resolves due to the elimination of the causative and/or initiating factors, the deposition of matrix proteins continues on the tissues with eventual scar formation. Recently, the role of cytokines in controlling connective tissue metabolism and remodeling during wound healing and fibrosis has been emphasized. Henceforth, advances in cytokine research are

likely to lead to a new field of molecular medicine in which cytokines and their antagonists should provide innovative therapies for the prevention and treatment of fibrotic diseases.

Wound Healing and Fibrosis

During wound healing, inflammatory changes and revascularization are associated with proliferation and migration of mesenchymal cells into the injured area, followed by matrix remodeling, wound contraction and re-establishment of epithelial continuity. Several cytokines and growth factors including interleukins, platelet-derived growth factor (PDGF), transforming growth factor (TGF)-␤1 and vascular endothelial growth factor (VEGF) participate in the healing process. These factors increase the rate of healing and the breaking strength of the repaired tissue. Furthermore, they also enhance angiogenesis and subsequent blood flow to dermal wounds, partly by stimulating the local release of other growth factors, including TGF-␤1, basic fibroblast growth factor and VEGF [6, 7]. The phenotype and behavior of cells involved in the healing process are regulated by cytokines and growth factors, which exert their effects on cells through autocrine and/or paracrine mechanisms. During wound repair, fibroblasts accumulate in the injured area until the defect is filled with stratified layers of cells and matrix. TGF-␤1 plays a determinant role in fibroplasia during wound healing and fibroproliferative diseases, while myofibroblasts are implicated in wound contraction. In contrast to adult cutaneous wound healing, fetal wounds can heal without any histological evidence of scarring in early gestation. The mechanism of scarless repair in fetal wound is not clear, but intrinsic functions of dermal fibroblasts, ECM components, inflammatory responses, cytokine profiles and intracellular signal transduction of fetal wound healing differs from that of adult wound healing. Advanced knowledge of fetal scarless wound healing may help us to design therapeutic strategies to modify adult wound that mimics fetal scarless repair. The accelerated rate of healing, relative lack of acute inflammatory response, and absence of neovascularization distinguishes healing of fetal wounds from that of adult wounds. Two major cytokines involved in scarless healing process are interleukin (IL)-8 and TGF-␤; the low level of IL-8 is responsible for the lack of cellular recruitment and inflammation seen in fetal wound healing and may eventually contribute to scarless wound repair [8]. On the other hand, experimental studies suggest that fetal skin fibroblasts secrete and activate TGF-␤ to a lesser extent than adult skin fibroblasts, while exogenous addition of TGF-␤1 in fetal wounds resulted in healing with scar tissue formation [9]. Understanding the molecular mechanisms of scarless fetal wound healing and applying this knowledge as a basis

Razzaque/Taguchi

2

for developing new therapeutic strategies to manipulate the adult wounds and progressive fibrotic diseases to a scarless repair will be our challenge [10–12]. Fibrosis is a multistep process [13], usually begins with the release of cytokines from neighboring inflammatory cells, which in turn activate fibroblasts, induce their proliferation, contribute to fibroblastic trans-differentiation and excessive synthesis of ECM, and ultimately lead to fibrosis. Fibrosis or scar formation is the end result of a cascade of events that follow tissue injury and the subsequent healing process. Fibrosis could appear as a result of a physical trauma, immune injury or certain metabolic disorders that lead to the activation and proliferation of fibroblasts with accumulation of matrix proteins, affecting the structures and functions of the organs and tissues. It is well known that in affected tissues, chronic inflammation generally precedes fibrosis and that inflammatory cell-derived cytokines are crucial mediators of fibrogenesis.

Factors Influencing and Regulating Fibrosis

The primary causes of fibrosis are diverse. Alcohol and viral infections are major causes of liver fibrosis, while glomerulosclerosis and tubulointerstitial fibrosis in the kidney result from glomerulonephritis, diabetic mellitus or hypertension. Prolonged hypertension and myocardial infraction can develop diffuse cardiac fibrosis with progressive heart failure. Toxic vapors, inorganic dusts and parquet have shown to induce pulmonary fibrosis, although the molecular mechanism remains mostly unknown. Certain drugs, including cisplatin, gentamicin and bleomycin could induce fibrosis of the lungs and kidneys [14–17]. Scar formation is also a common feature in keloids, hypertrophic dermal scars and scleroderma; again, the pathomechanisms of scarring are not yet completely known. The fibrotic diseases progress inexorably to result in impairment of organ function, increasing disability and eventual death, e.g., cryptogenic fibrosing alveolitis. Recent progress in our understanding of the cellular and molecular events of fibrotic diseases provides promising opportunities for developing novel therapies. For instance, blocking the biological activities of TGF-␤1, PDGF, IL-1 and monocyte chemoattractant protein (MCP)-1 seems to have an anti-scarring effect in various experimental models of renal fibrosis [18–21]. Cytokines regulate essential biologic events including inflammation and tissue repair. Factors that play specific roles in fibrogenesis are designated as the ‘fibrogenic factors’, which include, IL-4, IL-6, TGF-␤, insulin-like growth factor-I and endothelin-1. Other cytokines/growth factors involved in the fibrotic process include IL-1, connective tissue growth factor (CTGF), tumor necrosis factor (TNF)-␣ and PDGF. These cytokines influence wound healing,

Renal Fibrogenesis

3

tissue repair and matrix synthesis. Of these, TGF-␤1 and CTGF are expressed at high levels during tissue remodeling and greatly affect the formation of connective tissue by stimulating the transcription of collagen.

Renal Fibrosis

The excessive production of matrix proteins, by resident and phenotypically altered renal cells, and their disorganized accumulation, contribute to renal fibrosis [22–25]. Several in vivo as well as in vitro experiments have suggested that a close interaction among inflammatory cells, epithelial cells, fibroblasts and myofibroblasts is required for the initiation and progression of renal fibrosis. In figure 1 we schematically summarize some important molecules, which interact with each other during renal interstitial fibrosis. Such interaction results in the generation of profibrotic factors, including TNF-␣, IL-1, PDGF and TGF-␤1. Of these, PDGF secreted by macrophages is a potent mitogen and chemoattractant for a number of cell type, while TGF-␤1 is probably one of the most important factors that regulate increased synthesis of matrix proteins in the fibrotic kidneys. Excessive accumulation of ECM, especially fibrillar and basement membrane collagens (type I, III, and IV), is a hallmark of fibrosis in human and experimental renal fibrosis, and is possibly due to transcriptional activation of the corresponding genes. Excessive deposition of collagens has been found in the glomerulosclerotic lesions of human diabetic nephropathy, hypertensive nephrosclerosis and IgA nephropathy [26–30]. Studies using in situ hybridization have indicated that resident renal cells (mesangial and epithelial cells) are the source of these collagens in the fibrotic kidneys [27, 28]. Accumulation of type VI collagen with upregulation of its mRNA has also been detected in these human renal fibrotic diseases [29, 30]. As mentioned, various ‘profibrotic factors’, such as TGF-␤1, have been demonstrated to have the potential to mediate both human and experimental renal fibrosis. TGF-␤1 is an important regulator of cell growth, differentiation and formulation of the ECM. It plays a role in matrix remodeling by decreasing degradation of matrix proteins through a reduction in protease synthesis and by increasing the synthesis of protease inhibitors. TGF-␤1 is expressed at high levels during renal fibrosis, and contributes to the scarring, by stimulating the transcription of ECM genes [31]. Both in vitro and in vivo studies have convincingly shown that modulation of TGF-␤1 suppresses collagen production and subsequently modulates the fibrotic process [32, 33]. Besides fibrogenic effects, TGF-␤1 also exhibits diverse effects on cells; for example, it suppresses the growth of epithelial cells, inhibits keratinocyte proliferation, enhances neovascularization, acts as a chemoattractant for monocytes

Razzaque/Taguchi

4

Lymphocytes Endothelial cells

Differentiation

Differentiation Fibroblasts

C m he M CS mo IC IF F kin es VC AM AM

F G PD 1 ␤ 1 IL FTG

-1

IL-1/TNF-␣

ET

Macrophages

Myofibroblasts

Tubular epithelial cells

TGF-␤1/CTGF FGF/IL-4 ET-1/AT-II HSP47

Matrix production Capillary obliteration Apoptosis Tubular atrophy

Plasmin, MMPs, ?ADAMs PAI-1, TIMPs

Interstitial fibrosis

Fig. 1. Simplified schematic diagram showing possible molecular interactions during renal tubulointerstitial fibrosis. In addition to the limited number of factors included in the diagram, there are additional factors, which may have significant involvement in the fibrotic process (IL-1: interleukin-1; IL-4: interleukin-4; TNF-␣: tumor necrosis factor-␣; mCSF: macrophage colony-stimulating factor; MIF: macrophage migration inhibitory factor; ICAM: intercellular adhesion molecules; VCAM: vascular cell adhesion molecules; TGF-␤1: transforming growth factor-␤1; PDGF: platelet-derived growth factor; CTGF: connective tissue growth factor; FGF: fibroblast growth factor; ET-1: edothelin-1; AT-II: angiotensin II; HSP47: heat-shock protein 47; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; ADAM: a disintegrin and a metalloproteinase; PAI-1: plasminogen activator inhibitor-1).

and fibroblasts, and is involved in immunosuppression [34, 35]. Although inhibition of TGF-␤1 expression has protective effects on the fibrotic process, it could also disrupt other non-fibrotic physiologic functions. Thus, identification of the fibrosis-specific factors and their inhibition will be ideal for the design of future therapies.

Renal Fibrogenesis

5

CTGF is a heparin-binding 38-kDa cysteine-rich peptide that promotes proliferation, collagen synthesis and chemotaxis by mesenchymal cells [36]. The bioactivity of CTGF has shown to be partly controlled by TGF-␤1, and a brief exposure of fibroblasts to TGF-␤1 resulted in high level of CTGF expression [37]. This upregulation is dependent on sequences present in the 5⬘ upstream region of the CTGF promoter. CTGF is overexpressed in various fibrotic renal disorders, including diabetic nephropathy [38]. Given its bioactivity, CTGF could be also considered as a fibrosis-specific factor in the kidney. Future studies will determine whether inhibition of CTGF bioactivity could alter the fibrotic events in the kidney; this would be an important strategy in designing site-specific therapies or developing agents that could prevent or slow down fibrotic process in clinical situations where its likelihood is high. Heat-shock protein 47 (HSP47), a 47-kDa stress protein, is localized in the endoplasmic reticulum of collagen-producing cells and thought to act as a collagen-specific molecular chaperon during the biosynthesis and secretion of procollagen [39–41]. The formation of a triple-helical structure is one of the post-translational events in collagen synthesis. In the endoplasmic reticulum, HSP47 binds to the ␣-polypeptide chains, possibly to assist in the alignment and folding of the triple helix. Subsequently, HSP47 dissociates from the procollagen molecule once it enters the Golgi apparatus and is secreted to the cell surface as a procollagen. Several in vivo studies have emphasized the role of HSP47 as an important factor in the fibrotic process in various organs, including skin, eye, lung, liver and kidney [41–47]. Significantly high levels of HSP47 mRNA and protein are found in dermal fibroblasts of patients with cicatricial pemphigoid, relative to those of normal healthy individuals, and TGF-␤1 has found to induce increased expression of HSP47 in dermal fibroblasts [47]. The same pattern of cytokine-regulated expression was observed for type I collagen levels [47]. These results suggest a possible role of HSP47 in the abundant production of type I procollagen by dermal fibroblasts in cicatricial pemphigoid [47]. Overexpression of HSP47 with an increased accumulation of collagen was also seen during human and experimental lung, liver and renal fibrosis, and modulation of HSP47 expression could slow down the renal fibrotic process [48]. In addition to the aforementioned factors, certain adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), appear to play a role in fibroblast and/or epithelium-T-cell interaction in the early inflammatory events of renal fibrotic diseases [49]. The pathogenic role of chemokines in the recruitment of interstitial inflammatory cells during renal fibrosis has been suggested in numerous studies. For instance, tubular epithelial cells derived MCP-1, osteopontin, regulated upon activation, normal T-cell expressed and secreted (RANTES) and endothelin are involved in inflammatory events in a variety of

Razzaque/Taguchi

6

human and experimental tubulointerstitial diseases. A role of macrophage colony-stimulating factor and macrophage migration inhibitory factor in the recruitment and local proliferation of macrophages has been suggested in various inflammatory diseases, including renal fibrotic diseases [50–53]. Early events of fibrosis comprise inflammatory changes, including proliferation of matrix-producing cells and inflammatory infiltrates, while in the late stages, most of these proliferating and infiltrating cells are cleared (possibly by apoptosis), leaving mostly acellular fibrotic mass. Increased rate of apoptosis has been identified during the development of glomerular scarring in both human and experimental studies [54–56]. Similarly, increased rate of apoptosis has been shown to be associated with pulmonary fibrosis, and suppression of apoptosis by caspase inhibitors could attenuate fibrotic process in the lung [57], suggesting that modulation of apoptosis might have potential therapeutic implication in fibrosis.

Matrix Remodeling

Recent studies have suggested that proteolytic enzymes (i.e., matrix metalloproteinases (MMPs)) and their inhibitors (tissue inhibitors of metalloproteinases (TIMPs)) are actively involved in the orchestrated process of renal fibrosis [58]. Production of ECM is predominantly achieved through the excessive synthesis of collagens whereas degradation of the ECM is mediated predominantly by MMPs. TIMPs are specific inhibitors of MMP activity. A fine balance of these two classes of molecules helps in maintaining normal structural integrity of tissues and organs. Disrupted balance results in pathological states, such as excessive matrix accumulation (i.e., fibrotic diseases) or tissue destruction (e.g., rheumatoid arthritis). It has been shown that glomerular resident cells express and secrete several types of MMPs, including gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). MMP-3 has been detected in both animal and human kidneys. In passive a Heymann nephritis model, McMillan et al. [59] demonstrated a marked increase in MMP-9 synthesis within glomerular epithelial cells. Altered expression of MMP-3 and TIMP-1 has been detected in renal biopsy sections in various renal diseases including IgA nephropathy [58]. Current information suggests a potential role for MMPs and TIMPs in matrix remodeling in both human and experimental renal fibrotic diseases. Recently it has been demonstrated that transcription factor Ets-1 enhance the promoter activity of such genes as MMP-1, MMP-3 and u-PA. A coordinated upregulation of MMP-3 and Ets-1 has been reported in experimentally induced crescentic glomerulonephritis. The expression pattern and cellular distribution

Renal Fibrogenesis

7

of Ets-1 and MMP-3 were similar, and Ets-1-positive renal cells often expressed MMP-3 in the diseased kidney, reflecting a close association between Ets-1 and MMP-3 expression [60]. It is likely that Ets-1 might participate in matrix remodeling in renal diseases by upregulating expression of proteolytic enzymes, including of MMP-3 [60]. Adamalysins (ADAMs) are type I transmembrane glycoproteins containing a disintegrin and a metalloproteinase domain. ADAMs are thought to be involved in important cellular events, including cell adhesion and membrane protein shedding [61]. Approximately half of the ADAMs has shown to be metzincin superfamily of metalloproteinases, which also includes the MMPs. Recently, a subgroup of ADAMs have been identified, designated ADAMthrombospondin (TS) proteinases. The ADAM-TS group, unlike typical membrane-anchored ADAMs, lacks a transmembrane domain and a cytoplasmic domain at the C terminus. Instead, these contain a variable number of TS type-1 domains [62]. ADAMs and ADAM-TSs may involve in the proteolysis of matrix proteins. Identifying the specific members of MMPs, ADAMs, ADAM-TSs and TIMPs involved in matrix remodeling will help in determining their role(s) in renal scarring. If specific members of these molecules can be identified, a therapeutic manipulation can be developed to restore their balance to modulate fibrotic process. Recently, a pathologic role of ADAM-TS-1 has suggested in liver fibrosis in cirrhotic rats [63].

Conclusion

Over the past decade or more, significant progress has been made in our understanding and identifying the molecules involved in renal fibrosis. However, further studies are needed to determine the coordinate link among these molecules during renal fibrosis. A better understanding of the regulation and interactions of these molecules should allow the development of strategies for specific intervention or modification of renal fibrosis. We realize that renal scarring is a multistep and multifactorial process. To that end, we have tried to provide basic information that could be important, relevant and useful in this narrow, yet very important field of fibrogenesis.

Acknowledgements The technical assistance of staff members of Department of Pathology, Nagasaki University Graduate School of Medical Sciences is gratefully acknowledged. Our apology goes to all authors whose work could not be cited due to the limitation of space.

Razzaque/Taguchi

8

References 1 2 3 4 5 6

7 8 9

10 11

12 13 14

15 16 17 18

19

20 21 22 23

Zhang K, Phan SH: Cytokines and pulmonary fibrosis. Biol Signals 1996;5:232–239. Stenback F, Wasenius VM, Sotaniemi EA: Interstitial collagen in alcoholic human liver. Histol Histopathol 1987;2:401–411. Razzaque MS, Taguchi T: Pulmonary Fibrosis: cellular and molecular events. Pathol. Int 2003;53: 133–145. Mutsaers SE, Bishop JE, McGrouther G, Laurent GJ: Mechanisms of tissue repair: From wound healing to fibrosis. Int J Biochem Cell Biol 1997;29:5–17. Razzaque MS, Foster CS, Ahmed AR: Tissue and molecular events in human conjunctival scarring in ocular cicatricial pemphigoid. Histol Histopathol 2001;16:1203–1212. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPietro LA: Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998;152:1445–1452. Tonnesen MG, Feng X, Clark RA: Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5:40–46. Liechty KW, Crombleholme TM, Cass DL, Martin B, Adzick NS: Diminished interleukin-8 production in the fetal wound healing response. J Surg Res 1998;77:80–84. Krummel TM, Michna BA, Thomas BL, Sporn MB, Nelson JM, Salzberg AM, Cohen IK, Diegelmann RF: Transforming growth factor-␤ induces fibrosis in a fetal wound model. J Pediatr Surg 1988;23:647–652. Stelnicki EJ, Chin GS, Gittes GK, Longaker MT: Fetal wound repair: Where do we go from here? Semin Pediatr Surg 1999;8:124–130. Cass DL, Bullard KM, Sylvester KG, Yang EY, Sheppard D, Herlyn M, Adzick NS: Epidermal integrin expression is upregulated rapidly in human fetal wound repair. J Pediatr Surg 1998;33: 312–316. Mackool RJ, Gittes GK, Longaker MT: Scarless healing. The fetal wound. Clin Plast Surg 1998;25:357–365. Razzaque MS, Taguchi T: Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc 2002;35:68–80. Yamate J, Ishida A, Tsujino K, Tatsumi M, Nakatsuji S, Kuwamura M, Kotani T, Sakuma S: Immunohistochemical study of rat renal interstitial fibrosis induced by repeated injection of cisplatin, with special reference to the kinetics of macrophages and myofibroblasts. Toxicol Pathol 1996;24:199–206. Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat-shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. Hay J, Shahzeidi S, Laurent G: Mechanisms of bleomycin-induced lung damage. Arch Toxicol 1991;65:81–94. Razzaque MS, Taguchi T: Localization of HSP47 in cisplatin-treated rat kidney: Possible role in tubulointerstitial damage. Clin Exp Nephrol 1999;3:222–228. Karkar AM, Tam FW, Steinkasserer A, Kurrle R, Langner K, Scallon BJ, Meager A, Rees AJ: Modulation of antibody-mediated glomerular injury in vivo by IL-1ra, soluble IL-1 receptor, and soluble TNF receptor. Kidney Int 1995;48:1738–1746. Johnson RJ, Raines EW, Floege J, Yoshimura A, Pritzl P, Alpers C, Ross R: Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor. J Exp Med 1992;175:1413–1416. Chou DH, Lee W, McCulloch CA: TNF-␣ inactivation of collagen receptors: Implications for fibroblast function and fibrosis. J Immunol 1996;156:4354–4362. Yokoyama H, Wada T, Furuichi K: Chemokines in renal fibrosis; in Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 66–89. Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14:1199–1212. Badid C, Mounier N, Costa AM, Desmouliere A: Role of myofibroblasts during normal tissue repair and excessive scarring: Interest of their assessment in nephropathies. Histol Histopathol 2000;15:269–280.

Renal Fibrogenesis

9

24

25 26

27 28

29

30 31 32

33

34 35 36 37

38 39 40 41

42 43 44 45

46

Ina K, Kitamura H, Tatsukawa S, Takayama T, Fujikura Y, Shimada T: Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy. Med Electron Microsc 2002;35:87–95. Moriyama T, Imai E: Role of myofibroblasts in progressive renal diseases; in Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 120–140. Razzaque MS, Kumatori A, Harada T, Taguchi T: Coexpression of collagens and collagen-binding heat-shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80:434–443. Razzaque MS, Koji T, Taguchi T, Harada T, Nakane PK: In situ localization of type III and type IV collagen-expressing cells in human diabetic nephropathy. J Pathol 1994;174:131–138. Razzaque MS, Koji T, Kawano H, Harada T, Nakane PK, Taguchi T: Glomerular expression of type III and type IV collagens in benign nephrosclerosis: Immunohistochemical and in situ hybridization study. Pathol Res Pract 1994;190:493–499. Razzaque MS, Koji T, Harada T, Taguchi T: Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections. Histochem Cell Biol 1999;111:1–6. Razzaque MS, Koji T, Harada T, Taguchi T: Identification of type VI collagen synthesizing cells in human diabetic glomerulosclerosis using renal biopsy sections. Anal Cell Pathol 1997;15:175–181. Tamaki K, Okuda S: Role of TGF-␤ in the progression of renal fibrosis; in Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 44–65. Border WA, Noble NA, Yamamoto T, Harper JR, Yamaguchi Y, Pierschbacher MD, Ruoslahti E: Natural inhibitor of transforming growth factor-␤ protects against scarring in experimental kidney disease. Nature 1992;360:361–364. Isaka Y, Brees DK, Ikegaya K, Kaneda Y, Imai E, Noble NA, Border WA: Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat Med 1996;2: 418–423. Cerwenka A, Swain SL: TGF-␤1: Immunosuppressant and viability factor for T lymphocytes. Microbes Infect 1999;1:1291–1296. Reinhold D, Wrenger S, Kahne T, Ansorge S: HIV-1 Tat: Immunosuppression via TGF-␤1 induction. Immunol Today 1999;20:384–385. Grotendorst GR: Connective tissue growth factor: A mediator of TGF-␤ action on fibroblasts. Cytokine Growth Factor Rev 1997;8:171–179. Grotendorst GR, Okochi H, Hayashi N: A novel transforming growth factor-␤ response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ 1996;7:469–480. Gupta S, Clarkson MR, Duggan J, Brady HR: Connective tissue growth factor: Potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 2000;58:1389–1399. Nagata K: Expression and function of heat-shock protein 47: A collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 1998;16:379–386. Razzaque MS, Ahsan N, Taguchi T: Heat-shock protein 47 in renal scarring. Nephron 2000;86: 339–341. Razzaque MS, Hossain MA, Kohno S, Taguchi T: Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat-shock protein 47. Virchows Arch 1998;432:455–460. Liu D, Razzaque MS, Nazneen A, Naito T, Taguchi T: Role of heat-shock protein 47 on tubulointerstitium in experimental radiation nephropathy. Pathol Int 2002;52:340–347. Razzaque MS, Taguchi T: Collagen-binding heat-shock protein 47 expression in anti-thymocyte serum-induced glomerulonephritis. J Pathol 1997;183:24–29. Liu D, Razzaque MS, Cheng M, Taguchi T: The renal expression of heat-shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J 2001;33:623–630. Naitoh M, Hosokawa N, Kubota H, Tanaka T, Shirane H, Sawada M, Nishimura Y, Nagata K: Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid. Biochem Biophys Res Commun 2001;280:1316–1322. Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heat-shock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183–1188.

Razzaque/Taguchi

10

47

48

49

50

51 52

53 54 55 56

57

58

59

60

61 62

63

Razzaque MS, Ahmed AR: Collagens, collagen-binding heat-shock protein 47 and transforming growth factor-␤1 are induced in cicatricial pemphigoid: Possible role(s) in dermal fibrosis. Cytokine 2002;17:311–316. Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T: Life-long dietary restriction modulates the expression of collagens and collagen-binding heat-shock protein 47 in aged Fischer 344 rat kidney. Histochem J 1999;31:123–132. Matsumura R, Umemiya K, Nakazawa T, Ochiai K, Kagami M, Tomioka H, Tanabe E, Sugiyama T, Sueishi M: Expression of cell adhesion molecules in tubulointerstitial nephritis associated with Sjögren’s syndrome. Clin Nephrol 1998;49:74–81. Isbel NM, Hill PA, Foti R, Mu W, Hurst LA, Stambe C, Lan HY, Atkins RC, Nikolic-Paterson DJ: Tubules are the major site of m-CSF production in experimental kidney disease: Correlation with local macrophage proliferation. Kidney Int 2001;60:614–625. Razzaque MS, Foster CS, Ahmed AR: Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2002;43:2977–2983. Lan HY, Yang N, Nikolic-Paterson DJ, Yu XQ, Mu W, Isbel NM, Metz CN, Bucala R, Atkins RC: Expression of macrophage migration inhibitory factor in human glomerulonephritis. Kidney Int 2000:57:499–509. Lan HY, Yang N, Metz C, Mu W, Song Q, Nikolic-Paterson DJ, Bacher M, Bucala R, Atkins RC: TNF␣ up-regulates renal MIF expression in rat crescentic glomerulonephritis. Mol Med 1997;3:136–144. Razzaque MS, Ahsan N, Taguchi T: Role of apoptosis in fibrogenesis. Nephron 2002;90:365–372. Savill J, Smith J, Sarraf C, Ren Y, Abbott F, Rees A: Glomerular mesangial cells and inflammatory macrophages ingest neutrophils undergoing apoptosis. Kidney Int 1992;42:924–936. Kashihara N, Sugiyama H, Makino H: Implication of apoptosis in progression of renal diseases; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 156–172. Wang R, Ibarra-Sunga O, Verlinski L, Pick R, Uhal BD: Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor. Am J Physiol Lung Cell Mol Physiol 2000;279:L143–L151. Miyazaki M, Nishino T, Abe K, Furusu A, Koji T, Kohno S: Regulation of renal extracellular matrix metabolism; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 141–155. McMillan JI, Riordan JW, Couser WG, Pollock AS, Lovett DH: Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy. J Clin Invest 1996;97:1094–1101. Naito T, Razzaque MS, Nazneen A, Liu D, Nihei H, Koji T, Taguchi T: Renal expression of Ets-1 protooncogene during progression of rat crescentic glomerulonephritis. J Am Soc Nephrol 2000;11:2243–2255. Huovila AP, Almeida EA, White JM: ADAMs and cell fusion. Curr Opin Cell Biol 1996;8:692–699. Hurskainen TL, Hirohata S, Seldin MF, Apte SS: ADAM-TS5, ADAM-TS6 and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family. J Biol Chem 1999;274:2555–2563. Diamantis I, Luthi M, Hosli M, Reichen J: Cloning of the rat ADAMTS-1 gene and its downregulation in endothelial cells in cirrhotic rats. Liver 2000;20:165–172. Takashi Taguchi, MD, PhD, Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4, Sakamoto, Nagasaki 852–8523 (Japan) E-Mail [email protected]

Renal Fibrogenesis

11

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 12–19

Disturbance of Syncytial Cell Function in Glomerular Mesangial Cells Involved in the Progressive Glomerular Diseases Takashi Oite, Jian Yao, Tetsuo Morioka Department of Cellular Physiology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Introduction

Until now it has been proposed that glomerular hemodynamic changes or glomerular growth response may promote the development of glomerulosclerosis, irrespective of etiology [1, 2]. However, the cellular and molecular mechanisms leading to progressive glomerulosclerosis still remain unclear. Here, we present a new concept of the pathogenesis of progressive glomerulosclerosis, based on the accumulating evidence obtained from our work and that of others.

Modulation of Mesangial Cell Behavior by Cell-Matrix and/or Cell-Cell Interaction (in vitro Approach for Clarifying the Mechanism Leading to Progressive Glomerulosclerosis)

In the normal glomerulus, the endocapillary region is a closed room surrounded by the glomerular basement membrane comprised of a capillary lumen, endothelial cells, mesangial cells and mesangial matrices. Communication circuits between cells, or cells and matrices, are considered to be pivotal for the maintenance of normal tissue physiology and for the initiation and progression of pathophysiologic abnormalities such as glomerular inflammation and glomerulosclerosis. As shown in figure 1, mesangial cell growth and

Regulation by extracellular matrix and/or vascular endothelial cells (⫹)

Stable (like physiological state in vivo)

(⫺)

Stimulated (like pathological state in vivo)

Fig. 1. Mesangial cell behavior in vitro versus in vivo.

turnover are very limited in the physiological state in vivo. Mesangial cells form a tree-like branching network between the glomerular capillary loops, and extend their cytoplasm within a three-dimensional mesangial matrix which contains type IV, V and VI collagens, laminin, fibronectin, entactin and proteoglycans. When transferred to an in vitro culture system, mesangial cells change their phenotypes, exemplified by expression of ␣-smooth muscle cell actin and production of the interstitial collagen type I. In addition, they proliferate actively on the plastic plate [3]. We have investigated the interaction between cells, and cells and matrices in the in vitro culture system, as described below. To elucidate the role of glomerular endothelial cells, we examined the effect of mesangial cell proliferation under a condition of cell-cell contact or without contact in the co-culture system [4]. In the cell contact culture system, the growth of rat mesangial cells was significantly inhibited up to day 3 of culture. This inhibition of growth could not be detected in co-culture of fibroblasts with mesangial cells. For this experiment, we used cultured human umbilical vein endothelial cells as a source of vascular endothelial cells due to the methodological ease. The same pattern of suppression in rat mesangial cell proliferation was found preliminarily using cultured rat aortic endothelial cells obtained in our laboratory [5]. Interstitial type I and III collagens are known to be localized in the mesangium or sclerotic lesions in a variety of glomerular diseases [6–8]. We investigated the effect of various extracellular matrix components on the behavior of mesangial cells in a three-dimensional gel culture system [9]. When cultured on a reconstituted basement membrane, Matrigel, mesangial cells aggregated and formed isolated colonies, and thereafter extended an array of cell bipolar processes to form a dendritic network structure. Interestingly, they proliferated very slowly as the culture time progressed, like in vivo physiological condition. Taken together, we propose that mesangial cell behavior without cell contact with endothelial cells and/or extracellular matrices in vitro is analogue to

New Aspects for Clarifying the Mechanisms of Progressive Glomerulosclerosis

13

that of mesangial cells in the pathological condition in vivo [10], as shown in figure 1.

Function of Glomerular Mesangial Cells as a Syncytium

It is about a quarter of century ago that a high density of gap junctional intercellular communication (GJIC) was found first between both glomerular and extraglomerular mesangial cells using a freeze-fracture technique [11]. Until now, however, the knowledge about the functional role of GJIC in mesangial cells is very limited. Most of the work concerning gap junction protein, named by connexins, has been done in the fields of development, cancer, neuroscience and cardiology. Recently, we have reconsidered the functional role of GJIC in mesangial cells [12], because there is little information on the specific biological details of how gap junctions work to coordinate mesangial cell behavior. The gap junction protein, connexin 43, expressed ubiquitously in a variety of cell types, was examined by immunohistochemistry, immunoprecipitation and Western blot to reconfirm its presence in the mesangial cells. Next, the strength of GJIC was evaluated by single cell injection of Lucifer yellow (fig. 2). Microinjection of this fluorescent dye into a single mesangial cell resulted in the transmission of a network of about 10–20 cells. Third, we attempted to examine GJIC function in the pathological condition such as in response to the inflammatory cytokines. Platelet-derived growth factor (PDGF) is the most potent mitogen for mesangial cells and wellknown active player in mesangial cell contraction and extracellular matrix synthesis as well [13]. The addition of PDGF into mesangial cell culture induced a rapid and transient inhibition of GJIC, followed by increase of tyrosine phosphorylation of connexin 43. This response to PDGF was remarkably inhibited by pretreatment of mesangial cells with phosphatidylinositol 3⬘-kinase inhibitor. In addition, PDGF also caused activation of extracellular signalregulated kinase (ERK). The relevant chain reactions produced by PDGF are summarized in figure 3. We can imagine that a sophisticated cellular communication may exist between extraglomerular and glomerular mesangial cells, mesangial cells and vascular endothelial and mesangial cells of both afferent and efferent arteries, mesangial cells and renin-secreting granular cells, and so on. And these mutual cell communications may provide the mesangium with the characteristics of a functional syncytium through GJIC, exemplified by the potential role of the gap junctions in the regulation of renal hemodynamics in vivo. Further investigation containing mesangial cell contraction via GJIC signaling is ongoing in our laboratory.

Oite/Yao/Morioka

14

Single cell microinjection Lucifer yellow Ethidium bromide

a

b

c

Fig. 2. Lucifer yellow diffusion from a microinjected MC. a–c Arrow indicates the microinjected mesangial cell.

PDGF

src activation

*

MAPK activation

*Phosphorylation of tyrosine residues

*

PKC activation

Phosphorylation of serine/threonine Connexin 43

Conformational change of connexin Disclosure of gap junctional communication

Fig. 3. Potential mechanisms responsible for the disruption of GJIC. Blockade sites of PI3K inhibitors are indicated by asterisks.

Experimental Models of Progressive Glomerulosclerosis (Approach from in vivo Study)

There are very few experimental models of glomerulosclerosis with an irreversible course. The 5/6 ablation model has been considered to be most famous and reliable. We have learned a great deal from this experimental

New Aspects for Clarifying the Mechanisms of Progressive Glomerulosclerosis

15

Male Wistar rat MoAb1-22-3 0.5mg intravenous injection After 30min Uninephrectomy ( i.v. ⫹Nx group)

Sham operation ( i.v.⫹ sham group)

Sacrificed at days 3, 14, 56 and 84 after injection

Fig. 4. Experimental schedule: progressive glomerulosclerosis induced in the rat by a one-shot injection of anti-Thy-1.1 monoclonal antibody followed by unilateral nephrectomy.

model, e.g., disease association of glomerular hyperfiltration, hyperfusion, hypertension and hypertrophy [2]. However, a few problems exist in this model that there is some variability in disease severity and glomerular lesions among the animal strains used and still among the same strain of rats used in the same experimental schedule. Therefore, further experimental models with high reproducibility are needed to clarify the cellular and molecular mechanisms. Very recently, we presented details that progressive glomerulosclerosis could be induced in the rat by a one-shot injection of anti-Thy-1.1 monoclonal antibody followed by unilateral nephrectomy, as shown in figure 4 [14]. This experimental model has several advantages for analyzing the progression factors leading to the irreversible glomerulosclerosis. First, we can compare the disease course between the nephrectomized (one-kidney) and sham-operated (twokidney) groups in which the same dose of nephritogenic antibody is bound to each kidney. Second, there is a sharp difference in the prognosis of disease between one- and two-kidney models. We already know that the two-kidney model is fundamentally reversible [8]. In contrast, progressive glomerulosclerotic lesions with renal insufficiency occurred in the one-kidney model. Third, we can use the different rat strains for inducing glomerulonephritis, e.g. in Wistar rats and Munich-Wistar rats. In Munich-Wistar rats, we found similar glomerular lesions such as mesangiolysis with segmental microaneurysm-like ballooning of the capillary loop 3 days after injection of the same antibody [15]. The advantage of using this special strain is that many glomeruli are located directly under the capsular surface of the kidney cortex, allowing us to examine easily not only glomerular hemodynamic changes, but also morphological recovery of the glomerular capillary, using a confocal laser scanning microscope. A real-time and three-dimensional analysis of glomerular hemodynamics revealed that images of the repaired vessel were notched and blood flow

Oite/Yao/Morioka

16

was retarded, suggesting that regeneration of glomerular capillary was not completed. Here, we focus on the nephritogenic epitope determined by the monoclonal antibody against rat mesangial cells, which has been used for the induction of the relevant glomerular injury in our laboratory. An epitope recognized by anti-Thy-1 monoclonal antibody, named by 1-22-3, was concentrated specifically on the mesangial cell surfaces facing the neighboring endothelial cells in situ and in vitro co-culture system [16–18]. Impressively, there was a sharp difference in localization and cell signaling between this antibody and the commercially available monoclonal anti-Thy-1 antibody, OX-7. OX-7 bound to mesangial cell around all cell surfaces and extracellular components as well. These two different epitopes were determined to be presented on the whole molecule of Thy-1.1 which was expressed on COS-1 cells transfected with the entire coding region of cDNA for Thy-1.1. From a cell physiopathological aspect, it should be emphasized that there is a 2–3 times higher inositol triphosphate production and a greater response in Thy-1.1-mediated increase in intracellular free calcium concentration [19] and a physical association of protein tyrosine kinases [20] by 1-22-3-treated than OX-7-treated mesangial cells. Taken together with the circumstantial evidence showing the stronger nephritogenicity of 1-22-3 than that of OX-7 [8], we believe that the epitope recognized by 1-22-3 is important in the initiation and progression of antibody-induced glomerulonephritis. Using this one-kidney model induced by anti-Thy-1 antibody injection, we have shown that there was a positive association between impairment of vascular regeneration and development of glomerulosclerosis. In brief, semiquantitative analysis revealed that the capillary density and mRNA expression of PECAM-1, VCAM-1 and VEGF were significantly lower in the one-kidney group compared to the two-kidney group on day 14 after the disease induction. On day 84, progressive glomerulosclerosis lesions were found, followed by a decrease of the capillary density in the one-kidney group, while glomerular structure recovered to an almost normal state in the two-kidney group. Assuming the process of glomerular repair in the one-shot, reversible model of the anti-Thy-1 model, a return to the normal architecture of the glomerular capillary tuft may need well-balanced recruitment of mesangial and endothelial cells, and extracellular matrix. As described in the section of in vitro study (fig. 1), mesangial cell behavior was regulated by the intercellular contact with endothelial cells, and by the mesangial matrix. Without cell contact with endothelial cells or physiological components of the extracellular matrix, mesangial cells showed some pathological features such as an increased proliferative activity and production of interstitial collagen, which is often used as a marker in progressive glomerulosclerosis.

New Aspects for Clarifying the Mechanisms of Progressive Glomerulosclerosis

17

Concluding Remarks

Our final goal is to stop the progression of glomerulosclerosis leading to renal insufficiency, or to repair the sclerotic lesions, returning to the non-perplexed condition in chronically progressive glomerular diseases such as diabetic nephropathy and IgA nephritis. Key issues in future studies will be to confirm whether disturbance of syncytial cell function in mesangial cells persists, leading to irreversible glomerulosclerosis, and whether the vicious circle resulting in irreversible glomerulosclerosis can be suppressed or returned to normal. For these purposes, we have not only done experiments in the in vitro system at cellular and molecular levels, but also performed integrated studies using in vivo studies as described above, or isolated glomeruli [21–24] as well as in an ex vivo system using isolated kidney perfusion. We will need further work and time to extrapolate our knowledge obtained from in vitro, ex vivo and in vivo experiments for the clarification of pathogenesis and therapy in humans.

Acknowledgments We thank Prof. Dr. A. Vogt and Prof. Dr. S. Batsford in Freiburg, Germany, for their continuous encouragement. This work was supported by research grants (04670199, 06670215, A11770597, A13770598, 10670988, 12671032) from the Ministry of Education, Science, Sports and Culture, and from the Study Group on IgA nephropathy, and a grant for The Promotion of Niigata University Research Projection.

References 1 2 3 4 5 6 7

8

Hostetter TH, Olson JL, Rennke HG, Venkatachalam MA, Brenner BM: Hyperfiltration in remnant nephrons: A potentially adverse response to renal ablation. Am J Physiol 1981;241:F85–F93. Fogo AB, Kon V: Pathophysiology of progressive renal disease; in Neilson EG, Couser WG (eds): Immunologic Renal Diseases. Philadelphia, Lippincott-Raven, 1997, pp 683–726. Floege J, Redeke HR, Johnson RJ: Glomerular cells in vitro versus the glomerulus in vivo. Kidney Int 1994;45:360–368. Saeki T, Morioka T, Arakawa M, Shimizu F, Oite T: Modulation of mesangial cell proliferation by endothelial cells in coculture. Am J Pathol 1991;139:949–957. Oite T, Suzuki Y, Morioka T, Saito M, Shimizu F: Efficient isolation of rat aortic endothelial cells by elimination of contaminating cells with a monoclonal antibody. Microvasc Res 1995;50:113–118. Yoshioka K, Takemura T, Akano N, Matsubara, Ooshima A, Maki S: Distribution of type I collagen in human disease in comparison with type III collagen. J Pathol 1990;162:141–148. Striker LM, Killen PD, Chi E, Striker GE: The composition of glomerulosclerosis. I. Studies in focal sclerosis, crescentic glomerulonephritis and membranoproliferative glomerulonephritis. Lab Invest 1984;51:181–191. Nakayama H, Oite T, Kawachi H, Morioka T, Kobayashi H, Orikasa M, Arakawa M, Shimizu F: Comparative nephritogenicity of two monoclonal antibodies of rat Thy-1.1 molecule. Nephron 1998;78:453–463.

Oite/Yao/Morioka

18

9

10

11 12 13 14

15

16

17 18 19

20 21 22 23

24

Saito K, Shimizu F, Sato T, Oite T: Modulation of human mesangial cell behaviour by extracellular matrix components – The possible role of interstitial type III collagen. Clin Exp Immunol 1993;91:510–515. Oite T, Saito M, Morioka T: Modulation of mesangial cell behavior by communication circuits between extrinsic and intrinsic cells or cells and matrices: Niigata Symposium of Nephrology 1995; in Arakawa M, Nakagawa Y (eds): Recent Advances in Molecular Nephrology. Niigata, Kohoko-Do, 1995, pp 98–112. Pricam C, Humbert F, Perrelet A, Orci L: Gap junctions in mesangial and lacis cells. J Cell Biol 1974;63:349–354. Yao J, Morioka T, Oite T: PDGF regulates gap junction communication and connexin 43 phosphorylation by PI 3-kinase in mesangial cells. Kidney Int 2000;31:1915–1926. Sterzel RB, Rupprecht HD: Glomerular mesangial cells; in Neilson EG, Couser WG (eds): Immunologic Renal Diseases. Philadelphia, Lippincott-Raven, 1997, pp 595–626. Wada Y, Morioka T, Oyanagi-Tanaka Y, Yao J, Suzuki Y, Gejyo F, Arakawa M, Oite T: Impairment of vascular regeneration proceeds progressive glomerulosclerosis in anti-Thy-1 glomerulonephritis. Kidney Int 2002;61:432–443. Oyanagi-Tanaka Y, Yao J, Wada Y, Morioka T, Suzuki Y, Gejyo F, Arakawa M, Oite T: Real-time observation of hemodynamic changes in glomerular aneurysms induced by anti-Thy-1 antibody. Kidney Int 2001;59:252–259. Kawachi H, Orikasa M, Matsui K, Iwanaga T, Toyabe S, Oite T, Shimizu F: Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen. Clin Exp Immunol 1992;88:399–404. Kawachi H, Oite T, Shimizu F. Quantitative study of mesangial injury with proteinuria induced by monoclonal antibody 1-22-3. Clin Exp Immunol 1993;92:342–346. Oite T, Saito M, Suzuki Y, Arii T, Morioka T, Shimizu F: A specific Thy-1 molecular epitope expressed on rat mesangial cells. Exp Nephrol 1996;4:350–360. Narisawa-Saito M, Kimura S, Fujiwara N, Oite T, Shimoji K, Shimizu F: Thy-1-mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell. J Cell Physiol 1996;168:705–710. Narisawa-Saito M, Yamanashi Y, Morioka T, Oite T, Shimizu F: Thy-1 molecule associates with protein tyrosine kinase in rat mesangial cells. Clin Exp Immunol 1996;106:86–90. Omori S, Morioka T, Wakasugi M, Kawachi H, Arakawa M, Shimizu F, Oite T: Quantification of apoptotic cells using isolated glomeruli. Nephron 1997;77:474–478. Oite T: Sensitive analysis of apoptosis using confocal laser scan microscopy. Exp Nephrol 2000;8:312–314. Li B, Yao J, Hoshiyama M, Morioka T, Suzuki Y, Oite T: Nitric oxide increases albumin permeability of isolated rat glomeruli via a phosphorylation-dependent mechanism. J Am Soc Nephrol 2001;12:2616–2624. Yao J, Morioka T, Li B, Oite T: Coordination of mesangial cell contraction by gap junctionmediated intercellular Ca2⫹ wave. J Am Soc Nephrol 2002;13:2018–2026.

Dr. Takashi Oite, Department of Cellular Physiology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Niigata 951–8510 (Japan) Tel. ⫹81 25 2272156, Fax ⫹81 25 2270769, E-Mail [email protected]

New Aspects for Clarifying the Mechanisms of Progressive Glomerulosclerosis

19

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 20–31

Proteinuria and Tubulointerstitial Injury: The Causative Factors for the Progression of Renal Diseases Seiichi Matsuo, Yoshiki Morita, Shoichi Maruyama, Liu Manchang, Yukio Yuzawa Division of Clinical Immunology, Department of Internal Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

Introduction

Tubulointerstitial injury is important because it is more closely related not only to the present renal function, but also to the functional outcome independent of the disease type than the glomerular injury. Tubulointerstitial injury takes place under various conditions. Proteinuria is now thought to be an important factor, and a meritorious number of studies concerning the causative relationship between proteinuria and tubulointerstitial injury have been published to date. It is now widely accepted that proteinuria is not only the marker of glomerular injury, but also the independent factor to promote tubulointerstitial injury. Although the precise mechanism has not been fully elucidated, it is the case in both experimental animal models and in humans with proteinuric renal diseases that persistent proteinuria stimulates proximal tubular epithelial cells (PTECs) to release various cytokines, chemokines and growth factors resulting in the leukocyte infiltration and fibrosis in the interstitial tissue. This hypothesis is also supported by the in vitro experiments. Relationship between Proteinuria and Tubulointerstitial Injury

Tubulointerstitial tissue accounts for about 80% of the whole kidney, and the presence and severity of tubulointerstitial injury has been thought to be an

important factor predicting the bad prognosis of various renal diseases. In 1970, Schainuck et al. [1] reported that the degree of tubulointerstitial injury inversely correlated with the glomerular filtration rate better than that of glomerular injury. Bohle et al. [2] did an elaborate study elucidating that the presence of interstitial fibrosis at the time of biopsy is a good predictor of the worse outcome of renal function in various glomerulonephritides. These studies indicated the importance of tubulointerstitial injury in the progression of renal injury. Nath [3] has proposed the hypothetical mechanism by which tubulointerstitial injury proceeds automatically as the number of working nephrons decreases. Subsequently, various studies have been performed to test this hypothesis [4]. Among the various factors causing tubulointerstitial injury, proteinuria and the plasma proteins leaking from the glomerular barrier into the tubular lumen have been thought to be important candidates of the culprits [5–7]. In the 1990s, many studies focusing on the various plasma proteins contained in the proteinuria were done to show the importance of individual plasma protein. On the other hand, large-scale clinical trials for glomerular diseases revealed that the persistent proteinuria was the predicting factor for the progression of renal injury. For example, the MDRD [8] and GISEN [9] studies showed that the degree of proteinuria positively correlated with the reduction of renal function, especially the nephrotic range of proteinuria was the strong predictor of renal death. The renoprotective effects of angiotensin-converting enzyme (ACE) inhibitor and the selective blocker of type I angiotensin II receptor (ATI blocker) as well as the low protein diet are partially attributable to the reduction of proteinuria.

Evidence Obtained in the Experimental Models Which Supports the Role of Proteinuria in the Tubulointerstitial Injury

There have been many reports concerning the mechanisms of tubulointerstitial injury in animal models with proteinuria. In puromycin aminonucleoside nephrosis models in rats, interstitial infiltration of macrophages and T cells was observed as proteinuria emerged while cellular infiltration disappeared with regression of proteinuria [10]. Low protein diet and ACE inhibitor reduced interstitial infiltration [11]. In this model, various chemoattractants including macrophage chemoattracting protein-1 (MCP-1) [12], osteopontin [13, 14], interferon-inducible protein (IP-10) [15] were upregulated. Transforming growth factor-␤ (TGF-␤) and metalloproteinase inhibitor, both promoting interstitial fibrosis, are reported to increase [16]. The protein overload nephropathy is a model of nephrotic syndrome induced by the repeated injection of bovine serum albumin, and proteinuria occurs via non-immune mechanisms. In this model, too, there was an interstitial infiltration of macrophages and an increase of extracellular matrix,

Proteinuria and Tubulointerstitial Injury

21

and upregulation of MCP-1 and osteopontin expression in PTECs [17]. Apoptosis, which is thought to be one of the important factors of tubulointerstitial injury, increased 30-fold 1 week after induction of disease in the corticomedullary layer [18]. In adriamycin nephropathy, a progressive renal injury in rats characterized by heavy proteinuria and tubulointerstitial injury, expression of latent TGF-␤-binding protein in the glomerular cells, infiltrating macrophages in the renal interstitium and interstitial fibroblasts has been reported [19]. In this model, IP-10 production from tubulointerstitium increased when proteinuria became maximal [20]. Since nuclear factor-␬B (NF-␬B) is a key molecule which mediates production of various cytokines and chemokines at the transcription level, it is reasonable to expect the inhibition of renal injury by inhibiting NF-␬B. Indeed, suppression of NF-␬B activity by pyrradine dithiocarbamate (PDTC) by 73% significantly inhibited macrophage infiltration, tubular atrophy and expansion of interstitial volume in adriamycin nephrosis rats [21]. Taken together, in these experimental findings, proteinuria induces interstitial fibrosis via the common pathway of interstitial infiltration of macrophages and T cells, increased production of extracellular matrices, and dysregulation of extracellular matrix degradation.

Reabsorption and Degradation of Urinary Protein: Mechanisms of Tubulointerstitial Injury Associated with Proteinuria

Under normal conditions, only low-molecular-weight proteins are filtered into the tubular lumen. These proteins are reabsorbed by PTECs through receptor-mediated or non-mediated endocytosis. Part of the reabsorbed proteins are conveyed from the cytoplasm into the systemic circulation, and the rest are transferred to lysosomes and degraded by lysosomal enzymes into amino acids. These amino acids return to the systemic circulation via the basolateral membrane of tubular cells [22]. Under proteinuric conditions, larger sized plasma proteins pass through the glomerular filtration barrier and reach the tubular lumen. Exposure to a large amount of plasma proteins, which are absent under normal conditions, is extensive stress for PTECs. PTECs reabsorb a large amount of filtered proteins by the lysosomal pathway, concentrate them, and form reabsorption droplets. When proteinuria persists and the tubular reabsorption continues, lysosomes are enlarged and finally blow up. As a consequence, cytoplasm is exposed to the lysosomal enzymes and reactive oxygen species emerge resulting in tubular cell injury. Thus, an excessive amount of protein traffic through the glomerular barrier is harmful to the tubular cells. Figure 1 depicts this hypothesis. Although the individual protein contained in

Matsuo/Morita/Maruyama/Manchang/Yuzawa

22

Plasma proteins Celullar effect

Biochemical effect ET-1

ROS

Tf-irons

ROS

Complement

Cytolysis Cytoskeletal alteration

Macrophage infiltration

MCP-1 TNF-␣ IL-6

Lipid chemoattractant Albumin

RANTES

MCP-1

ET-1

NH3

ROS

Lipoproteins

ROS

Proteins

ET-1 Fibroblast activation Osteopontin

Lysosomal rupture

Fig. 1. Relationship between proteinuria and proximal tubular cells.

the urinary proteins is the candidate for the culprit causing tubulointerstitial injury, albumin, transferrin, iron ions, complement proteins and lipoproteins have all been extensively studied. Exposure of these substances to PTECs makes these cells proliferate, increase expression and production of cytokines, chemokines and extracellular matrices, resulting in the promotion of inflammation and fibrosis of renal interstitium. In the following, the role of individual protein in the development of tubulointerstitial injury is reviewed.

Urinary Albumin and Tubulointerstitial Injury

Albumin has been most extensively studied as a causal factor inducing tubular injury. Exposure of albumin to PTECs induces increased expression of various cytokines and chemokines, increased production of extracellular matrix, and mitogenic responses. Normally, the albumin concentration in the glomerular filtrate is 2–3 mg/ml, and the filtered albumin returns to the renal veins by the transtubular mechanism [23]. Under proteinuric conditions, urinary albumin concentration increases and albumin is reabsorbed, transferred to the lysosomes and degraded into amino acids in PTECs. Albumin binds to two receptors, megalin

Proteinuria and Tubulointerstitial Injury

23

[24] and cubulin [25], through which it is reabsorbed. Megalin and cubulin are the multi-ligand receptors and they bind not only albumin but also other proteins. In vitro experiments using cultured PTECs revealed that albumin stimulated and transformed these cells into potent proinflammatory cells. In the nephrotic urine of rats with protein overload nephropathy, a novel nonpolar lipid with potent macrophage chemoattracting activity was detected. Tubular uptake of fatty acid bearing albumin resulted in the release of a novel nonpolar lipid, which had a potent chemoattracting activity, while lipid-free albumin did not induce production of a such lipid in cultured PTECs [26]. Among several fatty acids contained in albumin, palmitate and oleate had a different metabolic destiny. Since palmitate profoundly inhibited the OK cell growth while oleate was stimulatory, certain fatty acids seem to have pathological effects on the PTECs [27]. This hypothesis was challenged by the work showing that rat PTECs in culture, when challenged by the delipidated bovine serum albumin, increased the mRNA expression of MCP-1 with its peak at 4 h and duration for at least 24 h. The MCP-1 expression on the basolateral side was 2.4 times higher than that on the apical portion [28]. MCP-1 expression was mediated by NF-␬B activation [29]. Similar results were reported from a different group where rat PTECs in culture released MCP-1, RANTES (regulated on activation, normal T-cell expressed and secreted) and endothelin-1 by exposure to albumin in a dose-dependent manner. These substances were released mainly on the basolateral side. These phenomena were observed both by lipid-repleted and -depleted albumin, and the increased expression of MCP-1 and RANTES was NF-␬B-dependent [30, 31]. Endothelin-1 is involved in the ischemic injury via peritubular capillary contraction, resulting in the inflammation and fibrosis of renal interstitium. Concerning the mitogenic effect of albumin, increased activity of phosphatidylinositide 3-kinase has been reported to be involved [32].

Complement Activation and Proteinuria-Associated Tubulointerstitial Injury

Tubular injury by the urinary complement activation was first reported by Sato and Ullrich [33, 34]. In the 1980s, it was demonstrated using frozen sections of normal rat kidney that complement was activated via an alternative pathway on the brush border of PTEC [35]. The same group demonstrated that the urine samples of proteinuric patients contained a large amount of C3 and the amount of urinary C3 correlated with C3 deposition in the renal tubules [36]. In 1991, the pioneer work by Ogrodowski et al. [37] showed that a membrane attack complex (MAC) deposited in the luminal side of renal tubular cells, and that MAC was detected in the urine of patients with complement-independent

Matsuo/Morita/Maruyama/Manchang/Yuzawa

24

diseases such as focal glomerulosclerosis and diabetic nephropathy. In experimental animals, it was reported that urinary MAC was detected characteristically in Heymann’s nephritis, a rat model of human membranous nephropathy, but not in other models of glomerulonephritis [38]. In Heymann’s nephritis, urinary MAC excretion was thought to derive from the glomerular epithelial cells but not from complement activation in the tubular lumen. In recent years it was demonstrated that complement activation occurred in the tubular lumen of proteinuric rats, and that tubular activation of complement caused the subsequent tubulointerstitial injury [39, 40]. Complement depletion by cobra venom factor and suppression of complement activation by soluble complement receptor type 1 (sCR1) successfully lessened the tubulointerstitial injury while the level of proteinuria was not altered. In support of this notion, Nangaku et al. [41] demonstrated that tubulointerstitial injury and osteopontine expression were significantly impaired in proteinuric rats with C6 deficiency. These facts strongly suggested that the complement activation in the tubular lumen or on the luminal surface of PTECs is deeply involved in the tubulointerstitial injury associated with proteinuria. It is known that C3 molecules, when reacted with H2O or ammonia, are transformed into C3i or amidated C3 in the fluid phase by cleavage of internal thiol ester bonds. These ‘activated’ C3 molecules degrade other intact C3 molecules into C3a and C3b, resulting in the activation of alternative pathway [42]. Thus, complement activation occurs naturally in our body. Once complement activation occurs, the reaction is enhanced in the C3 accelerating loop. The host cells usually express complement regulatory proteins (membranetype complement regulators) on their surface, and these molecules protect host cells from the autologous complement attack. It was reported in the normal human kidneys that brush borders of PTECs lacked or only weakly expressed, if any, these complement regulators [43]. Ammonia has been thought to be the key molecule for complement activation [44]. Ammonia production in the proximal tubular cells is upregulated in various conditions such as systemic acidosis, significant nephron loss, and proteinuria. Administration of bicarbonate to 5/6 nephrectomized rats reduced ammonia production in the kidney, reduced complement deposition in the tubulointerstitial area and improved the tubulointerstitial injury [45]. Although most of the complement components are produced in the liver, it is now well known that various extrahepatic tissues also synthesize and release various complement components. PTECs produce C3, C4 and factor B, and the production of C3 and C4 is increased by interferon-␥ and other cytokines [46]. It was also reported that C3 expression in the renal cortex was upregulated in patients with membranous nephropathy, lupus nephritis, IgA nephropathy and interstitial nephritis [47]. Factor D, which is necessary for activation of the alternative pathway, is increased in the urine of patients with renal failure [48]. The locally produced complement components might contribute to

Proteinuria and Tubulointerstitial Injury

25

Glomerular damage Local complement production

Complement activation

Glomerular scarring

Non-selective proteinuria

Glomerular hyperfiltration

NH3 increase

Nephronloss

Acidosis Tubulointerstitial injury

NH3 increase per single nephron

Fig. 2. Hypothesis of tubulointerstitial injury by complement activation.

the tubulointerstitial injury. In patients with non-selective proteinuria, complement activation products such as iC3b, Bb and MAC were increased in the urine. The amount of complement activation products in the urine is correlated with the level of proteinuria and the decline rate of renal function. Administration of sodium bicarbonate to the proteinuric patients with mild acidosis and renal insufficiency significantly reduced urinary excretion of complement activation products without affecting proteinuria [49]. In vitro experiments using PTECs in culture revealed that there was an increased generation of reactive oxygen species and disruption of cytoskeletal fibers [50], increased C3 production with polarity [51] and production of TNF-␣ and interleukin-6 [52] when challenged by complement. In the remnant kidney model, IgG and C3 deposition in PTECs was observed 1 week after 5/6 nephrectomy, and macrophages positive for MHC class II antigen infiltrated around the C3 deposition. These abnormalities were significantly inhibited by the ACE inhibitor [53]. Summing up these observations, it is strongly suggested that complement activation in the tubular lumen or on the surface of PTECs mediates proteinuriaassociated tubulointerstitial injury [54]. The possible mechanism is depicted in figure 2.

Iron Ions and Tubulointerstitial Injury

Transferrin and iron generate iron ions in the cytosol when the intracellular circumstance is acidic. The transferrin contained in the urinary protein is reabsorbed by the PTECs and liberates iron ions in the cytosol resulting in the

Matsuo/Morita/Maruyama/Manchang/Yuzawa

26

generation of reactive oxygen species which causes tubular cell injury [55]. In rat models of nephrotoxic serum nephritis [56] and remnant kidney model [57], substances which chelated iron ions significantly improved tubulointerstitial injury caused by the reactive oxygen species [58]. It was also reported that low iron diet partially reduced injury. An in vitro study using cultured PTECs showed that holo-transferrin, but not bovine serum albumin or apo-transferrin, caused a functional alteration because only holo-transferrin induced generation of malondialdehyde, a product of lipid peroxidation [59]. These data suggested the importance of transferrin in the urinary protein as the source of irons and reactive oxygen species which causes tubular damage.

Lipoproteins, Cytokines and Growth Factors

Among the various lipoproteins that leak into the urinary space in patients with proteinuria, high-density lipoprotein and low-density lipoprotein are thought to generate cytotoxic radicals after reabsorption and oxidation in PTECs [60]. PTECs stimulated by high-density lipoprotein produced endothelin-1 [61]. Furthermore, lysophosphatidic acid, which had a growth factor-like activity as a intercellular lipid mediator, showed mitogenic effects on OK cells [62]. Since hyperlipidemia and lipiduria are characteristic in nephrotic patients, lipoproteins seem to have important roles in the development of tubulointerstitial injury. PTECs not only respond to various cytokines and growth factors, they also generate these substances. PTECs from patients with renal diseases showed an increased expression of granulocyte/monocyte colony-stimulating factor and platelet-derived growth factor [63]. The supernatant of these PTECs activated fibroblasts to upregulate the production of extracellular matrices [64]. It was reported that the content of insulin-like growth factor-1 (IGF-1) was increased in the urine of nephrotic patients and rats, and PTECs stimulated by the nephrotic urine or human recombinant IGF-1 revealed proliferation and increased production of type IV collagen. These effects were lessened by the anti-IGF-1 antibody [65]. Mouse PTECs, when challenged with the urine of diabetic nephropathy mice or by recombinant human hepatic growth factor, generated MCP-1 and RANTES [66]. Recently, connective tissue growth factor has been implicated to promote interstitial fibrosis at the downstream of TGF-␤ [67, 68]. Thus, cytokines and growth factors play central roles in tubulointerstitial inflammation and fibrosis. In summary, in patients with chronic glomerulonephritis and diabetic nephropathy associated with non-selective proteinuria, proteinuria itself is the common cause of tubulointerstitial injury. Alteration and damage of PTECs by urinary proteins and subsequent production of various cytokines, growth factors

Proteinuria and Tubulointerstitial Injury

27

and other basoactive substances by PTECs are the common pathway of tubulointerstitial injury [69, 70]. Although it is not well evaluated which protein contributes more to the development of tubulointerstitial injury, many clinical studies carried out on a large scale revealed that the level of proteinuria correlates very well with the poor prognosis of renal function. Therefore, it is very important to reduce urinary protein leak by the combination of ACE inhibitors, AII receptor antagonists, low protein diet, and specific treatment to the glomerular injury such as steroids and immunosuppressants.

References 1 2

3 4 5 6 7 8

9

10 11 12 13

14 15 16 17

Schainuck LI, Striker GE, Cutler RE, Benditt EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1970;1:631–640. Bohle A, Wehrmann M, Bogenschutz O, Batz C, Vogl W, Schmitt H, Muller CA, Muller GA: The long-term prognosis of the primary glomerulonephritis. A morphological and clinical analysis of 1,747 cases. Pathol Res Pract 1992;188:908–924. Nath KA: Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 1992;20:1–17. Eddy AA: Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. J Am Soc Nephrol 1994;5:1273–1287. Burton C, Harris KPG: The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis 1996;27:765–775. Remuzzi G, Ruggenenti P, Benigni A: Understanding the nature of renal disease progression. Kidney Int 1997;51:2–15. Bruzzi I, Benigni A, Remuzzi G: Role of increased glomerular protein traffic in the progression of renal failure. Kidney Int 1997;52(suppl 62):29–31. Peterson JC, Adler S, Burkart JM, Greene T, Hebert LA, Hunsicker L, King AJ, Klahr S, Massry SG, Seifter JL, for the MDRD Study Group: Blood pressure control, proteinuria and the progression of renal disease. Ann Intern Med 1995;123:754–762. Ruggenenti P, Perna A, Mosconi L, Pisoni R, Remuzzi G, on behalf of the Gruppo Italino di Studi Epidemiologici in Nefrologi (GISEN): Urinary protein excretion rate is the best independent predictor of ESRF in non-diabetic proteinuric chronic nephropathies. Kidney Int 1998;53: 1209–1216. Eddy AA, Michael AF: Acute tubulointerstitial nephritis associated with aminonucleoside nephrosis. Kidney Int 1988;33:14–23. Eddy AA, McCulloch L, Liu E, Adams J: A relationship between proteinuria and acute tubulointerstitial disease in rats with experimental nephrotic syndrome. Am J Pathol 1991;138:1111–1123. Eddy AA, Warren JS, Liu E, McCulloch L: Expression and function of monocyte chemoattractant protein-1 in experimental nephrotic syndrome. Clin Immunol Immunopathol 1996;78:140–151. Pichler R, Giachelli CM, Lombardi D, Pippin J, Gordon K, Alpers CE, Schwartz SM, Johenson RJ: Tubulointerstitial disease in glomerulonephritis: Potential role of osteopontin (uropontin). Am J Pathol 1994;144:915–926. Magil AB, Pichler RH, Johnson RJ: Osteopontin in chronic puromycin aminonucleoside nephrosis. J Am Soc Nephrol 1997;8:1383–1390. Tang WW, Qi M, Warren JS, Van GY: Chemokine expression in experimental tubulointerstitial nephritis. J Immunol 1997;159:870–876. Jones CL, Buch S, Post M, McCulloch L, Liu E, Eddy AA: Pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis. Kidney Int 1991;40:1020–1031. Eddy AA, Giachelli CM, McCulloch L, Liu E: Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 1995;47:1546–1557.

Matsuo/Morita/Maruyama/Manchang/Yuzawa

28

18

19 20

21 22

23 24 25

26 27 28 29

30

31

32

33 34 35

36

37 38 39

Thomas ME, Brunskill NJ, Harris KPG, Bailey E, Pringle JH, Furness PN, Walls J: Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy. Kidney Int 1999;55: 890–898. Tamaki K, Okuda S, Ando T, Iwamoto T, Nakayama M, Fujishima M: TGF-␤1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy. Kidney Int 1994;45:525–536. Gomez-Chiarri M, Ortiz A, Gonzalez-Cuadrado S, Seron D, Emancipator SN, Hamilton TA, Barat A, Plaza JJ, Gonzalez E, Egido J: Interferon-inducible protein-10 is highly expressed in rats with experimental nephrosis. Am J Pathol 1996;148:301–311. Rangen GK, Wang Y, Tay YC, Harris DCH: Inhibition of nuclear factor-␬B activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int 1999;56:118–134. Maack T, Park CH, Camargo MJF: Renal filtration, transport and metabolism of proteins; in Seldin DW, Giebisch G (eds): The Kidney: Physiology and Pathophysiology. New York, Raven Press, 1985, pp 1773–1803. Eppel GA, Osicka TM, Pratt LM, Jablonski P, Howden B, Glasgow EF, Comper WD: The return of glomerular-filtered albumin to the rat renal vein. Kidney Int 1999;55:1861–1870. Cui S, Verroust PJ, Moestrup SK, Christensen EI: Megalin/gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol 1996;271:F900–F907. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, Willnow TE, Moestrup SK, Christensen EI: Cubulin is an albumin-binding protein important for renal tubular albumin reabsorption. J Clin Invest 2000;105:1353–1361. Kees-Folts D, Sadow JL, Schreiner GF: Tubular catabolism of albumin is associated with the release of an inflammatory lipid. Kidney Int 1994;45:1697–1709. Thoms ME, Schreiner GF: Contribution of proteinuria to progressive renal injury: Consequences of tubular uptake of fatty acid bearing albumin. Am J Nephrol 1993;13:385–398. Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DCH: Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 1997;8:1537–1545. Wang Y, Rangan GK, Chen J, Tay YC, Wang Y, Harris DCH: Induction of monocyte chemoattractant protein-1 by nuclear factor-␬B in proximal tubule cells. J Am Soc Nephrol 1999;10: 1204–1213. Zoja C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, Ronco P, Remuzzi G: Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 1995;26:934–941. Zoja C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, Remuzzi G: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-␬B activation. Kidney Int 1998;53:1608–1615. Dixon R, Brunskill NJ: Activation of mitogenic pathways by albumin in kidney proximal tubule epithelial cells: Implications for the pathophysiology of proteinuric states. J Am Soc Nephrol 1999;10:1487–1497. Sato K, Ullrich KJ: Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. I. Mechanism of inhibition. Biochim Biophys Acta 1974;343:609–614. Sato K, Ullrich KJ: Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. II. Evidence that complement is involved. Biochim Biophys Acta 1974;343:182–187. Camussi G, Rotunno M, Segoloni G, Brentjens JR, Andres GA: In vitro alternative pathway activation of complement by the brush border of proximal tubules of normal rat kidney. J Immunol 1982;128:1659–1663. Camussi G, Stratta P, Mazzucco G, Gaido M, Tetta C, Casteollo R, Rotunno M, Vercellone A: In vivo localization of C3 on the brush border of proximal tubules of kidneys from nephrotic patients. Clin Nephrol 1985;23:134–141. Ogrodowski JL, Herbert LE, Sedmak D, Cosio FG, Tamerius J, Kolb W: Measurement of SC5b-9 in urine patients with the nephrotic syndrome. Kidney Int 1991;40:1141–1147. Pruchno CJ, Burns MW, Schulze M, Johnson RJ, Baker PJ, Couser WG: Urinary excretion of C5b-9 reflects disease activity in passive Heymann’s nephritis. Kidney Int 1989;36:65–71. Morita Y, Nomura A, Yuzawa Y, Nishikawa K, Hotta N, Shimizu F, Matsuo S: The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis. J Am Soc Nephrol 1997;8:1363–1372.

Proteinuria and Tubulointerstitial Injury

29

40

41 42 43 44 45

46 47 48 49 50

51 52

53

54 55 56 57 58 59 60 61 62 63 64

Nomura A, Morita Y, Maruyama S, Hotta N, Nadai M, Wang L, Hasegawa T, Matsuo S: Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis. Am J Pathol 1997;151:539–547. Nangaku M, Pippin J, Couser WG: Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome. J Am Soc Nephrol 1999;10:2323–2331. Hostetter MK, Gordon DL: Biochemistry of C3 and related thioester proteins in infection and inflammation. Rev Infect Dis 1987;9:97–109. Ichida S, Yuzawa Y, Okada H, Yoshioka K, Matso S: Localization of the complement regulatory proteins in the normal human kidney. Kidney Int 1994;46:89–96. Nath KA, Hostetter MK, Hostetter TH: Increased ammoniagenesis as a determinant of progressive renal injury. Am J Kidney Dis 1991;17:654–657. Nath KA, Hostetter MK, Hostetter TH: Pathophysiology of chronic tubulointerstitial disease in rats. Interaction of dietary acid load, ammonia and complement component C3. J Clin Invest 1985;76:667–675. Sacks SH, Zhou W, Sheerin N: Complement synthesis in the injured kidney: Does it have a role in immune complex glomerulonephritis? J Am Soc Nephrol 1996;7:2314–2319. Sacks SH, Zhou W, Andrews PA, Hartley B: Endogenous complement C3 synthesis in immune complex nephritis. Lancet 1993;342:1273–1274. Volanakis JE, Barnum SR, Guddens M, Galla JH: Renal filtration and catabolism of complement protein D. N Engl J Med 1985;312:395–399. Morita Y, Ikeguchi H, Nakamura J, Hotta N, Yuzawa Y, Matsuo S: Complement activation products in the urine from proteinuric patients. J Am Soc Nephrol 2000;11:700–707. Biancone L, David S, Pietra DV, Montrucchio G, Cambi V, Camussi G: Alternative pathway activation of complement by cultured human proximal tubular epithelial cells. Kidney Int 1994;45:451–460. Tang S, Sheerin NS, Zhou W, Brown Z, Sacks SH: Apical proteins stimulate complement synthesis by cultured human proximal tubular epithelial cells. J Am Soc Nephrol 1999;10:69–76. David S, Biancone L, Caserta C, Bussolati B, Cambi V, Camussi G: Alternative pathway complement activation induces proinflammatory activity in human proximal tubular epithelial cells. Nephrol Dial Transplant 1997;12:51–56. Abbate M, Zoja C, Rottoli D, Corna D, Perico N, Bertani T, Remuzzi G: Antiproteinuric therapy while preventing the abnormal protein traffic in proximal tubule abrogates protein- and complement-dependent interstitial inflammation in experimental renal disease. J Am Soc Nephrol 1999;10:804–813. Matsuo S, Morita Y, Mizuno M, Nishikawa K, Yuzawa Y: Proteinuria and damage to tubular cells – Is complement a culprit?. Nephrol Dial Transplant 1998;13:2723–2726. Shah S: Role of iron in progressive renal disease. Am J Kidney Dis 2001;3(suppl):30–33. Alfrey AC, Froment DH, Hammond WS: Role of iron in the tubulointerstitial injury in nephrotoxic serum nephritis. Kidney Int 1989;36:753–759. Nankivell BJ, Tay YC, Boadle RA, Harris DC: Dietary protein alters tubular iron accumulation after partial nephrectomy. Kidney Int 1994;45:1006–1013. Remuzzi A, Puntorieri S, Brugnetti B, Bertani T, Remuzzi G: Renoprotective effect of low iron diet and its consequence on glomerular hemodynamics. Kidney Int 1991;39:647–652. Chen L, Wang Y, Tay YC, Harris DCH: Proteinuria and tubulointerstitial injury. Kidney Int Suppl 1997;61:60–62. Ong ACM, Moorhead JF: Tubular lipidosis: Epiphenomenon or pathogenetic lesions in human renal disease? Kidney Int 1994;45:753–762. Ong ACM, Jowett TP, Moorhead JF, Owen JS: Human high density lipoproteins stimulate endothelin-1 release by cultured human proximal tubular cells. Kidney Int 1994;46:1315–1321. Dixon RJ, Brunskill NJ: Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI3-kinase and ERK. Kidney Int 1999;56:2064–2075. Frank J, Engler-Blum G, Rodemann HP, Muller GA: Human renal tubular cells as a cytokine source: PDGF-B, GM-CSF and IL-6 mRNA expression in vitro. Exp Nephrol 1993;1:26–35. Frank J, Engler-Blum G, Rodemann HP, Muller GA: The role of interstitial cells in the progression of renal diseases. J Am Soc Nephrol 1992;2(suppl):198–205.

Matsuo/Morita/Maruyama/Manchang/Yuzawa

30

65

66 67

68 69 70

Hirschberg R: Bioactivity of glomerular ultrafiltrate during heavy proteinuria may contribute to renal tubulointerstitial lesions. Evidence for a role for insulin-like growth factor I. J Clin Invest 1996;97:116–124. Wang SN, LaPage J, Hirschberg R: Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy. Kidney Int 2000;57:1002–1014. Ito Y, Goldschmeding R, Bende RJ, Claessen N, Chand M, Kleij L, Rabelink TJ, Weening JJ, Aten J: Kinetics of connective tissue growth factor expression during experimental proliferative glomerulonephritis. J Am Soc Nephrol 2001;12:472–484. Ito Y, Aten J, Bende RJ, Osmar BS, Rabelink TJ, Weening JJ, Goldschmeding R: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998;53:853–861. Wall J: Relationship between proteinuria and progressive renal disease. Am J Kidney Dis 2001;37(suppl 2):13–16. Eddy AA: Role of cellular infiltrates in response to proteinuria. Am J Kidney Dis 2001;37 (suppl 2):25–29.

Dr. S. Matsuo, Division of Clinical Immunology, Department of Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan) Tel. ⫹81 52 7442192, Fax ⫹81 52 7442209, E-Mail [email protected]

Proteinuria and Tubulointerstitial Injury

31

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 32–43

Molecular Mechanism of Cell Injury Yohei Maeshima, Hirofumi Makino Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Introduction

Recent progress in molecular biology and approaches to artificially alter the gene expression, such as gene transfer or gene targeting, greatly contributed in the investigation of the mechanism involved in chronic progressive renal disorders. Some of these milestone studies in recent nephrology will be reviewed and future direction will be discussed. The central features observed in various human glomerular diseases are the proliferation of glomerular mesangial cells as well as the expansion of glomerular extracellular matrix [1–3]. Mesangial cell proliferation and activation (phenotypic alteration) characterizes many types of glomerulonephritis (GN) and is central to the development of glomerulosclerosis and renal failure [1, 2, 4, 5]. Mesangial cells proliferate in response to various growth factors or cytokines such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), IL-1 and IL-6 [6–11]. In this review, the mechanism of the renal involvement of representative growth factors, cell cycle protein, transcription factors and interventional therapeutic approaches for chronic renal disorders will be summarized.

PDGF

Since proliferation of mesangial cells is an important pathologic feature that precedes glomerulosclerosis, several reagents have been tested for their potential capacity to regulate mesangial cell proliferation. PDGF is associated with proliferation of mesangial cells and mesangial matrix expansion [4, 12].

Isaka et al. [13] used an in vivo transfection technique with hemagglutinating virus of Japan (HVJ)-liposome, and introduced either transforming growth factor-␤ (TGF-␤) or PDGF-B gene alone into the kidney. Introduction of PDGF-B mainly induced glomerular cell proliferation, and transfection of TGF-␤ resulted in matrix accumulation in mesangium, and both resulted in glomerulosclerosis [13]. Johnson et al. [14] reported that anti-PDGF-BB antibody inhibited mesangial cell proliferation in vivo using an anti-Thy-1.1induced GN model. The impact of inhibiting PDGF signaling was confirmed later by Nakamura et al. [15] using a gene transfer approach. They generated a chimeric cDNA encoding an extracellular domain of the ␤-PDGF receptor fused with IgG-Fc (PDGFR/Fc). Chimeric PDGFR/Fc molecule completely inhibited the tyrosine phosphorylation of ␤-PDGF receptors and cellular proliferation induced by PDGF in vitro. Then, PDGFR/Fc expression vector was introduced into the muscle of an anti-Thy-1.1 GN rat model by electroporation. A previous report by Tsujie et al. [16] demonstrated the advanced efficiency of electroporation method over the HVJ-liposome method. Plasmid DNA expressing ␤-galactosidase (pCAGGS-LacZ) or luciferase was injected into renal artery followed by in vivo electroporation using a pair of tweezer-type electrodes, resulting in transgene expression in 75% of glomeruli after 4 days [16]. Nakamura et al. [15] employed this technique for PDGFR/Fc gene transfer which significantly reduced the glomerular cell proliferation as well as matrix expansion further confirming the important role of PDGF-B in proliferation of mesangial cells.

TGF-␤1

TGF-␤1 is a multifunctional growth factor with diverse biological actions and plays a central role in accumulation of extracellular matrix in glomerular disorders [17, 18]. TGF-␤1 strongly stimulates the synthesis of extracellular matrix components such as proteoglycan, fibronectin by cultured mesangial and glomerular epithelial cells [19]. TGF-␤1 possesses the ability to suppress the activity of proteases and induce protease inhibitor activity [20]. As previously described, in vivo transfection of TGF-␤1 cDNA-induced expansion of mesangial matrix leading to glomerulosclerosis [13]. Border et al. [21] used an anti-Thy-1.1 GN rat model and administered anti-TGF-␤1 antibody at the time of disease induction. The increased production of extracellular matrix was markedly suppressed and histological manifestations of the disease were dramatically attenuated providing direct evidence for a causal role of TGF-␤1 in the pathogenesis of the experimental GN. These several evidence revealing the pivotal role of TGF-␤1 in the progression of glomerulosclerosis prompted the development of gene therapeutic approaches to regulate the activity of TGF-␤1.

Molecular Mechanism

33

Antisense technology utilizes oligodeoxynucleotides (ODN) complementary to mRNA of the target gene and was developed to inhibit expression of specific genes to evaluate the functional significance of those genes [22, 23]. Akagi et al. [24] introduced antisense ODN (AS-ODN) for TGF-␤1 into the nephritic kidney induced by anti-Thy-1.1 antibody by the HVJ-liposome-mediated gene transfer method. They confirmed that transfected ODN accumulated mainly in the nuclei of mesangial cells in the glomeruli of transfected kidneys, and accumulation of mesangial matrix as well as the expression of TGF-␤1 were markedly attenuated by AS-ODN transfection. The proteoglycan decorin is a known inhibitor of TGF-␤1. Isaka et al. [25] transferred decorin cDNA into rat skeletal muscle using an HVJ-liposome technique to increase the amount of decorin messenger RNA and protein present in skeletal muscle and decorin present in kidney. Transfection of decorin into anti-Thy-1.1 GN-induced rats showed a significant reduction in levels of glomerular TGF-␤1 mRNA and protein, extracellular matrix accumulation and proteinuria [25]. Next, Isaka et al. [26] generated a chimeric cDNA encoding an extracellular domain of the TGF-␤ type II receptor fused to the IgG-Fc domain (TGF-␤-RII/Fc). TGF-␤-RII/Fc diminished the TGF-␤1-induced production of EIIIA-positive fibronectin in cultured normal rat kidney cells. Then, TGF-␤-RII/Fc was introduced into the muscle of the anti-Thy-1.1 GN-induced rats by the HVJliposome-mediated gene transfer method, in order to block the TGF-␤ activity through systemic delivery of chimeric molecules. Treatment with TGF-␤-RII/Fc gene transfection suppressed both the glomerular TGF-␤ mRNA expression and mesangial extracellular matrix accumulation [26]. TGF-␤1 has a wide variety of biological functions and it also acts as the macrophage deactivator potentially attenuating macrophage-mediated acute glomerular injury. Kitamura [27] investigated the role of endogenous TGF-␤1 in modulating the glomerular cell activation triggered by macrophages using stromelysin as an indicator, utilizing an in vivo transfer system of LPS-stimulated reporter macrophages into either normal rat glomeruli or nephritic glomeruli expressing active TGF-␤1. In the normal glomeruli, stromelysin expression was markedly induced in resident cells after the transfer of activated macrophages in contrast to marked repression in those glomeruli producing active TGF-␤1, confirming the hypothesis that TGF-␤1 is an endogenous defender that attenuates certain actions of infiltrating macrophages in the glomerulus [27]. This group previously developed systems to use mesangial cells as well as macrophages as vectors for gene transfer into glomeruli [28, 29]. They further reported an in vivo cytocensor system using the CArG box element as a molecular sensor for glomerular injury [30]. This approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has

Maeshima/Makino

34

subsided [30]. This technique may be applied in combination with AS-ODN for TGF-␤1 or TGF-␤-RII/Fc as novel therapeutic approaches to regulate accumulation of extracellular matrix in progressive GN.

Proliferating Cell Nuclear Antigen (PCNA)/Cyclin/ Cyclin-Dependent Kinase (CDK)

PCNA is a co-factor of DNA polymerase-␦ and is essential for DNA replication [31]. PCNA is required for the progression of the cell cycle from the G1 to S phase. Expression of PCNA is low in quiescent cells but it increases once cells are stimulated by mitogens such as PDGF and bFGF [32]. PCNA has been widely used as a marker of cell proliferation in experimental nephritis models and renal biopsy specimens [7, 33, 34]. Ki-67 is a nuclear protein defined by the monoclonal antibody Ki-67, and is associated with cell proliferation [35]. Intervention of growth factors at the cell surface level to inhibit mesangial cell proliferation has some limitations because there are multiple mitogens and redundant transduction pathways. However, these mitogenic intracellular transduction pathway converges at the G1-S boundary of the cell cycle. We utilized AS-ODN against PCNA and Ki-67 to suppress proliferation of mesangial cells by the intervention at the level of G1-S boundary of the cell cycle, the final common pathway of the mitogenic signals [36]. The antisense and sense phosphorothioate-ODN complementary to PCNA and Ki-67 mRNA, including the initiation codon, were synthesized. Both PCNA and Ki-67 AS-ODN significantly inhibited human mesangial cell proliferation as compared with sense ODN. The expression of target protein and mRNA was inhibited by treating mesangial cells with each AS-ODN. Cell cycle transitions are orchestrated by cyclin and their catalytic partners, cyclin-dependent-kinases (CDK) [37]. In mammalian cells, the cyclin E-CDK2 and cyclin D-CDK4 complexes are catalytically active during G1 phase and phosphorylate Rb leading to the transition of cell cycle into S phase [38]. CDK inhibitory proteins (CKI) prevent proliferation by inhibiting cell cycle progression. Shankland et al. [39–42] extensively studied the changes and the role of CDK and CKI in renal diseases. Normal quiescent rat glomeruli have a differential expression for CKIs; p27Kip1 is highly expressed, and the levels of p21 (Cip1, Waf1, Sdi1) are low [39]. Mesangial cell proliferation in Thy-1 GN is associated with reduced p27Kip1 levels, an increase in glomerular cyclin A and CDK2 activity. The resolution of mesangial cell proliferation was associated with a return to baseline levels for p27Kip1, while the expression for p21 substantially increased. Mesangial p21 expression was maintained following the resolution of proliferation. A complex interplay of cell cycle

Molecular Mechanism

35

regulatory proteins was observed during the glomerular response to injury in vivo. They also examined the changes of CDK and CKI using a remnant kidney model, experimental membranous nephropathy model and human renal biopsy specimens [40–42]. These findings implicate novel biological roles for cell cycle proteins in the regulation of cell hypertrophy and apoptosis [43]. Terada et al. [44] overexpressed p16INK4 (AxCAp16) and p21Cip1 (AxCAp21) into mesangial cells utilizing an adenovirus vector system. AxCAp16 and AxCAp21 caused a significant inhibition in the [3H]thymidine incorporation and mitogen-induced increase in CDK4 kinase activity, and reduced the percentage of cells in S phase. Overexpression of cyclin D1 reduced cell size and increased the percentage of the cells in S and G2 phase. These findings suggest that p16INK4 and p21Cip1 function as inhibitors of the proliferation of mesangial cells induced by growth-promoting factors and that adenovirus-mediated gene transfer of p16INK4 and p21Cip1 may serve as a potential therapeutic approach to mesangial proliferative GN [44]. Increasing understanding of the role for cell cycle regulatory proteins in renal disease may potentially lead to the establishment of novel therapeutic interventions.

E2F

E2F is a transcription factor that was originally identified through its role in the transcriptional activation of the adenovirus E2 promoter [45]. Subsequently, sequences homologous to the E2F-binding site were identified in the upstream regions of several genes encoding proteins involved in the G1-S transition of the cell cycle. These include the genes for dihydrofolate reductase, DNA polymerase, thymidine kinase, thymidilate synthase, c-Myc, c-Myb, cyclin A, cdc2, retinoblastoma gene product and PCNA [46–49]. During the G0 and early G1 phase of the cell cycle, E2F exists in a complex with unphosphorylated Rb or the related protein p107. In this period, E2F does not activate the transcription of its target genes. As the cells progress into the S phase, Rb and p107 are phosphorylated in cooperation with G1 cyclins (cyclins D and E), resulting in the release of E2F from the complex. The released E2F is transcriptionally active, and induces the coordinated transactivation of genes required for DNA synthesis, resulting in cell cycle progression [48]. This cell cycle regulatory role and the finding that E2F was involved in the signaling pathways initiated by several mitogenic signals suggested that suppression of E2F activity may potentially inhibit mesangial cell proliferation. Inoshita et al. [50] studied the role of E2F in the regulation of cultured mesangial cells. The E2F family consists of a group of five closely related proteins (E2F1 through E2F5). In primary cultured mesangial cells, the protein expression of E2F1

Maeshima/Makino

36

a E2F

Rb

Promoter Rb

P DNA polymerase ␣ thymidine kinase cdc2 PCNA

E2F Promoter

b E2F-decoy ODN E2F E2F

Promoter

DNA polymerase ␣ thymidine kinase cdc2 PCNA

Fig. 1. Inhibition of E2F activity by decoy ODN. a In quiescent cells, E2F is bound to Rb protein. Upon stimulation by various mitogens, Rb protein will be phosphorylated and released E2F binds to the consensus E2F-binding site located in the promoter region of downstream genes, resulting in transactivation of genes involved in cell cycle progression. b Double-stranded E2F decoy ODN containing the consensus E2F-binding sequence, binds to free E2F and inhibits its binding to the promoter of downstream genes resulting in the inhibition of transcription of these genes.

through E2F3 was induced by fetal calf serum stimulation, especially E2F1 was strongly induced among them. They then used adenovirus-mediated gene transfer to investigate the function of E2F1. Overexpression of E2F1-induced protein expression of cyclin D1 and cyclin E and increased promoter activity. They concluded that E2F1 plays an important role in the G1/S transition phase [50]. Transcription factor decoy technology has been developed to inhibit the expression of several genes using synthetic double-stranded ODN containing the consensus binding sequence of a specific transcription factor [51, 52] (fig. 1). We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy ODN that contained the consensus E2F-binding site sequence (E2F decoy) [33]. E2F decoy inhibited proliferation of human mesangial cells in a concentration-dependent manner. Electrophoretic mobility shift assays demonstrated that the decoy’s inhibitory effect was due to the specific binding of the decoy ODN to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy-1.1 GN model. E2F decoy ODN

Molecular Mechanism

37

a

b Fig. 2. Effect of E2F decoy treatment on glomerular cell proliferation and matrix expansion. E2F decoy ODN-treated rats with GN (b) exhibited significantly decreased glomerular cellularity and mesangial matrix deposition as compared to animals treated with mismatch control ODN (a). Periodic acid-Schiff staining. ⫻200.

was introduced into the left kidney 36 h after the induction of GN using a cationic multilamellar liposome. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71% [33] (fig. 2). Similar results using cultured mesangial cells were reported by Tomita et al. [53].

Nuclear Factor-␬B (NF-␬B)

NF-␬B comprises a family of dimeric transcription factors that regulate the expression of genes involved in inflammation and cell proliferation [54]. In resting cells, NF-␬B dimers remain in the cytoplasm as an inactive form bound to the inhibitory subunit I␬B. Upon stimulation, I␬B is phosphorylated, ubiquitinated and ultimately degraded by proteolytic cleavage by the proteasome system. Released NF-␬B dimers translocate into the nuclei and activate the transcription of downstream target genes such as IL-1, IL-2, IL-6, TNF-␣, ICAM-1, VCAM-1, metalloproteinases and I␬B␣ [54]. NF-␬B has been found to be activated in experimental GN models [55, 56] as well as in a hydronephrosis model [57]. Activation of NF-␬B can be modulated by pharmacological interventions in vitro and in vivo. It is now widely acknowledged that the antiinflammatory action of steroids is based on the inhibition of the transactivation of NF-␬B-dependent genes [54]. Application of transcription factor decoy to inhibit NF-␬B activation in experimental GN was reported by Tomita et al. [58]. In an anti-glomerular basement membrane (GBM) disease model, the left kidney was perfused with NF-␬B decoy ODN mixed with HVJ-liposome complex. NF-␬B decoy ODN treatment substantially inhibited the renal pathologic

Maeshima/Makino

38

changes with a 50% reduction in proteinuria, a 3-fold reduction in histologic damage such as crescent formation, a 50% reduction in mononuclear leukocyte infiltration, and reduction in the renal expression of IL-1, TNF-␣ and ICAM-1. This study demonstrated the possibility of NF-␬B decoy ODN treatment as a novel therapeutic approach in rapidly progressive GN [58]. Several reports demonstrated that angiotensin II (AII) activates NF-␬B, resulting in inflammatory renal diseases. Angiotensin-converting enzyme inhibitors (ACEI) are known to reduce macrophage infiltration in several models of renal injury. Ruiz-Ortega et al. [59] reported that AII activates NF-␬B, leading to increased synthesis of monocyte chemoattractant protein-1 (MCP-1) and mononuclear cell infiltration in an immune complex nephritis model. They further showed that systemic infusion of AII into normal rats increased renal NF-␬B and AP-1 activity associated with inflammatory cell infiltration and tubular damage [60]. The AT1 receptor antagonist diminished NF-␬B activity in glomerular and tubular cells and abolished renal AP-1 activity, improved tubular damage and normalized the arterial blood pressure. The AT2 receptor antagonist diminished mononuclear cell infiltration and NF-␬B activity in glomerular and inflammatory cells, without any effect on AP-1 and blood pressure. These data suggest that AT1 receptor mainly mediates tubular injury via activation of AP-1/NF-␬B, whereas AT2 receptor activates NF-␬B resulting in the inflammatory cell infiltration in the kidney [60]. Unilateral ureteral obstruction (UUO), an experimental hydronephrosis model, exhibits interstitial inflammatory cell infiltration and interstitial fibrosis. NF-␬B and AP-1 activity increases in the early stage after disease induction contributing to infiltration of inflammatory cells [57]. Pharmacological blockade of AII signaling by ACEI or AT1 receptor antagonist (ARB) attenuated activation of NF-␬B resulting in improved tubulointerstitial damage induced by UUO [61, 62]. Interestingly, the therapeutic effect by ACEI (enalapril) was more remarkable than ARB (SC-51316), and this difference was attributed to the different VCAM-1 levels influencing on monocyte/macrophage infiltration of the kidney during UUO [62]. Recently, Satoh et al. [64] induced a UUO model in AT1a receptor-deficient mice [63]. The AT1a receptor-deficient mice exhibit hypotension and increased renin activity as compared to wild-type mice. UUO in wild-type mice resulted in a marked expansion of tubulointerstitium with increased deposition of type III and IV collagen and a number of infiltrating monocytes/macrophages in the interstitium [63]. In contrast, these changes were significantly attenuated in UUO-induced AT1a-deficient mice. UUO induction resulted in the activation of NF-␬B in wild-type mice but was inhibited in AT1a-deficient mice. The results provide direct evidence that AII acting via the AT1a receptor plays a pivotal role in the development of tubulointerstitial fibrosis in UUO potentially via activation of NF-␬B.

Molecular Mechanism

39

Conclusion

Recent advances in the research of the mechanism of chronic progressive renal diseases were reviewed. Although not included in this review, the role of glomerular epithelial cells (podocytes) and endothelial cells as well as the involvement of apoptosis [65–69] have been also studied extensively. Increasing knowledge on endothelial cells including the mechanism of angiogenesis [70–73] might be suggestive for the understanding of the renal disease progression. Clarification of the mechanism of the disease progression as well as the development of a tissue-targeted drug delivery system may potentially lead to the invention of novel beneficial therapeutic approaches for chronic renal disorders. Acknowledgments This study was supported in part by research grant 09671167 from a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Y.M. is the recipient of a research grant from the Okayama Medical Foundation and the KANAE Foundation for Life & Socio-Medical Science.

References 1 2 3 4 5

6 7

8

9 10 11

Striker LJ, Peten EP, Elliot SJ, Doi T, Striker GE: Mesangial cell turnover: Effect of heparin and peptide growth factors. Lab Invest 1991;64:446–456. Klahr S, Schreiner G, Ichikawa I: The progression of renal disease. N Engl J Med 1988;318:1657–1666. Alpers CE, Hudkins KL, Gown AM, Johnson RJ: Enhanced expression of ‘muscle-specific’ actin in glomerulonephritis. Kidney Int 1992;41:1134–1142. Floege J, Eng E, Young BA, Johnson RJ: Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo. Kidney Int Suppl 1993;39:S47–S54. Makino H, Kashihara N, Sugiyama H, Kanao K, Sekikawa T, Okamoto K, Maeshima Y, Ota Z, Nagai R: Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes. Diabetes 1996;45:488–495. Shultz PJ, DiCorleto PE, Silver BJ, Abboud HE: Mesangial cells express PDGF mRNAs and proliferate in response to PDGF. Am J Physiol 1988;255:F674–F684. Floege J, Eng E, Young BA, Alpers CE, Barrett TB, Bowen-Pope DF, Johnson RJ: Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 1993;92:2952–2962. Floege J, Eng E, Lindner V, Alpers CE, Young BA, Reidy MA, Johnson RJ: Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis and mitogenicity in mesangial proliferative glomerulonephritis. J Clin Invest 1992;90:2362–2369. Abbott F, Ryan JJ, Ceska M, Matsushima K, Sarraf CE, Rees AJ: Interleukin-1␤ stimulates human mesangial cells to synthesize and release interleukins-6 and -8. Kidney Int 1991;40:597–605. Kashihara N, Watanabe Y, Makino H, Kanwar YS: Interleukin-1-induced alterations in glomerular proteoglycans: Biochemical and tissue autoradiographic aspects. J Am Soc Nephrol 1992;3:203–213. Ruef C, Budde K, Lacy J, Northemann W, Baumann M, Sterzel RB, Coleman DL: Interleukin-6 is an autocrine growth factor for mesangial cells. Kidney Int 1990;38:249–257.

Maeshima/Makino

40

12

13

14

15

16 17

18 19 20

21 22 23 24

25 26

27 28

29 30

31 32

33

Yoshimura A, Gordon K, Alpers CE, Floege J, Pritzl P, Ross R, Couser WG, Bowen-Pope DF, Johnson RJ: Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. Kidney Int 1991;40:470–476. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: Glomerulosclerosis induced by in vivo transfection of transforming growth factor-␤ or platelet-derived growth factor gene into the rat kidney. J Clin Invest 1993;92:2597–2601. Johnson RJ, Raines EW, Floege J, Yoshimura A, Pritzl P, Alpers C, Ross R: Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor. J Exp Med 1992;175:1413–1416. Nakamura H, Isaka Y, Tsujie M, Akagi Y, Sudo T, Ohno N, Imai E, Hori M: Electroporationmediated PDGF receptor-IgG chimera gene transfer ameliorates experimental glomerulonephritis. Kidney Int 2001;59:2134–2145. Tsujie M, Isaka Y, Nakamura H, Imai E, Hori M: Electroporation-mediated gene transfer that targets glomeruli. J Am Soc Nephrol 2001;12:949–954. Okuda S, Languino LR, Ruoslahti E, Border WA: Elevated expression of transforming growth factor-␤ and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix. J Clin Invest 1990;86:453–462. Tamaki K, Okuda S: Role of TGF-␤ in the progression of renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 44–65. Border WA, Okuda S, Languino LR, Ruoslahti E: Transforming growth factor-␤ regulates production of proteoglycans by mesangial cells. Kidney Int 1990;37:689–695. Laiho M, Saksela O, Andreasen PA, Keski-Oja J: Enhanced production and extracellular deposition of the endothelial- type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-␤. J Cell Biol 1986;103:2403–2410. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor-␤1. Nature 1990;346:371–374. Wagner RW: Gene inhibition using antisense oligodeoxynucleotides. Nature 1994;372:333–335. Kashihara N, Maeshima Y, Makino H: Antisense oligonucleotides. Exp Nephrol 1998;6:84–88. Akagi Y, Isaka Y, Arai M, Kaneko T, Takenaka M, Moriyama T, Kaneda Y, Ando A, Orita Y, Kamada T, Ueda N, Imai E: Inhibition of TGF-␤1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1996;50:148–155. Isaka Y, Brees DK, Ikegaya K, Kaneda Y, Imai E, Noble NA, Border WA: Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat Med 1996;2:418–423. Isaka Y, Akagi Y, Ando Y, Tsujie M, Sudo T, Ohno N, Border WA, Noble NA, Kaneda Y, Hori M, Imai E: Gene therapy by transforming growth factor-␤ receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1999;55:465–475. Kitamura M: TGF-␤1 as an endogenous defender against macrophage-triggered stromelysin gene expression in the glomerulus. J Immunol 1998;160:5163–5168. Kitamura M, Taylor S, Unwin R, Burton S, Shimizu F, Fine LG: Gene transfer into the rat renal glomerulus via a mesangial cell vector: Site-specific delivery, in situ amplification, and sustained expression of an exogenous gene in vivo. J Clin Invest 1994;94:497–505. Kitamura M, Suto TS: Transfer of genetically engineered macrophages into the glomerulus. Kidney Int 1997;51:1274–1279. Kitamura M, Kawachi H: Creation of an in vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity. J Clin Invest 1997;100:1394–1399. Prelich G, Stillman B: Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell 1988;53:117–126. Jaskulski D, Gatti C, Travali S, Calabretta B, Baserga R: Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors. J Biol Chem 1988;263:10175–10179. Maeshima Y, Kashihara N, Yasuda T, Sugiyama H, Sekikawa T, Okamoto K, Kanao K, Watanabe Y, Kanwar YS, Makino H: Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo. J Clin Invest 1998;101:2589–2597.

Molecular Mechanism

41

34

35

36

37 38 39

40 41

42

43 44

45 46

47 48

49 50 51 52 53

54 55

Witzgall R, Brown D, Schwarz C, Bonventre JV: Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 1994;93:2175–2188. Schluter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, Gerdes J: The cell proliferation-associated antigen of antibody Ki-67: A very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol 1993;123:513–522. Maeshima Y, Kashihara N, Sugiyama H, Makino H, Ota Z: Antisense oligonucleotides to proliferating cell nuclear antigen and Ki-67 inhibit human mesangial cell proliferation. J Am Soc Nephrol 1996;7:2219–2229. Hartwell L: Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1992;71:543–546. Sherr CJ: Mammalian G1 cyclins. Cell 1993;73:1059–1065. Shankland SJ, Hugo C, Coats SR, Nangaku M, Pichler RH, Gordon KL, Pippin J, Roberts JM, Couser WG, Johnson RJ: Changes in cell-cycle protein expression during experimental mesangial proliferative glomerulonephritis. Kidney Int 1996;50:1230–1239. Shankland SJ, Hamel P, Scholey JW: Cyclin- and cyclin-dependent kinase expression in the remnant glomerulus. J Am Soc Nephrol 1997;8:368–375. Shankland SJ, Floege J, Thomas SE, Nangaku M, Hugo C, Pippin J, Henne K, Hockenberry DM, Johnson RJ, Couser WG: Cyclin kinase inhibitors are increased during experimental membranous nephropathy: Potential role in limiting glomerular epithelial cell proliferation in vivo. Kidney Int 1997;52:404–413. Shankland SJ, Eitner F, Hudkins KL, Goodpaster T, D’Agati V, Alpers CE: Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: Role in podocyte proliferation and maturation. Kidney Int 2000;58:674–683. Shankland SJ, Wolf G: Cell cycle regulatory proteins in renal disease: Role in hypertrophy, proliferation and apoptosis. Am J Physiol Renal Physiol 2000;278:F515–F529. Terada Y, Yamada T, Nakashima O, Tamamori M, Ito H, Sasaki S, Marumo F: Overexpression of cell cycle inhibitors (p16INK4 and p21Cip1) and cyclin D1 using adenovirus vectors regulates proliferation of rat mesangial cells. J Am Soc Nephrol 1997;8:51–60. Kovesdi I, Reichel R, Nevins JR: Identification of a cellular transcription factor involved in E1A trans-activation. Cell 1986;45:219–228. Thalmeier K, Synovzik H, Mertz R, Winnacker EL, Lipp M: Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev 1989;3:527–536. Dalton S: Cell cycle regulation of the human cdc2 gene. EMBO J 1992;11:1797–1804. Chittenden T, Livingston DM, Kaelin WG Jr: The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell 1991;65: 1073–1082. Watson RJ, Dyson PJ, McMahon J: Multiple c-myb transcript cap sites are variously utilized in cells of mouse haemopoietic origin. EMBO J 1987;6:1643–1651. Inoshita S, Terada Y, Nakashima O, Kuwahara M, Sasaki S, Marumo F: Regulation of the G1/S transition phase in mesangial cells by E2F1. Kidney Int 1999;56:1238–1241. Kashihara N, Maeshima Y, Makino H: Therapeutic intervention in glomerulonephritis by oligonucleotides. Exp Nephrol 1997;5:126–131. Tomita N, Morishita R, Higaki J, Ogihara T: A novel strategy for gene therapy and gene regulation analysis using transcription factor decoy oligonucleotides. Exp Nephrol 1997;5:429–434. Tomita N, Horiuchi M, Tomita S, Gibbons GH, Kim JY, Baran D, Dzau VJ: An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro. Am J Physiol 1998;275:F278–F284. Guijarro C, Egido J: Transcription factor-␬B (NF-␬B) and renal disease. Kidney Int 2001;59: 4154–24. Sakurai H, Hisada Y, Ueno M, Sugiura M, Kawashima K, Sugita T: Activation of transcription factor NF-␬B in experimental glomerulonephritis in rats. Biochim Biophys Acta 1996;1316:132–138.

Maeshima/Makino

42

56

57 58

59

60

61 62 63

64

65 66 67

68 69 70

71 72

73

Sakurai H, Shigemori N, Hisada Y, Ishizuka T, Kawashima K, Sugita T: Suppression of NF-␬B and AP-1 activation by glucocorticoids in experimental glomerulonephritis in rats: Molecular mechanisms of anti-nephritic action. Biochim Biophys Acta 1997;1362:252–262. Morrissey J, Klahr S: Transcription factor NF-␬B regulation of renal fibrosis during ureteral obstruction. Semin Nephrol 1998;18:603–611. Tomita N, Morishita R, Lan HY, Yamamoto K, Hashizume M, Notake M, Toyosawa K, Fujitani B, Mu W, Nikolic-Paterson DJ, Atkins RC, Kaneda Y, Higaki J, Ogihara T: In vivo administration of a nuclear transcription factor-␬B decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol 2000;11:1244–1252. Ruiz-Ortega M, Bustos C, Hernandez-Presa MA, Lorenzo O, Plaza JJ, Egido J: Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-␬B activation and monocyte chemoattractant protein-1 synthesis. J Immunol 1998;161:430–439. Ruiz-Ortega M, Lorenzo O, Ruperez M, Blanco J, Egido J: Systemic infusion of angiotensin II into normal rats activates nuclear factor-␬B and AP-1 in the kidney: Role of AT(1) and AT(2) receptors. Am J Pathol 2001;158:1743–1756. Morrissey JJ, Klahr S: Rapid communication. Enalapril decreases nuclear factor-␬B activation in the kidney with ureteral obstruction. Kidney Int 1997;52:926–933. Morrissey JJ, Klahr S: Differential effects of ACE and AT1 receptor inhibition on chemoattractant and adhesion molecule synthesis. Am J Physiol 1998;274:F580–F586. Satoh M, Kashihara N, Yamasaki Y, Maruyama K, Okamoto K, Maeshima Y, Sugiyama H, Sugaya T, Murakami K, Makino H: Renal interstitial fibrosis is reduced in angiotensin II type 1a receptor-deficient mice. J Am Soc Nephrol 2001;12:317–325. Sugaya T, Nishimatsu S, Tanimoto K, Takimoto E, Yamagishi T, Imamura K, Goto S, Imaizumi K, Hisada Y, Otsuka A, et al.: Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia. J Biol Chem 1995;270:18719–18722. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota A: Apoptosis in glomerular sclerosis. Kidney Int 1996;49:103–111. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z: Reactive oxygen species induce apoptosis in cultured human mesangial cells. J Am Soc Nephrol 1996;7:2357–2363. Sugiyama H, Kashihara N, Maeshima Y, Okamoto K, Kanao K, Sekikawa T, Makino H: Regulation of survival and death of mesangial cells by extracellular matrix. Kidney Int 1998;54: 1188–1196. Makino H, Kashihara N, Sugiyama H, Sekikawa T, Ota Z: Role of apoptosis in the progression of glomerulosclerosis. Contrib Nephrol. Basel, Karger, 1996, vol 118, pp 41–47. Makino H, Sugiyama H, Kashihara N: Apoptosis and extracellular matrix-cell interactions in kidney disease. Kidney Int Suppl 2000;77:S67–S75. Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, Hopfer H, Xiao Y, Stillman IE, Kalluri R: Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J Biol Chem 2000;275:21340–21348. Maeshima Y, Colorado PC, Kalluri R: Two RGD-independent ␣v␤3 integrin-binding sites on tumstatin regulate distinct anti-tumor properties. J Biol Chem 2000;275:23745–23750. Maeshima Y, Manfredi M, Reimer C, Holthaus KA, Hopfer H, Chandamuri BR, Kharbanda S, Kalluri R: Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin. J Biol Chem 2001;276:15240–15248. Maeshima Y, Sudhakar A, Lively JC, Ueki K, Kharbanda S, Kahn CR, Sonenberg N, Hynes RO, Kalluri R: Tumstatin, an endothelial cell specific inhibitor of protein synthesis. Science 2002; 295:140–143.

Hirofumi Makino, Okayama University Graduate School of Medicine and Dentistry, Department of Medicine and Clinical Science, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan) Tel. ⫹81 86 2357235, Fax ⫹81 86 2225214, E-Mail [email protected]

Molecular Mechanism

43

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 44–65

Role of TGF-␤ in the Progression of Renal Fibrosis Kiyoshi Tamaki, Seiya Okuda Department of Nephrology, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Introduction

Major causes of most glomerular diseases leading to end-stage renal failure, are immunological disorder (primary or secondary glomerulonephritis), metabolic disorder (diabetic nephropathy), and vascular damage (hypertensive nephrosclerosis). Although the mechanisms of the initial injury in patients suffering these renal diseases are different, the final histological feature of chronic progressive renal disorder is uniformly characterized by glomerulosclerosis and interstitial fibrosis [1]. This histopathological feature is common to most cases of progressive renal disease and it is termed ‘renal fibrosis’. Normal renal structures are replaced by fibrotic tissues, which impair renal function and lead to renal failure. Recent advances in molecular biology have revealed that tissue fibrosis is the result of excessive accumulation of extracellular matrix (ECM) [2]. The progression of glomerular diseases is affected by several factors including hyperfiltration, glomerular hypertrophy, glomerular hypertension, proteinuria, hyperlipidemia, and so on [1]. These factors activate glomerular cells, tubular epithelial cells, interstitial cells and infiltrating cells, and make them secrete humoral factors, including growth factors, which induce cell infiltration/proliferation and differentiation. Among a lot of growth factors, transforming growth factor- (TGF-) has the most powerful fibrogenic action (fig. 1) [3–5]. TGF- increases the synthesis of matrix proteins and decreases their degradation in vitro. Overexpression of TGF- has been reported to be found in areas of progressive accumulation of ECM causing kidney fibrosis. Some experiments have shown that a blockade of the TGF- action reduces

Initial pathogenic insult

Normal repair & healing

Renal injury 1. Persistence of initial insult Diabetes mellitus HIV Systemic hypertension 2. Factors affecting the process of glomerular injury Glomerular hypertension Hyperfiltration Proteinuria Angiotensin II Dietary protein Dietary salt intake Hyperlipidemia AGE Glomerular cells Tubular cells Interstitial cells Infiltrating cells

Activation

Sustained up-regulation of TGF- gene expression

Differentiation

Secretion of latent TGF-

Activation of latent TGF-

Accumulation of ECM

Glomerulosclerosis / tubulointerstitial fibrosis Renal fibrosis

End-stage renal failure

Fig. 1. The role of TGF- in the progression of renal fibrosis. AGE, advanced glycation end product; HIV, human immunodeficiency virus; ECM, extracellular matrix.

renal sclerotic and fibrotic changes. Furthermore, recent evidence obtained from experiments in TGF-1 transgenic mice has suggested that TGF- plays a key role in the development of fibrosis in progressive kidney disease. Therefore, therapies that may disrupt renal TGF- activity may be promising in slowing the progression to end-stage renal disease.

TGF- in Renal Fibrosis

45

In this review, we summarized the role of TGF- in the progression of renal diseases to renal fibrosis. In addition, we reviewed recent evidence showing the therapeutic strategy of the fibrotic disease by blocking the effect of TGF-.

Structure of Latent TGF-␤

There are three isoforms of TGF-: TGF-1, TGF-2 and TGF-3 [6]. Each isoform is encoded by a distinct gene and expressed in a tissue-specific and development-regulated fashion [7–11]. Although the three isoforms are highly similar in their biological activities in vitro, each has a distinct biological function in vivo. Among these isoforms, TGF-1 seems to be involved in tissue fibrosis [3]. TGF- is usually secreted as a biologically inactive or latent form with a high-molecular-weight complex (fig. 2), which is required for activation before its action [6, 12]. Latent TGF- is composed of three components: mature TGF-, which is Mr 25,000 disulfide-bonded dimer and biologically active, latency-associated peptide (LAP), which is sufficient for the latency of TGF-, and latent TGF- binding protein (LTBP). Latent TGF- is synthesized as a precursor form. LAP is cleaved from the TGF- precursor by proteolytic processing but remains associated with mature TGF-. Dissociation of LAP from mature TGF- renders TGF- biologically active. LAP is linked to a single molecule of LTBP by a disulfide bond. Latent TGF- complex composed of mature TGF- and LAP is termed ‘small latent TGF- complex’, whereas the latent TGF- complex composed of mature TGF-, LAP and LTBP is termed ‘large latent TGF- complex’. LTBP is involved in functional difference between these two latent complexes. LTBP functions to enhance secretion of latent TGF-, target it to the ECM, act as an anchor for tissue storage of the latent growth factor, and concentrate latent TGF- to the cell surface where activation takes place [6, 12, 13]. In normal rat kidney, glomerular cells and vascular cells produce latent TGF-1 with LTBP whereas tubular epithelial cells secrete latent TGF-1 without LTBP [14]. Immunostaining of LTBP has been shown to localize extracellularly in glomeruli and vessel walls, suggesting that a large latent TGF-1 complex is associated with glomerular and vascular capillary walls. The matrix-associated latent TGF-1 may be necessary for the turnover of matrix proteins to maintain the integrity of the structure of glomeruli and vessels. TGF-1 is thought to be involved in the glomerular vascularization during renal development since the infusion of neutralizing anti-TGF-1 antibody into the kidney of neonatal rats inhibits the organization of endothelial

Tamaki/Okuda

46

TGF- precursor LAP

LTBP

Mature TGF-

Signal peptide

Signal peptide

LAP (80 kDa)

Mature TGF- (25kDa) (125–205kDa)

Small latent TGF- complex (SLTGF-)

Large latent TGF- complex (LLTGF-)

Fig. 2. Structure of latent TGF- (from Miyazawa et al. [148], with permission).

cells into capillaries, capillary lumen formation and endothelial cell fenestration [15].

The TGF-␤/Smad Signaling

Mature TGF- binds to cell-surface receptors termed type I receptor, type II receptor and type III receptor (TR-I, TR-II, TR-III, respectively) [16, 17]. TR-III has a short cytoplasmic domain and has no ability to transduce the TGF- signal. TR-III acts as a modulator of TGF- signaling by binding the ligand and presenting it to TR-II. TR-I and TR-II are serine-threonine kinase receptors. TR-II kinases are constitutively active without ligand binding. Upon ligand binding and formation of type I and type II receptor complexes, followed by receptor conformational changes, type I receptor is phosphorylated and activated by type II receptor kinases. TGF- acts through the Smad pathway [16–20]. The eight Smad proteins (Smad1 to Smad8) have been identified and classified into three groups, i.e. receptor-regulated Smads (R-Smads), common-mediated Smads (Co-Smad), and inhibitory Smads (I-Smad). Smad2 and Smad3 act as R-Smad for TGF- signaling. Ligand-activated TGF- receptor complexes phosphorylate Smad2 and Smad3, which bind to Co-Smad, Smad4. The Smad complexes then go to the nucleus, where they interact with various transcriptional factors to regulate the transcription of many TGF- target genes. I-Smads (Smad6 and Smad7) interfere with the phosphorylation of R-Smads, thereby inhibiting TGF- signaling. I-Smads are also the target gene of TGF-, indicating that I-Smads act as ligand-induced negative regulators of TGF- signals.

TGF- in Renal Fibrosis

47

In addition, there is growing evidence that other pathways, distinct from Smad-mediated modulation of gene transcription, are initiated downstream of the receptors, such as the mitogen-activated and stress-activated protein kinase pathways. In cultured mesangial cells, both of the Smad pathway and the mitogen-activated protein kinase pathway are present and involved in TGF-1-induced type I collagen gene transcription [21–23].

Role of TGF-␤ in Fibrogenesis

TGF- is a multifunctional growth factor, which regulates cell growth, cell differentiation, ECM metabolism, the immune system and induction of apoptosis. TGF- plays a major role in regulating ECM synthesis and degradation. In cultured glomerular mesangial and epithelial cells, and tubular epithelial cells, TGF- stimulates the synthesis of ECM components, including collagen, fibronectin, laminin and proteoglycan [24–27]. It also suppresses matrix degeneration by downregulating the expression of proteases, such as plasminogen activator and collagenase, and by inducing protease inhibitors, such as plasminogen activator inhibitor (PAI) and tissue inhibitor of metalloprotease [28, 29]. Furthermore, TGF- causes a striking increase in the expression of ECM receptor-like integrin on the cell surface [30]. The net effect of TGF- on matrix metabolism is the accumulation of ECM proteins. Although TGF- is essential for wound healing, sustained overexpression of TGF- results in excessive deposition of ECM proteins resulting in tissue sclerosis and fibrosis. Another important role of TGF- in renal fibrosis is its effect on cell differentiation. Fibroblasts are just one of the candidates that participate in tissue fibrosis. In damaged tissues, fibroblasts become activated by known as well as by unknown stimuli, and they secrete matrix proteins and humoral factors including growth factors. Such fibroblasts are called myofibroblasts since they express -smooth muscle actin (-SMA) after its activation [31]. Myofibroblasts found in diseased glomeruli originate from mesangial cells [32]. This phenotype change is induced by TGF-, which increases the expression of -SMA in cultured mesangial cells [33]. In addition, recent evidence has shown that one of the origins of myofibroblasts observed in injured tubulointerstitium is tubular epithelial cells [34, 35]. Tubular epithelial cells can convert to myofibroblasts at the site of injury and express fibroblast-specific protein. The phenotype change from epithelial cells to mesenchymal cells like fibroblasts is called epithelial-mesenchymal transdifferentiation (EMT) [35]. In cultured tubular epithelial cells, treatment with TGF- alone or in combination with epidermal growth factor induces phenotypic changes in fibroblasts to express -SMA

Tamaki/Okuda

48

and/or produce interstitial collagen [35, 36]. Thus, TGF- plays a role in (trans)differentiation during tissue fibrosis and sclerosis.

TGF-␤ and Tissue Repair in Renal Diseases

The original observation that TGF- has a critical role in mesangial matrix expansion is made in a model of acute mesangioproliferative glomerulonephritis induced by the injection of anti-thymocyte serum (ATS), which is termed anti-Thy-1 glomerulonephritis. The injection of ATS induces mesangiolysis, followed by transient and significant mesangial cell proliferation and matrix production. TGF- overexpression is associated with the pathological accumulation of ECM proteins such as fibronectin and small proteoglycan including decorin and biglycan [37]. The intravenous injection of anti-TGF- neutralizing antibody to this model dramatically prevents excessive matrix expansion [38]. Thus, TGF- acts as a key mediator for mesangial matrix production in this model. In addition, the accumulation of ECM has also been shown to be the result of decreased matrix degradation by the induction of protease inhibitor such as PAI-1, which is also induced by TGF- [29]. Furthermore, TGF--induced expression of integrin has also been observed [30]. Taken together, the net effect of the TGF- action is linked to mesangial matrix accumulation in Thy-1 glomerulonephritis. However, the matrix expansion observed in this model is transient and reversible. After the peak of the mesangial expansion, the cell proliferation and matrix accumulation dissolve with the decrease in expression of TGF- as well as matrix proteins, and thereafter glomerular structure returns to normal. Thus, TGF- seems to be necessary for tissue repair after injury in this model.

TGF-␤ and Tissue Fibrosis in Renal Diseases

In contrast to acute reversible glomerulonephritis, studies done using the rat model of progressive renal fibrosis with deterioration of kidney function have shown that sustained overexpression of TGF- is associated with pathological accumulation of ECM proteins, such as collagen, fibronectin, laminin and proteoglycan [3]. The causal injury (glomerulonephritis, diabetes, hypertension) and/or other factors, such as angiotensin II (Ang-II), proteinuria, and dietary protein intake may induce TGF- overproduction, which, in turn, increases matrix production and attenuates its degradation, leading to sclerosis and fibrosis [1, 3, 39]. In animal models of glomerulosclerosis and tubulointerstitial fibrosis [40–48], hypertensive nephrosclerosis [49–51] and diabetic glomerulosclerosis [52], and in human renal diseases, including IgA nephropathy or

TGF- in Renal Fibrosis

49

other types of glomerulonephritis [53, 54], focal glomerulosclerosis [53], diabetic glomerulosclerosis [52], and HIV nephropathy [55, 56], overexpression of TGF- gene or protein have been associated with their pathological findings. Thus, in the progressive renal disease, TGF- acts as a mediator of renal fibrosis. Therefore, TGF- is considered as ‘fibrogenic growth factor’. The most striking evidence that TGF- is a fibrogenic growth factor was obtained from transgenic mice with the TGF-1 gene. Some transgenic mice that overexpress a constitutively active TGF-1 under the control of a specific promoter have been made. Transgenic mice overexpressing the transgene under the control of rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver and kidney [57]. Expression of the transgene is localized to the proximal tubular cells, which are associated with tubulointerstitial fibrosis followed by glomerulosclerosis. In mice transgenic for active TGF-1 under the control of albumin promoter and enhancer, which show liver fibrosis due to the hepatic overproduction of TGF- in a paracrine fashion, the high plasma level of active TGF- protein causes renal fibrosis [58, 59]. In mice bearing the TGF-1 gene under the control of Ren-1c promoter, overproduction of TGF-1, which can be induced by captopril and is limited to the juxtaglomerular cells, results in thickening of the glomerular basement membrane [60]. In addition, gene transfer of TGF- to rat kidney using the HVJ-liposome method causes an overexpression of TGF- in the glomeruli leading to glomerular matrix accumulation [61]. Although the overexpression of TGF- observed in these mice and rats transgenic for TGF-1 may not be physiological, overexpression of TGF- itself might be essential for the progression of organ fibrosis. TGF- overexpression may cause activation of residual cells or infiltrating cells, which, in turn, produce more TGF- leading to a vicious circle of tissue fibrosis.

Activation of Latent TGF-␤

In mice transgenic for TGF-1 it has been shown that TGF-1 plays a central role in tissue fibrosis. However, it is questionable whether the transgenic model can be considered as a prototype of the organ fibrosis observed in human disease and conventional animal models, since TGF- secreted in the transgenic mice is mostly active. Because TGF- is secreted as a latent form, the activation step is one of the key steps for TGF- to exert its function. The activation may be regulated by a strict mechanism to prevent excessive action of TGF- in vivo [6, 12]. In vitro, TGF- is known to be activated by LTBP, thrombospondin-1, SDS, extreme pH (acid or alkali), and proteases such as plasmin and cathepsin [6, 12]. Thrombospondin-1 seems to be an important activator during fetal development by inducing a conformational change of

Tamaki/Okuda

50

latent TGF- to activate it [62]. In some animal models of renal diseases, such as aminonucleoside nephrosis and passive Heymann nephritis, expression of thrombospondin-1, which always precedes tubulointerstitial fibrosis, is closely associated with an increased expression and activation of TGF-1 [47, 63]. However, the precise interrelation between thrombospondin-1 and activation of latent TGF- in renal fibrosis has not been elucidated. Lipoprotein (Lp(a)) inhibits activation of plasminogen to plasmin and thereby negatively affects the activation of latent TGF- [64]. Although Lp(a) seems to be involved in the inhibition of latent TGF- activation in atherosclerosis [64], there is no relation between latent TGF- activation and the degree of atherosclerosis in hemodialysis patients [65]. A recent report has shown that integrin 6 binds to LAP and activates latent TGF-1 by inducing a conformational change of the latent growth factor [66, 67]. Mice lacking the integrin are protected from bleomycininduced pulmonary fibrosis in which TGF- plays a role [67]. In acute glomerulonephritis models, such as Thy-1 glomerulonephritis [37] and anti-glomerular basement membrane nephritis in rabbits [68], mature TGF- is detected in association with matrix production. In contrast, in rat models of chronic and progressive renal fibrosis induced by adriamycin, progressive increase in latent TGF- produced by the damaged tissue is linked to matrix accumulation while the amount of active TGF- is small [40]. The latent TGF- found in this model is associated with matrix proteins via LTBP and may be activated little by little [41]. This may be a possible explanation of the chronicity of adriamycin-induced nephropathy. Further studies are needed to elucidate how TGF- is activated in renal fibrosis.

TGF-␤ and Angiotensin II in Renal Fibrosis

Considerable evidence suggests that the intrarenal renin-angiotensin system plays an important role in the progression of renal fibrosis and sclerosis [69, 70]. Angiotensin-converting enzyme inhibitors (ACEI) and Ang-II receptor blockers (ARB) can attenuate progressive renal fibrosis in animal models [49] and can slow disease progression in human renal diseases [71–73]. Since the agents that interfere with Ang-II activity may decrease glomerular injury without altering glomerular pressure, it has been suggested that Ang-II has direct effects on glomerular cells and induces sclerosis independently of its hemodynamic actions. The effects of Ang-II on matrix metabolism in mesangial cells, tubular cells, fibroblasts and other cells have been shown to be mediated, at least in part, by TGF- [27, 74–79]. Exposure of these cells to Ang-II in vitro increases TGF- expression and secretion as well as its bioactivity. There is large body of evidence showing that Ang-II-induced TGF- expression

TGF- in Renal Fibrosis

51

plays an critical role in the progression of the renal diseases, including progressive mesangial glomerulonephritis [80], unilateral ureteral obstruction [81–83], chronic cyclosporine-induced nephropathy [84], a number of models of hypertensive nephropathy [49, 51, 85] and diabetic nephropathy [86]. ACEI and ARB may interfere with the matrix accumulation induced by Ang-IIinduced TGF- expression. Therefore, these drugs should not be used only as anti-hypertensive medications, but should also be considered as renoprotective agents that modulate tissue remodeling by preventing local TGF- biosynthesis.

TGF-␤ and Diabetic Nephropathy

TGF- has also been shown to act as a key factor for renal fibrosis in experimental models of diabetic kidney disease [87, 88] as well as in patients with diabetic nephropathy [52]. In cultured mesangial cells or tubular epithelial cells, a high concentration of glucose stimulates TGF- expression leading to the production of matrix proteins such as collagen IV [89–91]. In streptozotocin (STZ)induced diabetic rats, the expression of glomerular TGF-1 mRNA increases immediately after the onset of hyperglycemia, followed by a sustained increase of glomerular TGF-1 protein and matrix expansions [92, 93]. Treatment of diabetic rats with intensive insulin normalizes the blood glucose concentration and attenuates the increase of glomerular TGF- expression [92]. Upregulation of renal TGF-1 mRNA expression in renal hypertrophy associated with early diabetic nephropathy is also observed in diabetic NOD mice and BB rats, which are the models of type I diabetes [94]. In addition, TGF-1 mRNA and proteins are also increased in overt diabetic nephropathy in humans [52, 95]. Furthermore, treatment of STZ-induced diabetic mice with neutralizing anti-TGF- antibody prevents glomerular hypertrophy, reduces the increment in kidney weight and attenuates the increase in gene expression of matrix proteins without any effect on blood glucose [96]. Chronic inhibition of TGF- with the neutralizing antibody against the growth factor in db/db mice, a model of type II diabetes, also prevents glomerulosclerosis and renal dysfunction [97]. In this model, TGF- receptor as well as Smad3 may be involved in the progression to renal fibrosis [98]. Ang-II also plays an important role in the development of diabetic nephropathy [99]. Glucose increases expression of the angiotensinogen gene in proximal tubule cells [100]. Treatment with high glucose increases Ang-II production by mesangial cells, which results in stimulation of TGF- secretion, decreases matrix degradation, and increases matrix accumulation [101]. Ang-II type I receptor antagonist, losartan, blocks glucose-induced TGF- secretion [102]. These observations in vitro indicate that high glucose can activate the local renin-angiotensin system. Treatment with an ACE inhibitor, ramipril, of

Tamaki/Okuda

52

the rats with STZ-induced diabetes in vivo prevents tubulointerstitial damage as well as tubular expression of both TGF-1 and collagen IV [103]. Thus, in diabetes, the high levels of glucose stimulate the production of TGF- indirectly, in part, via the renin-angiotensin system. Treatment with high glucose activates protein kinase C (PKC) system which is followed by an enhancement of TGF- activity in cultured mesangial cells [104]. Activated PKC and 1 isoforms are observed in the glomeruli of rats with STZ-induced diabetes [105]. The addition of PKC -selective inhibitor, LY333531, to cultured mesangial cells inhibits the increase in PKC activity induced by high glucose [105]. Treatment of diabetic rats with the PKC inhibitor also decreases PKC activities in vivo and prevents the increase in glomerular mRNA expression of TGF-1 and ECM components, such as fibronectin and collagen IV [105, 106]. A new biological role of hyperglycemia that is emerging is that high glucose induces the accumulation of matrix proteins through the process of non-enzymatic glycation. In culture, Amadori-modified albumin, which is a major glycated protein found in plasma, plays a role in matrix turnover in renal mesangial and endothelial cells. Incubation with glycated albumin induces PKC activity, enhances TGF- bioactivity and stimulates the gene expression and production of matrix proteins, such as fibronectin and collagen IV [107–110]. Advanced glycosylation end product (AGE) accumulation precedes the increase of glomerular TGF-1 as well as the upregulation of ECM proteins, such as tenascin and laminin, in NOD mice [111]. Intravenous administration of AGE-modified albumin to normal rats induces glomerular gene expression of TGF-1 and ECM proteins accompanied with glomerulosclerosis [112, 113]. Neutralization of glycated albumin with anti-glycated albumin antibody, in db/db mice, ameliorates proteinuria, the accumulation of matrix proteins and renal insufficiency [114]. Furthermore, long-term treatment of diabetic OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats, a model of type II diabetic glomerulosclerosis, with AGE inhibitor, OPB-9195, attenuates the increased renal expression of TGF-1 and collagen IV and diminishes the increase of albuminuria [115]. Thus, high glucose-induced activation of the renin-angiotensin system, PKC activity and generation of AGE play key roles in the development of diabetic nephropathy through overexpression of TGF-.

Therapeutic Implication of blocking bioactivities of TGF-␤

Since overexpression of TGF- is the final common pathway leading to end-stage renal disease, blocking the effects of excessive TGF- activity has

TGF- in Renal Fibrosis

53

1. Regulation of TGF-␤ gene expression

Transcription/ translation

TGF- gene

Low protein diet Salt restriction Blockade of renin-angiotensin system Control of blood glucose PKC inhibitor AGE inhibitor Antisense oligonucleotide

Latent TGF-

Activation of latent TGF-

Mature TGF- (active TGF-)

2. Regulation of latent TGF-␤ activation Camostat mesilate (?)

3. Neutralization of mature TGF-␤ Neutralizing antibody · Decorin Soluble receptors · LAP

Receptor/signaling molecules

4. Regulation at the level of receptor Pseudo-receptor Inhibitory Smad

Smad MAPK Transcription of target genes

5. Modulation of intracellular signaling and of promoter of the target genes (?)

Fig. 3. Therapeutic implication of renal fibrosis by modulating TGF- action.

been extensively attempted. Action of TGF- requires that cells produce and secrete this growth factor, that TGF- be activated from latent complexes before binding to its signaling receptor, and that intracellular signaling to the nucleus through Smads or other signaling pathway. Each of these steps may have the potential to be a therapeutic target (fig. 3). Here we summarized the recent experimental data showing that a blockade of TGF- is a possible method to attenuate the progression of renal fibrosis. Regulation of TGF-b Expression As described above, Ang-II induces the production of ECM proteins via TGF- expression. Therefore, it is reasonable that ACEI and ARB be used in

Tamaki/Okuda

54

patients with primary/secondary glomerular diseases and diabetic nephropathy with or without hypertension. A high intake of salt also induces glomerulosclerosis through glomerular TGF- production in rats with normal blood pressure or hypertension [116, 117]. Salt restriction may thus be important to prevent glomerulosclerosis. Protein restriction, which is one of the most common therapies for patients with chronic renal failure, has been shown to delay kidney fibrosis by reducing TGF- expression in animal models of chronic progressive renal diseases [118, 119]. As described above, ACE inhibition, PKC regulation and AGE inhibition could normalize renal TGF- expression, suggesting development of these treatments would be useful to prevent the progression of diabetic nephropathy in the future. In addition to these therapies, direct suppression of TGF- gene expression using antisense oligodeoxynucleotides has been employed to block glomerular matrix expansion in Thy-1 glomerulonephritis [120] as well as interstitial fibrosis in rats with unilateral ureteral obstruction [121]. Regulation of Latent TGF-b Activation If secreted TGF- remains in a latent form, it does not have any effect. Protease inhibitor, camostat mesilate, inhibits generation of active TGF- by suppressing plasmin activity in hepatic stellate cells in vitro [122]. In the in vivo model of hepatic fibrosis, the inhibitor attenuates an increase in hepatic plasmin and active TGF- level, and hepatic fibrosis. Thus, development of a drug that regulates latent TGF- activation can be a possible therapeutic strategy for progressive renal disease or other organ fibrosis. Neutralization of Mature TGF-b Some reagents that neutralize mature (active) TGF- have been used to show the specificity of the TGF- action. Active TGF- can be neutralized by anti-TGF- antibody, decorin [123], LAP [124], soluble TGF- receptor [125–128], and so on. Neutralizing anti-TGF- antibody suppresses glomerular matrix expansion in Thy-1 glomerulonephritis [37] and it also prevents glomerular hypertrophy and renal insufficiency in diabetic animals [96, 97]. Decorin is a small heparan-sulfate proteoglycan, which is induced by TGF- in vitro [24] as well as in anti-Thy-1 glomerulonephritis in rats [37]. Decorin binds to TGF- and neutralizes its action, suggesting that decorin may be the extracellular component of a negative feedback function of the TGF- system [123]. Intravenous infusion of decorin or gene transfer of decorin into rat skeletal muscle, which increases the amount of decorin in muscle and then in plasma, can block glomerular matrix accumulation in anti-Thy-1

TGF- in Renal Fibrosis

55

glomerulonephritis [129, 130]. Gene transfer of chimeric cDNA expressing an extracellular domain of TGF- type II receptor fused to the Fc portion of IgG, into rat skeletal muscles delivers the chimeric molecules systemically, which acts as an soluble receptor and prevents mesangial expansion in anti-Thy-1.1 glomerulonephritis [131]. This evidence suggests the possibility that a blockade of the TGF- activity may prevent matrix deposition. However, the period of these animal models used is short term and anti-Thy-1.1 glomerulonephritis is a reversible glomerular disease model as described above. Similar inhibition studies should be done using experimental animals with long-term and progressive renal fibrosis, similar to human renal diseases, to clarify whether prolonged inhibition of TGF- action attenuates matrix accumulation and prevents the deterioration of renal function. Regulation at the Level of Receptor/Intracellular Signaling A truncated TGF- type II receptor lacking the intracellular domain can bind ligand but cannot transduce the signal leading to the inhibition of the TGF- effect as a dominant-negative receptor. Qi et al. [132] and Nakamura et al. [133] have reported that adenovirus-mediated truncated TGF- type II receptor prevents liver fibrosis [132, 133]. I-Smads are negative regulators of TGF- signaling which bind to type I receptor and block R-Smads activation. Nakao et al. [134] have showed that, in bleomycin-induced lung fibrosis, adenovirus-mediated gene transfer of Smad7, which interferes with Smad2 phosphorylation in vivo, prevents the gene expression of collagen and fibrosis. Modulation of Intracellular TGF-b Signaling and of the Promoter of the Target Gene Antagonizing intracellular TGF- signaling is another strategy to prevent TGF- action. Recently, the intracellular mechanism of TGF- signaling to the nucleus has been intensively investigated. Smad proteins seem to act in concert with other transcription factors, and co-activators and co-repressors, to control the expression of a certain set of TGF- target genes in a cell-specific manner. Regulation of the genes, such as PAI-1 or some collagens, is well understood [17–20]. However, there are many unanswered questions regarding how TGF-/Smad signaling or the mitogen-activated and stress-activated protein kinase pathways are regulated. In the future, the signaling pathway shall be fully elucidated and then the development of drugs, which modulate the expression of TGF- target genes, including ECM proteins, can be expected to follow. Such a drug might be useful as anti-fibrogenic therapy not only in the case of kidney fibrosis but also in fibrosis of other organs.

Tamaki/Okuda

56

Implication for the Diagnosis and Prognosis of Human Renal Diseases

The level of TGF- in plasma or urine may be used as diagnostic or prognostic marker for human renal diseases. Urinary excretion of TGF- is increased in patients with focal glomerulosclerosis [135], membranous nephropathy [136], IgA nephropathy [137], and crescentic glomerulonephritis [137]. Steroid treatment reduces the amount of proteinuria and urinary TGF- excretion in patients with IgA nephropathy [137]. An increase in plasma TGF- has been reported in patients with diabetic nephropathy [138], chronic renal failure [65], and chronic allograft nephropathy [139, 140]. Ang-II receptor antagonists decreases plasma levels of TGF-1 in diabetic patient [141] or transplant patients with chronic allograft nephropathy [139, 140]. The plasma or urine TGF- level may reflect renal TGF- expression in these diseases. Thus, plasma or urinary TGF- level can be a clinically useful parameter to evaluate disease activity or the therapeutic effect of a certain medication. Suthanthiran et al. [142] demonstrated a significantly higher concentration of TGF-1 in African-American patients under dialysis as compared to Caucasian patients. Different polymorphisms of the TGF-1 genes, which is associated with the increase in TGF-1 mRNA in peripheral blood mononuclear cells and plasma TGF-1 concentration, have also been reported to be linked with hypertension in African-American patients [143]. Polymorphisms in the TGF- gene, which is related to the progression of renal diseases, have not been identified yet in Asian or in European people. However, further investigation should be made in this field to show the relationship between the specific gene and disease progression.

Conclusion

In summary, TGF- is a key mediator of renal fibrosis as well as in other fibrotic and sclerotic diseases. Optimizing maximal TGF- reduction may be a promising therapeutic goal in progressive fibrotic diseases. However, TGF-1 knockout mice that are born alive undergo early postnatal death due to excessive inflammatory response with infiltration of lymphocytes and macrophages in many organs, which resemble those observed in human autoimmune diseases [7, 8]. Certain Smads or TGF- receptors are somatically mutated at high frequency in some types of human cancers, suggesting a potential function for components in TGF- signal transduction pathway as tumor suppressor [144–147]. Thus, long-term employment of neutralizing agent or inhibitor of the signal pathway leading to the elimination of the TGF- effects can bring

TGF- in Renal Fibrosis

57

unfavorable consequences and may not be safe for clinical usage. It is important to understand that TGF- is necessary for tissue repair whereas uncontrolled overexpression of TGF- causes ECM deposition resulting in renal or other organ dysfunctions. We must pay attention to the evidence that the regulation of TGF- expression depends on the repair or healing process of the original diseases.

References 1 2 3 4 5 6 7

8

9

10

11

12 13 14

15 16 17 18 19

Klahr S, Schreiner G, Ichikawa I: The progression of renal disease. N Engl J Med 1988;318: 1657–1666. Border WA, Okuda S, Nakamura T: Extracellular matrix and glomerular disease. Semin Nephrol 1989;9:307–317. Border WA, Noble NA: Transforming growth factor- in tissue fibrosis. N Engl J Med 1994;331: 1286–1292. Roberts AB, McCune BK, Sporn MB: TGF-: Regulation of extracellular matrix. Kidney Int 1992;41:557–559. Eddy AA: Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 1996;7:2495–2508. Miyazono K, Ichijo H, Heldin CH: Transforming growth factor-: Latent forms, binding proteins and receptors. Growth Factors 1993;8:11–22. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D: Targeted disruption of the mouse transforming growth factor-1 gene results in multifocal inflammatory disease. Nature 1992;359:693–699. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, Karlsson S: Transforming growth factor-1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 1993;90:770–774. Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell EL, Doetschman T: TGF-2 knockout mice have multiple developmental defects that are non-overlapping with other TGF- knockout phenotypes. Development 1997;124:2659–2670. Proetzel G, Pawlowski SA, Wiles MV, Yin M, Boivin GP, Howles PN, Ding J, Ferguson MW, Doetschman T: Transforming growth factor-3 is required for secondary palate fusion. Nat Genet 1995;11:409–414. Kaartinen V, Voncken JW, Shuler C, Warburton D, Bu D, Heisterkamp N, Groffen J: Abnormal lung development and cleft palate in mice lacking TGF-3 indicates defects of epithelial-mesenchymal interaction. Nat Genet 1995;11:415–421. Taipale J, Saharinen J, Keski-Oja J: Extracellular matrix-associated transforming growth factor-: Role in cancer cell growth and invasion. Adv Cancer Res 1998;75:87–134. Flaumenhaft R, Kojima S, Abe M, Rifkin DB: Activation of latent transforming growth factor-. Adv Pharmacol 1993;24:51–76. Ando T, Okuda S, Tamaki K, Yoshitomi K, Fujishima M: Localization of transforming growth factor- and latent transforming growth factor- binding protein in rat kidney. Kidney Int 1995;47: 733–739. Liu A, Dardik A, Ballermann BJ: Neutralizing TGF-1 antibody infusion in neonatal rat delays in vivo glomerular capillary formation. Kidney Int 1999;56:1334–1348. Heldin CH, Miyazono K, ten Dijke P: TGF- signalling from cell membrane to nucleus through SMAD proteins. Nature 1997;390:465–471. Massague J: TGF- signal transduction. Annu Rev Biochem 1998;67:753–791. Piek E, Heldin CH, ten Dijke P: Specificity, diversity, and regulation in TGF- superfamily signaling. FASEB J 1999;13:2105–2124. Miyazono K, ten Dijke P, Heldin CH: TGF- signaling by Smad proteins. Adv Immunol 2000; 75:115–157.

Tamaki/Okuda

58

20 21 22 23

24 25 26 27

28

29 30

31 32

33 34 35 36

37

38 39 40 41

42

Itoh S, Itoh F, Goumans MJ, ten Dijke, P: Signaling of transforming growth factor- family members through Smad proteins. Eur J Biochem 2000;267:6954–6967. Poncelet AC, de Caestecker MP, Schnaper HW: The transforming growth factor-/SMAD signaling pathway is present and functional in human mesangial cells. Kidney Int 1999;56:1354–1365. Hayashida T, Poncelet AC, Hubchak SC, Schnaper HW: TGF-1 activates MAP kinase in human mesangial cells: A possible role in collagen expression. Kidney Int 1999;56:1710–1720. Poncelet AC, Schnaper HW: Sp1 and Smad proteins cooperate to mediate transforming growth factor-1-induced 2(I) collagen expression in human glomerular mesangial cells. J Biol Chem 2001;276:6983–6992. Border WA, Okuda S, Languino LR, Ruoslahti E: Transforming growth factor- regulates production of proteoglycans by mesangial cells. Kidney Int 1990;37:689–695. Nakamura T, Miller D, Ruoslahti E, Border WA: Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-1. Kidney Int 1992;41:1213–1221. Pawluczyk IZ, Harris KP: Cytokine interactions promote synergistic fibronectin accumulation by mesangial cells. Kidney Int 1998;54:62–70. Wolf G, Mueller E, Stahl RA, Ziyadeh FN: Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-. J Clin Invest 1993;92:1366–1372. Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJ, Angel P, Heath JK: Transforming growth factor- modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987;6:1899–1904. Tomooka S, Border WA, Marshall BC, Noble NA: Glomerular matrix accumulation is linked to inhibition of the plasmin protease system. Kidney Int 1992;42:1462–1469. Kagami S, Border WA, Ruoslahti E, Noble NA: Coordinated expression of 1 integrins and transforming growth factor--induced matrix proteins in glomerulonephritis. Lab Invest 1993;69: 68–76. Sappino AP, Schurch W, Gabbiani G: Differentiation repertoire of fibroblastic cells: Expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest 1990;63:144–161. Johnson RJ, Iida H, Alpers CE, Majesky MW, Schwartz SM, Pritzi P, Gordon K, Gown AM: Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. -Smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest 1991;87: 847–858. Stephenson LA, Haney LB, Hussaini IM, Karns LR, Glass WF 2nd: Regulation of smooth muscle -actin expression and hypertrophy in cultured mesangial cells. Kidney Int 1998;54:1175–1187. Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewski JE, Neilson EG: Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 1995;130:393–405. Okada H, Danoff TM, Kalluri R, Neilson EG: Early role of Fsp1 in epithelial-mesenchymal transformation. Am J Physiol 1997;273:F563–F574. Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, Lan HY: Transforming growth factor- regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 1999; 56:1455–1467. Okuda S, Languino LR, Ruoslahti E, Border WA: Elevated expression of transforming growth factor- and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix. J Clin Invest 1990;86:453–462. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor- 1. Nature 1990;346:371–374. Ritz E, Orth SR: Nephropathy in patients with type 2 diabetes mellitus. N Engl J Med 1999;341: 1127–1133. Tamaki K, Okuda S, Ando T, Iwamoto T, Nakayama M, Fujishima M: TGF-1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy. Kidney Int 1994;45:525–536. Tamaki K, Okuda S, Miyazono K, Nakayama M, Fujishima M: Matrix-associated latent TGF- with latent TGF- binding protein in the progressive process in adriamycin-induced nephropathy. Lab Invest 1995;73:81–89. Jones CL, Buch S, Post M, McCulloch L, Liu E, Eddy AA: Renal extracellular matrix accumulation in acute puromycin aminonucleoside nephrosis in rats. Am J Pathol 1992;141:1381–1396.

TGF- in Renal Fibrosis

59

43 44 45 46 47

48

49

50 51

52

53

54 55 56

57 58

59

60

61

62 63

Eddy AA, Giachelli CM: Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 1995;47:1546–1557. Yamamoto T, Noble NA, Miller DE, Border WA: Sustained expression of TGF-1 underlies development of progressive kidney fibrosis. Kidney Int 1994;45:916–927. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 1993;44:313–321. Diamond JR, Kees-Folts D, Ding G, Frye JE, Restrepo NC: Macrophages, monocyte chemoattractant peptide-1, and TGF-1 in experimental hydronephrosis. Am J Physiol 1994;266:F926–F933. Hugo C, Shankland SJ, Pichler RH, Couser WG, Johnson RJ: Thrombospondin-1 precedes and predicts the development of tubulointerstitial fibrosis in glomerular disease in the rat. Kidney Int 1998;53:302–311. Moll S, Menoud PA, Fulpius T, Pastore Y, Takahashi S, Fossati L, Vassalli JD, Sappino AP, Schifferli JA, Izui S: Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis. Kidney Int 1995;48:1459–1468. Kim S, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Inada Y, Wada T, Ishimura Y, Chatani F, Iwao H: Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury. Kidney Int 1994;46:1346–1358. Tamaki K, Okuda S, Nakayama M, Yanagida T, Fujishima M: Transforming growth factor-1 in hypertensive renal injury in Dahl salt-sensitive rats. J Am Soc Nephrol 1996;7:2578–2589. Kashiwagi M, Shinozaki M, Hirakata H, Tamaki K, Hirano T, Tokumoto M, Goto H, Okuda S, Fujishima M: Locally activated renin-angiotensin system associated with TGF-1 as a major factor for renal injury induced by chronic inhibition of nitric oxide synthase in rats. J Am Soc Nephrol 2000;11:616–624. Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, Border WA: Expression of transforming growth factor- is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci USA 1993;90:1814–1818. Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H, Maki S: Transforming growth factor- protein and mRNA in glomeruli in normal and diseased human kidneys. Lab Invest 1993;68:154–163. Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border WA: Expression of transforming growth factor- isoforms in human glomerular diseases. Kidney Int 1996;49:461–469. Bodi I, Kimmel PL, Abraham AA, Svetkey LP, Klotman PE, Kopp JB: Renal TGF- in HIV-associated kidney diseases. Kidney Int 1997;51:1568–1577. Yamamoto T, Noble NA, Miller DE, Gold LI, Hishida A, Nagase M, Cohen AH, Border WA: Increased levels of transforming growth factor- in HIV-associated nephropathy. Kidney Int 1999;55:579–592. Clouthier DE, Comerford SA, Hammer RE: Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-1 transgenic mice. J Clin Invest 1997;100:2697–2713. Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB, Sporn MB, Thorgeirsson SS: Hepatic expression of mature transforming growth factor-1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA 1995;92:2572–2576. Kopp JB, Factor VM, Mozes M, Nagy P, Sanderson N, Bottinger EP, Klotman PE, Thorgeirsson SS: Transgenic mice with increased plasma levels of TGF-1 develop progressive renal disease. Lab Invest 1996;74:991–1003. Wogensen L, Nielsen CB, Hjorth P, Rasmussen LM, Nielsen AH, Gross K, Sarvetnick N, Ledet T: Under control of the Ren-1c promoter, locally produced transforming growth factor-1 induces accumulation of glomerular extracellular matrix in transgenic mice. Diabetes 1999;48:182–192. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: Glomerulosclerosis induced by in vivo transfection of transforming growth factor- or platelet-derived growth factor gene into the rat kidney. J Clin Invest 1993;92:2597–2601. Crawford SE, Stellmach V, Murphy-Ullrich JE, Ribeiro SM, Lawler J, Hynes RO, Boivin GP, Bouck N: Thrombospondin-1 is a major activator of TGF-1 in vivo. Cell 1998;93:1159–1170. Hugo CP, Pichler RP, Schulze-Lohoff E, Prols F, Adler S, Krutsch HC, Murphy-Ullrich JE, Couser WG, Roberts DD, Johnson RJ: Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo. Kidney Int 1999;55:2236–2249.

Tamaki/Okuda

60

64 65 66

67

68 69 70 71

72

73

74

75

76 77 78

79

80

81

82

83 84

Grainger DJ, Kirschenlohr HL, Metcalfe JC, Weissberg PL, Wade DP, Lawn RM: Proliferation of human smooth muscle cells promoted by lipoprotein (a). Science 1993;260:1655–1658. Fujisawa M, Haramaki R, Miyazaki H, Imaizumi T, Okuda S: Role of lipoprotein (a) and TGF-1 in atherosclerosis of hemodialysis patients. J Am Soc Nephrol 2000;11:1889–1895. Munger JS, Harpel JG, Giancotti FG, Rifkin DB: Interactions between growth factors and integrins: Latent forms of transforming growth factor- are ligands for the integrin v1. Mol Biol Cell 1998;9:2627–2638. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D: The integrin v6 binds and activates latent TGF-1: A mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999;96:319–328. Coimbra T, Wiggins R, Noh JW, Merritt S, Phan SH: Transforming growth factor- production in anti-glomerular basement membrane disease in the rabbit. Am J Pathol 1991;138:223–234. Border WA, Noble NA: Interactions of transforming growth factor- and angiotensin II in renal fibrosis. Hypertension 1998;31:181–188. Noble NA, Border WA: Angiotensin II in renal fibrosis: Should TGF- rather than blood pressure be the therapeutic target? Semin Nephrol 1997;17:455–466. Maschio G, Alberti D, Janin G, Locatelli F, Mann JF, Motolese M, Ponticelli C, Ritz E, Zucchelli P: Effect of the angiotensin-converting-enzyme inhibitor benazepril on the progression of chronic renal insufficiency. The Angiotensin-Converting-Enzyme Inhibition in Progressive Renal Insufficiency Study Group. N Engl J Med 1996;334:939–945. Ihle BU, Whitworth JA, Shahinfar S, Cnaan A, Kincaid-Smith PS, Becker GJ: Angiotensinconverting enzyme inhibition in nondiabetic progressive renal insufficiency: A controlled doubleblind trial. Am J Kidney Dis 1996;27:489–495. Giatras I, Lau J, Levey AS: Effect of angiotensin-converting enzyme inhibitors on the progression of nondiabetic renal disease: A meta-analysis of randomized trials. Angiotensin-Converting-Enzyme Inhibition and Progressive Renal Disease Study Group. Ann Intern Med 1997;127:337–345. Kagami S, Border WA, Miller DE, Noble NA: Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 1994;93:2431–2437. Wolf G, Mueller E, Stahl RA, Ziyadeh FN: Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-. J Clin Invest 1993;92:1366–1372. Ruiz-Ortega M, Egido J: Angiotensin II modulates cell growth-related events and synthesis of matrix proteins in renal interstitial fibroblasts. Kidney Int 1997;52:1497–1510. Kagami S, Kuhara T, Okada K, Kuroda Y, Border WA, Noble NA: Dual effects of angiotensin II on the plasminogen/plasmin system in rat mesangial cells. Kidney Int 1997;51:664–671. Wolf G, Ziyadeh FN, Zahner G, Stahl RA: Angiotensin II-stimulated expression of transforming growth factor- in renal proximal tubular cells: Attenuation after stable transfection with the c-mas oncogene. Kidney Int 1995;48:1818–1827. Wolf G, Zahner G, Schroeder R, Stahl RA: Transforming growth factor- mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells. Nephrol Dial Transplant 1996;11:263–269. Nakamura T, Obata J, Kimura H, Ohno S, Yoshida Y, Kawachi H, Shimizu F: Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis. Kidney Int 1999;55:877–889. Kaneto H, Morrissey J, McCracken R, Reyes A, Klahr S: Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed rat kidney. Kidney Int 1994;45: 1637–1647. Ishidoya S, Morrissey J, McCracken R, Reyes A, Klahr S: Angiotensin II receptor antagonist ameliorates renal tubulointerstitial fibrosis caused by unilateral ureteral obstruction. Kidney Int 1995; 47:1285–1294. Ishidoya S, Morrissey J, McCracken R, Klahr S: Delayed treatment with enalapril halts tubulointerstitial fibrosis in rats with obstructive nephropathy. Kidney Int 1996;49:1110–1119. Shihab FS, Bennett WM, Tanner AM, Andoh TF: Angiotensin II blockade decreases TGF-1 and matrix proteins in cyclosporine nephropathy. Kidney Int 1997;52:660–673.

TGF- in Renal Fibrosis

61

85 Hamaguchi A, Kim S, Ohta K, Yagi K, Yukimura T, Miura K, Fukuda T, Iwao H: Transforming growth factor-1 expression and phenotypic modulation in the kidney of hypertensive rats. Hypertension 1995;26:199–207. 86 Gilbert RE, Cox A, Wu LL, Allen TJ, Hulthen UL, Jerums G, Cooper ME: Expression of transforming growth factor-1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: Effects of ACE inhibition. Diabetes 1998;47:414–422. 87 Ziyadeh FN, Sharma K: Role of transforming growth factor- in diabetic glomerulosclerosis and renal hypertrophy. Kidney Int 1995;51:S34–S36. 88 Flyvbjerg A: Putative pathophysiological role of growth factors and cytokines in experimental diabetic kidney disease. Diabetologia 2000;43:1205–1223. 89 Ziyadeh FN, Sharma K, Ericksen M, Wolf G: Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-. J Clin Invest 1994;93:536–542. 90 Ziyadeh FN, Snipes ER, Watanabe M, Alvarez RJ, Goldfarb S, Haverty TP: High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule. Am J Physiol 1990;259:F704–F714. 91 Hoffman BB, Sharma K, Zhu Y, Ziyadeh FN: Transcriptional activation of transforming growth factor-1 in mesangial cell culture by high glucose concentration. Kidney Int 1998;54:1107–1116. 92 Shankland SJ, Scholey JW, Ly H, Thai K: Expression of transforming growth factor-1 during diabetic renal hypertrophy. Kidney Int 1994;46:430–442. 93 Park IS, Kiyomoto H, Abboud SL, Abboud HE: Expression of transforming growth factor- and type IV collagen in early streptozotocin-induced diabetes. Diabetes 1997;46:473–480. 94 Sharma K, Ziyadeh FN: Renal hypertrophy is associated with upregulation of TGF-1 gene expression in diabetic BB rat and NOD mouse. Am J Physiol 1994;267:F1094–F1101. 95 Iwano M, Kubo A, Nishino T, Sato H, Nishioka H, Akai Y, Kurioka H, Fujii Y, Kanauchi M, Shiiki H, Dohi K: Quantification of glomerular TGF-1 mRNA in patients with diabetes mellitus. Kidney Int 1996;49:1120–1126. 96 Sharma K, Jin Y, Guo J, Ziyadeh FN: Neutralization of TGF- by anti-TGF- antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 1996;45:522–530. 97 Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K: Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal anti-transforming growth factor- antibody in db/db diabetic mice. Proc Natl Acad Sci USA 2000; 97:8015–8020. 98 Hong SW, Isono M, Chen S, Iglesias-De La Cruz MC, Han DC, Ziyadeh FN: Increased glomerular and tubular expression of transforming growth factor-1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 2001;158:1653–1663. 99 Leehey DJ, Singh AK, Alavi N, Singh R: Role of angiotensin II in diabetic nephropathy. Kidney Int 2000;77:S93–S98. 100 Wang TT, Wu XH, Zhang SL, Chan JS: Effect of glucose on the expression of the angiotensinogen gene in opossum kidney cells. Kidney Int 1998;53:312–319. 101 Zhang SL, Filep JG, Hohman TC, Tang SS, Ingelfinger JR, Chan JS: Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells. Kidney Int 1999;55:454–464. 102 Singh R, Alavi N, Singh AK, Leehey DJ: Role of angiotensin II in glucose-induced inhibition of mesangial matrix degradation. Diabetes 1999;48:2066–2073. 103 Gilbert RE, Cox A, Wu LL, Allen TJ, Hulthen UL, Jerums G, Cooper ME: Expression of transforming growth factor-1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: Effects of ACE inhibition. Diabetes 1998;47:414–422. 104 Studer RK, Craven PA, DeRubertis FR: Antioxidant inhibition of protein kinase C-signaled increases in transforming growth factor- in mesangial cells. Metabolism 1997;46:918–925. 105 Koya D, Jirousek MR, Lin YW, Ishii H, Kuboki K, King GL: Characterization of protein kinase C isoform activation on the gene expression of transforming growth factor-, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. J Clin Invest 1997;100:115–126.

Tamaki/Okuda

62

106 Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J 2000;14:439–447. 107 Throckmorton DC, Brogden AP, Min B, Rasmussen H, Kashgarian M: PDGF and TGF- mediate collagen production by mesangial cells exposed to advanced glycosylation end products. Kidney Int 1995;48:111–117. 108 Ziyadeh FN, Han DC, Cohen JA, Guo J, Cohen MP: Glycated albumin stimulates fibronectin gene expression in glomerular mesangial cells: Involvement of the transforming growth factor- system. Kidney Int 1998;53:631–638. 109 Chen S, Cohen MP, Lautenslager GT, Shearman CW, Ziyadeh FN: Glycated albumin stimulates TGF-1 production and protein kinase C activity in glomerular endothelial cells. Kidney Int 2001;59:673–681. 110 Pugliese G, Pricci F, Romeo G, Pugliese F, Mene P, Giannini S, Cresci B, Galli G, Rotella CM, Vlassara H, Di Mario U: Upregulation of mesangial growth factor and extracellular matrix synthesis by advanced glycation end products via a receptor-mediated mechanism. Diabetes 1997; 46:1881–1887. 111 Yang CW, Hattori M, Vlassara H, He CJ, Carome MA, Yamato E, Elliot S, Striker GE, Striker LJ: Overexpression of transforming growth factor-1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice. J Am Soc Nephrol 1995;5:1610–1617. 112 Vlassara H, Striker LJ, Teichberg S, Fuh H, Li YM, Steffes M: Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc Natl Acad Sci USA 1994; 91:11704–11708. 113 Yang CW, Vlassara H, Peten EP, He CJ, Striker GE, Striker LJ: Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proc Natl Acad Sci USA 1994;91:9436–9440. 114 Cohen MP, Sharma K, Jin Y, Hud E, Wu VY, Tomaszewski J, Ziyadeh FN: Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists. A novel treatment strategy. J Clin Invest 1995;95:2338–2345. 115 Tsuchida K, Makita Z, Yamagishi S, Atsumi T, Miyoshi H, Obara S, Ishida M, Ishikawa S, Yasumura K, Koike T: Suppression of transforming growth factor- and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195. Diabetologia 1999;42:579–588. 116 Ying WZ, Sanders PW: Dietary salt modulates renal production of transforming growth factor- in rats. Am J Physiol 1998;274:F635–F641. 117 Yu HC, Burrell LM, Black MJ, Wu LL, Dilley RJ, Cooper ME, Johnston CI: Salt induces myocardial and renal fibrosis in normotensive and hypertensive rats. Circulation 1998;98:2621–2628. 118 Nakayama M, Okuda S, Tamaki K, Fujishima M: Short- or long-term effects of a low-protein diet on fibronectin and transforming growth factor- synthesis in adriamycin-induced nephropathy. J Lab Clin Med 1996;127:29–39. 119 Eddy AA: Protein restriction reduces transforming growth factor- and interstitial fibrosis in nephrotic syndrome. Am J Physiol 1994;266:F884–F893. 120 Akagi Y, Isaka Y, Arai M, Kaneko T, Takenaka M, Moriyama T, Kaneda Y, Ando A, Orita Y, Kamada T, Ueda N, Imai E: Inhibition of TGF-1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1996; 50:148–155. 121 Isaka Y, Tsujie M, Ando Y, Nakamura H, Kaneda Y, Imai E, Hori M: Transforming growth factor-1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int 2000;58:1885–1892. 122 Okuno M, Akita K, Moriwaki H, Kawada N, Ikeda K, Kaneda K, Suzuki Y, Kojima S: Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-. Gastroenterology 2001;120:1784–1800. 123 Yamaguchi Y, Mann DM, Ruoslahti E: Negative regulation of transforming growth factor- by the proteoglycan decorin. Nature 1990;346:281–284.

TGF- in Renal Fibrosis

63

124 Bottinger EP, Factor VM, Tsang ML, Weatherbee JA, Kopp JB, Qian SW, Wakefield LM, Roberts AB, Thorgeirsson SS, Sporn MB: The recombinant proregion of transforming growth factor-1 (latency-associated peptide) inhibits active transforming growth factor-1 in transgenic mice. Proc Natl Acad Sci USA 1996;93:5877–5882. 125 Yamamoto H, Ueno H, Ooshima A, Takeshita A: Adenovirus-mediated transfer of a truncated transforming growth factor- (TGF-) type II receptor completely and specifically abolishes diverse signaling by TGF- in vascular wall cells in primary culture. J Biol Chem 1996;271: 16253–16259. 126 Smith JD, Bryant SR, Couper LL, Vary CP, Gotwals PJ, Koteliansky VE, Lindner V: Soluble transforming growth factor- type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999;84:1212–1222. 127 Zheng H, Wang J, Koteliansky VE, Gotwals PJ, Hauer-Jensen M: Recombinant soluble transforming growth factor- type II receptor ameliorates radiation enteropathy in mice. Gastroenterology 2000;119:1286–1296. 128 George J, Roulot D, Koteliansky VE, Bissell DM: In vivo inhibition of rat stellate cell activation by soluble transforming growth factor- type II receptor: A potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA 1999;96:12719–12724. 129 Border WA, Noble NA, Yamamoto T, Harper JR, Yamaguchi Y, Pierschbacher MD, Ruoslahti E: Natural inhibitor of transforming growth factor- protects against scarring in experimental kidney disease. Nature 1992;360:361–364. 130 Isaka Y, Brees DK, Ikegaya K, Kaneda Y, Imai E, Noble NA, Border WA: Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat Med 1996;2:418–423. 131 Isaka Y, Akagi Y, Ando Y, Tsujie M, Sudo T, Ohno N, Border WA, Noble NA, Kaneda Y, Hori M, Imai E: Gene therapy by transforming growth factor- receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1999;55:465–475. 132 Qi Z, Atsuchi N, Ooshima A, Takeshita A, Ueno H: Blockade of type transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA 1999;96:2345–2349. 133 Nakamura T, Sakata R, Ueno T, Sata M, Ueno H: Inhibition of transforming growth factor- prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosaminetreated rats. Hepatology 2000;32:247–255. 134 Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I: Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999;104:5–11. 135 Kanai H, Mitsuhashi H, Ono K, Yano S, Naruse T: Increased excretion of urinary transforming growth factor- in patients with focal glomerular sclerosis. Nephron 1994;66:391–395. 136 Honkanen E, Teppo AM, Tornroth T, Groop PH, Gronhagen-Riska C: Urinary transforming growth factor-1 in membranous glomerulonephritis. Nephrol Dial Transplant 1997;12:2562–2568. 137 Haramaki R, Tamaki K, Fujisawa M, Ikedo H, Haramaki N, Okuda S: Steroid therapy and urinary transforming growth factor-1 in IgA nephropathy. Am J Kidney Dis 2001;38:1191–1198. 138 Sharma K, Ziyadeh FN, Alzahabi B, McGowan TA, Kapoor S, Kurnik BR, Kurnik PB, Weisberg LS: Increased renal production of transforming growth factor-1 in patients with type II diabetes. Diabetes 1997;46:854–859. 139 Inigo P, Campistol JM, Lario S, Piera C, Campos B, Bescos M, Oppenheimer F, Rivera F: Effects of losartan and amlodipine on intrarenal hemodynamics and TGF-1 plasma levels in a crossover trial in renal transplant recipients. J Am Soc Nephrol 2001;12:822–827. 140 Campistol JM, Inigo P, Jimenez W, Lario S, Clesca PH, Oppenheimer F, Rivera F: Losartan decreases plasma levels of TGF-1 in transplant patients with chronic allograft nephropathy. Kidney Int 1999;56:714–719. 141 Sharma K, Eltayeb BO, McGowan TA, Dunn SR, Alzahabi B, Rohde R, Ziyadeh FN, Lewis EJ: Captopril-induced reduction of serum levels of transforming growth factor-1 correlates with longterm renoprotection in insulin-dependent diabetic patients. Am J Kidney Dis 1999;34:818–823. 142 Suthanthiran M, Khanna A, Cukran D, Adhikarla R, Sharma VK, Singh T, August P: Transforming growth factor-1 hyperexpression in African-American end-stage renal disease patients. Kidney Int 1998;53:639–644.

Tamaki/Okuda

64

143 Li B, Khanna A, Sharma V, Singh T, Suthanthiran M, August P: TGF-1 DNA polymorphisms, protein levels and blood pressure. Hypertension 1999;33:271–275. 144 Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless P, Kim H, Tsui LC, Bapat B, Gallinger S, Andrulis IL, Thomsen GH, Wrana JL, Attisano L: MADR2 maps to 18q21 and encodes a TGF-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 1996; 86:543–552. 145 Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271:350–353. 146 Park K, Kim SJ, Bang YJ, Park JG, Kim NK, Roberts AB, Sporn MB: Genetic changes in the transforming growth factor- (TGF-) type II receptor gene in human gastric cancer cells: Correlation with sensitivity to growth inhibition by TGF-. Proc Natl Acad Sci USA 1994;91:8772–8776. 147 Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B: Inactivation of the type II TGF- receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336–1338. 148 Miyazawa K, Yokote K, Miyazono K: Biology of Growth Factor. Youdo-Sha, Inc, 2001, pp 1–93.

Kiyoshi Tamaki, MD, PhD, Department of Nephrology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011 (Japan) Tel. 81 942 353311/ext 5346, Fax 81 942 317763, E-Mail [email protected]

TGF- in Renal Fibrosis

65

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 66–89

Chemokines in Renal Fibrosis Hitoshi Yokoyama, Takashi Wada, Kengo Furuichi Department of Gastroenterology and Nephrology and Division of Blood Purification, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan

Introduction

Progressive renal diseases are the consequence of a process of glomerulosclerosis and destructive interstitial fibrosis. Since Henle et al.’s [1] first report, the strong correlation between the degree of interstitial fibrosis and renal dysfunction, even in glomerular diseases, has been well known [2–4]. In the first step of the interstitial fibrotic process, the cellular activation is caused in renal resident cells such as tubular epithelial cells, vascular endothelial cells and fibroblasts. Moreover, the interaction between infiltrated leukocytes and renal resident cells may provide the key roles to the progression of the inflammatory and fibrotic processes. Activated leukocytes and resident cells such as fibroblasts play a critical role in the switch from acute resolving to tissue repair or chronic persistent inflammation [5, 6]. Then, the process of renal interstitial fibrogenic cascade will progress step by step to the fibrogenic signaling phase, fibrogenic phase and the final phase of renal destruction as described by Eddy [7]. Recent investigations of the pathophysiological roles of chemokines and their cognate receptors have shed light on the detailed molecular mechanisms of leukocyte trafficking and activation in various diseases. In addition, these chemokines exert overlapping but distinct actions on specific cell types through interacting with the cognate G protein-coupled receptors with seven transmembrane domains [8–10]. Chemokine receptors are expressed not only on leukocytes, but also parenchymal cells, suggesting the possible autocrine/paracrine loop during the interaction of leukocytes and parenchymal cells. Therefore, detailed mechanisms of leukocyte recruitment and activation via chemokines/chemokine receptor

systems may provide a better understanding of the pathogenesis of the early phase during renal fibrosis. This review will focus on (1) the overview of chemokine and chemokine receptor systems, (2) chemokine and chemokine receptor systems in the pathogenesis of phase-specific renal disorders (‘chemokine cascade’) in experimental models and clinical renal diseases, and (3) the intervention of chemokines and chemokine receptor systems as the particular immunotherapeutic strategies to combat the specific phases of renal diseases for preventing renal fibrosis.

Chemokine Family

Chemotaxis and Leukocyte Activation Chemokine is the generic term for chemotactic cytokines. The chemokine family is divided into four groups (CXC, CC, C and CX3C) based on the location of the very conserved first two cysteine residues that form disulfide bonds in the chemokine tertiary structure [8–11]. In the CXC chemokine family, the first two cysteine residues in the primary amino acid sequence are separated by a single amino acid, whereas the cysteine residues lie next to each other in the CC chemokine family. Other two chemokine families each containing a single major molecule have been clarified in recent years. The C chemokine lymphotactin lacks the first and third cysteine of the 4-cysteine motif in chemokine family molecules. The final unique molecule fractalkine identified and characterized in 1997 has three intervening amino acids (CX3C motif) and exists as a membrane-bound glycoprotein with the chemokine domain atop and extended mucin-like stalk [12]. More than 44 chemokines (CXCL1–14, CCL1–27, XCL1–2 and CX3CL1) and 18 chemokine receptors (CXCR1–CXCR5, CCR1–CCR11, XCR1 and CX3CR1) have been identified until now (table 1) [11]. Leukocyte trafficking at the inflammatory site consists of two major functions, firstly the arrest, activation and firm adhesion of leukocytes on endothelial surfaces, and secondly the diapedesis and transmigration through endothelial cells with non-occluding junctions into the specific microenvironments [8–10]. Evidence is accumulating that chemokines through their cognate receptors are of importance for these steps. In addition, during the first step, a selectin-mediated rolling followed by an integrin-mediated firm adhesion are involved when leukocytes bind to endothelium [13, 14]. Chemokines expressed on the surface of endothelial cells interact with their cognate receptors on specific leukocytes, which trigger the activation of adhesion molecules and result in firm adhesion. Once leukocytes migrate into the interstitium, chemokines produced by both tissue cells and infiltrated cells induce a variety of biological

Chemokines and Renal Fibrosis

67

Table 1. Chemokine receptors, chemokines and their target cells Chemokine receptors

Ligands (chemokine*)

Major target cells

CXCR1 CXCR2

IL-8 IL-8 GROs, ENA-78, NAP-2 IP-10, Mig, I-TAC SDF-1, PBSF BLC, BCA-1

Neutrophil Neutrophil

MIP-1 RANTES MCP-2,3 MCP-1,2,3,4 Eotaxin RANTES MCP-2,3,4 TARC, MDC MIP-1, MIP-1 RANTES LARC, MIP-3 SLC, ELC, MIP-3 I-309 TECK ILC, CTACK MCP-1,2,4

Monocyte Dendritic cell (DC, immature)

T (Th2) Monocyte T (Th1) T (memory), DC (immature) T (naive), DC (mature) T (activated Th2?) T (47) T (CLA) Not expressed on leukocytes

XCR1

Lymphotactin ,

NK(?), T

CX3CR1

Fractalkine

NK, T (CD8), neuron

CXCR3 CXCR4 CXCR5 CCR1

CCR2 CCR3

CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10 CCR11

T (Th1) T (naive) B (activated)

Monocyte Eosinophil, basophil

*Selective chemokines are underlined. GROs, growth-related oncogene; ENA-78, epithelial cell-derived neutrophil-activating factor, 78 amino acids; NAP-2, neutrophil-activating peptide-2; IP-10, -interferoninducible protein-10; Mig, monokine induced by interferon-; I-TAC, interferon-inducible T cell -chemoattractant; SDF-1, stromal derived factor; PBSF, pre-B-cell-stimulatory factor; BLC, B-lymphocyte chemoattractant; BCA-1, B-cell-attracting chemokine-1; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T cell expressed and secreted; TARC, thymus- and activation-regulated chemokine; MDC, macrophage-derived chemokine; LARC, liver- and activation-regulated chemokine; SLC, secondary lymphoid tissue chemokine; ELC, Epstein-Barr virus-induced receptor ligand chemokine; TECK, thymus-expressed chemokine.

functions of leukocytes at the inflammatory sites (fig. 1). Selective expression of chemokine receptors contributes to cell specificity of chemokine actions. Thus, the chemokine systems provide sequential migratory patterns of the specific leukocytes with their cognate receptors in a multistep manner.

Yokoyama/Wada/Furuichi

68

Rolling Leukocyte

Leukocyte trafficking

Activation Spreading Diapedesis Endothelial cells

Transmigration Renal tubular epithelial cells Chemokines

Inflammation Complements: C3, C4 Chemokines: MCP-1, IL-8, RANTES, IP-10 Cytokines: IL-6, TNF- Growth factors: GM-CSF, CSF-1, PDGF, TGF-, VEGF

Fibrosis

Fig. 1. Chemokines in the leukocyte trafficking at the inflammatory site.

Other Biological Functions of Chemokines and Chemokine Receptors Recent studies revealed that chemokines through their cognate receptors act as growth factors for glomerular mesangial cells [15] and are involved in respiratory burst, hematopoiesis, angiogenesis, atherosclerosis, development and fibrogenesis [8–10]. Interferon-inducible protein (IP)-10 induced the proliferation of mesangial cells probably via the cognate receptor, CXCR3 [15]. In addition, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 (also termed as monocyte chemotactic and activating factor, MCAF) are closely related to the urinary excretion of protein in experimental models [16, 17] and human nephrotic syndrome [18], which may be related to the permeability of the glomerular capillaries. IL-8 has been shown to induce the migration of human umbilical endothelial cells and angiogenesis in rat cornea [19, 20]. Additional interest in chemokines and their cognate receptors is arising from recent identification of several chemokines as inhibitors and receptors as co-receptors for HIV infection [21]. The blockade of chemokine receptors as the clinical therapeutic approach has been anticipated in these fields. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes and endothelial cells of postcapillary venules in normal kidney is a highly promiscuous chemokine-binding protein, specific for the basic CC chemokines such as MCP-1 and normal T cell expressed and excreted (RANTES), as well as the CXC chemokines with the presence of the tripeptide motif glutamic acid-leucine-arginine (ELR) N-terminal to the first cysteine such as IL-8, growth-related oncogene (GRO) , platelet factor (PF)-4 and

Chemokines and Renal Fibrosis

69

neutrophil-activating protein (NAP)-2. DARC is not coupled to G proteins, however, which may act as a presentation-like structure for chemokines on the endothelial surfaces contributing to the leukocyte infiltration at the sites of postcapillary venules in kidney [22]. The chemokine receptor CCR5, which shares the ligand RANTES with DARC, is expressed by infiltrating T cells in the renal interstitium. DARC-positive interstitial venules were significantly increased during both acute transplant rejection and crescentic glomerulonephritis accompanied by an infiltration of CCR5-positive leukocytes. The increased number of DARC-positive venules in areas of interstitial injury and the colocalization with CCR5-positive infiltrating leukocytes may indicate a role for endothelial DARC expression during leukocyte adhesion and interstitial infiltration [23].

Chemokines and Chemokine Receptors in the Kidneys

In general, the interstitial infiltrate of mononuclear cells is a histological characteristic finding of progressive renal diseases. Mononuclear cells, monocytes and macrophages in particular, are thought to directly contribute to interstitial fibrosis as a source of profibrotic molecules. Most of these mononuclear cells are thought to infiltrate into the interstitium from the circulation through peritubular capillaries. Chemoattractant molecules produced by tubular epithelial cells are speculated to stimulate and enhance the influx of these cells. Chemokines such as MCP-1 and RANTES are thought to play an important role in this step. In this part, we overview the in vitro and in vivo chemokine production of renal resident cells and infiltrated cells focused on renal fibrosis. In vitro Expression of Chemokines by Glomerular Cells All types of renal parenchymal cells can express chemokines on stimulation, and mesangial cells can be induced chemokine receptors as well as its cognate ligand [6, 15, 24]. In vitro studies of mesangial cells revealed that proinflammatory stimuli such as IL-1, tumor necrosis factor- (TNF-), interferon- (IFN-) and lipopolysaccharide (LPS), growth factors including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), immune complexes or cross-linking of Fc receptors, and reactive oxygen species are able to induce IL-8, MCP-1, IP-10, macrophage inflammatory protein (MIP)-1 and RANTES from renal resident cells (table 2) [6]. In turn, these stimuli may induce the expression of CCR1 and CXCR3 on mesangial cells [15, 24]. These results suggest the certain roles of possible positive feedback loop dependent on

Yokoyama/Wada/Furuichi

70

Table 2. In vitro chemokine and chemokine receptor expression of renal resident cells Cells

Chemokines

Glomerulus Mesangial cells

MCP-1 RANTES, MIP-1 IL-8, GROs IP-10 MIP-2, CINC (mouse)

Endothelial cells

MCP-1 RANTES IL-8 IP-10

Podocytes

MCP-1 IP-10

Tubulointerstitium Epithelial cells (proximal)

Fibroblasts

Chemokine receptors

CCR1 CXCR3

MCP-1 MCP-4 RANTES IL-8, ENA-8, GROs MCP-1 IL-8 IP-10 SDF-1(?)

CINC, cytokine-induced neutrophil chemoattractants and activator; MIP-2, macrophage inflammatory protein-2.

chemokines and chemokine receptors, which results in progressive glomerular diseases. In fact, the mouse CXC chemokines, MIP-2 and KC upregulated MCP-1 and RANTES expression in mouse mesangial cells [25]. Autoinduction of MIP-2 and KC mRNA were also noted. These indicate chemokine amplification probably via their cognate receptors in mesangial cells, which may contribute to the maintenance and chronic course of glomerular inflammation resulting in glomerulosclerosis. In contrast, anti-inflammatory agents such as glucocorticoids and IL-1 receptor antagonist (IL-1RA), transforming growth factor- (TGF-), vitamin D, prostaglandins and antioxidants are able to inhibit the expression of chemokines [8–10]. Supporting this notion, we have reported that glucocorticoid therapy including methylprednisolone pulse therapy reduced the renal

Chemokines and Renal Fibrosis

71

expression of chemokines and their cognate receptors in accordance with the decrease in the clinical activities of human renal diseases [26–32]. Several recent studies have provided clear evidence that angiotensinconverting enzyme (ACE) inhibitors slow the progression of renal disease, even in immune-mediated renal injury [33]. These effects are mainly independent from a concomitant reduction in systemic blood pressure. Thus, vasoactive agents, especially angiotensin II (Ang II) exerts other effects on the kidney that are involved in the loss of renal function. Ang II induces proliferation of cultured mesangial and glomerular endothelial cells. In addition, Ang II can also induce chemokine, RANTES in glomerular endothelial cells through its AT2 receptor. Moreover, the administration of an AT2 receptor antagonist attenuated the influx of glomerular macrophages induced by the infusion of Ang II in vivo [34]. In vitro Expression of Chemokines by Renal Tubular Cells Proinflammatory stimuli such as IL-1, TNF- and IFN- are able to induce CC chemokines, MCP-1, MCP-4 and RANTES on proximal tubular epithelial cells in vitro [6, 35]. Proximal tubular epithelial cells can synthesize CXC chemokines, IL-8 stimulated by IL-1,, TNF-, LPS and CD40 ligand with IL-4 and IL-13, and ENA-78 by IL-1 (table 2). Cultured proximal tubular epithelial cells can also produce chemokines through protein loading, which is speculated to mimic the effects of proteinuria in glomerular diseases [36–39]. MCP-1 mRNA expression in proximal tubular epithelial cells increased in response to delipidated bovine serum albumin, holotransferrin and apotransferrin through nuclear factor (NF)-B activation, which was reduced by lysine, an inhibitor of protein uptake. Zoja et al. [37, 38] described a dose-dependent increase of RANTES expression by proximal tubular epithelial cells stimulated by the high concentration of bovine serum albumin (10–30 mg/ml). Wolf et al. [34, 40] also demonstrated that Ang II stimulates hypertrophy of cultured proximal tubular cells. Ang II exerts immunomodulatory effects on the kidney through the induction of chemokines such as MCP-1 and RANTES. In addition, Ang II stimulates bioactivation and expression of TGF- in tubular MCT cells. This Ang II-mediated expression of TGF- is due to an increase in transcriptional activity. A neutralizing anti-TGF- antibody attenuates the Ang II-induced increase in protein synthesis in tubular MCT cells suggesting that the hypertrophy is mediated by synthesis and activation of endogenous TGF-. Proximal tubular cells undergoing Ang II-mediated hypertrophy are arrested in the G1 phase of the cell cycle and express typical G1-phase-associated genes. Induction of such G1-phase-associated early growth response genes has been also described in vivo after infusion of Ang II into the renal artery. This G1phase arrest depends on the induction of the cyclin-dependent kinase (CdK)

Yokoyama/Wada/Furuichi

72

inhibitor p27Kip1. p27Kip1 expression is stimulated after incubation of LLCPK1 cells with Ang II or TGF- and binds to cyclin D1-CdK4 complexes, inhibits their kinase activity, and hampers G1-phase exit. Then, Ang II stimulates transcription of collagen type IV in tubular MCT cells. This stimulation is mediated by endogenous synthesis and autocrine action of TGF-, because a neutralizing anti-TGF- antibody as well as TGF- antisense oligonucleotides attenuate Ang II-induced collagen type IV transcription and synthesis. These findings indicate that Ang II has emerged as being multifunctional, acting as a growth factor and a profibrogenic cytokine in renal tubular epithelial cells. Chemokines/Chemokine Receptors in Animal Models Chemokines and Tubulointerstitial Lesions in Experimental Renal Injuries Accumulating data from experimental animal models of renal diseases suggest that chemokines and their cognate receptors expressed in the diseased kidneys govern critical aspects of acute and chronic renal inflammation leading to glomerulosclerosis and interstitial fibrosis [6, 7]. The classic model of nephrotoxic serum nephritis induced by antiglomerular basement membrane (GBM) antibodies was used for the studies of chemokine in mice or rats. In mice accelerated model, MCP-1, RANTES, IP-10 and MCP-3 mRNA expression increased in cortex or whole kidneys [summarized in 6]. Predominant tubular MCP-1 expression was associated with tubulointerstitial cell infiltration [41]. An upregulation of MCP-1, RANTES, MIP-1/, MCP-3, CINC, MIP-2, PF4, IP-10, TCA3, fractalkine and lymphotactin mRNA was reported in Wistar-Kyoko rat (WKY) model of nephrotoxic serum nephritis. MCP-1 protein was also detected on renal tubular epithelial cells as well as glomeruli and vascular endothelial cells in this model [17, 42–49]. In rats of puromycin aminonucleoside nephrosis, a marked macrophage predominant mononuclear interstitial infiltration occurred together with massive proteinuria and the upregulation of MCP-1, IP-10, MCP-3 and TCA3 mRNA expression [50, 51]. The site of IP-10 and MCP-1 mRNA production was localized to intrinsic tubulointerstitial cells and not to infiltrating monocytes or macrophages [50]. In addition, the numbers of interstitial monocytes and lymphocytes significantly increased, and the levels of the mRNA expression of the above chemokines were still higher than the control animals, whereas the levels of mRNAs for MIP-1/ and RANTES were not higher or were only slightly higher than the control ones on day 5 of this model [51]. In anti-tubular basement membrane (TBM) antibody-associated tubulointerstitial nephritis (TIN) induced by immunization of Brown Norway rats with bovine cortical TBM, increased MCP-1 mRNA appeared on day 7 when TIN

Chemokines and Renal Fibrosis

73

involved 10–40% of the cortex, and peaked rapidly on day 8 when there was 60–80% cortical involvement (at which time 75–80% of the infiltrating cells were neutrophils). The influx of mononuclear cells on day 8 was preceded by the expression of MCP-1 mRNA. The mRNA for MCP-1 was undetectable by day 10 (at which time 95% of the infiltrating cells were mononuclear). There is a temporal association in the expression of MCP-1 with the upregulation of intercellular adhesion molecule-1 and leukocyte infiltration within the tubulointerstitium in anti-TBM antibody-associated TIN [52]. Unilateral ureteral obstruction shows a prominent interstitial mononuclear cell infiltration and MCP-1 mRNA expression by tubular epithelial cells. Angiotensin-converting enzyme inhibitor and angiotensin receptor 1 (AT1) antagonist decreased MCP-1 expression and reduced cellular infiltration and fibrosis in this model. On the other hand, Ang II injection into the renal artery induced upregulation of MCP-1 and RANTES mRNA expression as observed in vitro experiments [34, 40]. In the renal ischemia and reperfusion model, ‘early response genes’, MCP-1/JE and KC/GRO were induced during renal ischemia. Upregulation of both RANTES and MCP-1 expression was observed 2 days after temporary renal pedicle occlusion with simultaneous right nephrectomy, which was prevented by the treatment with bioflavonoids or a soluble CTLA4Ig protein, a blocker of costimulatory molecule B7 [53, 54]. Rats with significant proteinuria induced by albumin injection develop interstitial inflammation and fibrosis concomitant with interstitial monocyte recruitment. In this model, MCP-1 and osteopontin mRNA levels were increased and the proteins showed distinct tubular patterns of distribution at 2 and 4 weeks as observed in vitro protein loading [36–39, 55]. Thus, immune- and non-immune-mediated injuries are potent inducers of chemokines, especially MCP-1, which appear to play a central role in renal tubulointerstitial lesions associated with leukocyte infiltration, regardless of the initiating insults (table 3). Lessons from Gene-Targeting Mice Recent data from gene-targeting mice can give rise to the identification of the actions of chemokines and their cognate receptors [6]. Accumulating data suggest that chemokines and their cognate receptors expressed in the diseased kidneys govern critical aspects of acute and chronic renal inflammation leading to glomerulosclerosis and interstitial fibrosis [6]. Lack of CCR1 enhanced Th1 responses and glomerular injury induced by the administration of nephrotoxic serum [56]. Considering the expression of CCR1 on mesangial cells [24] and the probable involvement of CCR1 in interstitial fibrosis [31], CCR1 may protect glomerular and interstitial injury after the initial insults. On the other hand,

Yokoyama/Wada/Furuichi

74

Table 3. In vivo expression of chemokine and chemokine receptor in renal tubulointerstitium (infiltrated cells or resident cells) Models Rat or mouse Anti-GBM glomerulonephritis

Chemokines (chemokine receptors)

MIP-2, CINC MCP-1/JE* RANTES Fractalkine

Anti-TBM nephritis

MIP-2, CINC MCP-1 (CCR2) MIP-1/

Puromycin aminonucleoside nephrosis

MCP-1, MCP-3 TAC3 IP-10

Unilateral ureteral obstruction

MCP-1

Renal ischemia

CINC, KC/GROs MCP-1/JE* RANTES

Angiotensin II injection

MCP-1 RANTES

Protein loading

MCP-1

Human Crescentic glomerulonephritis

MCP-1 (CCR2) MCP-4 (CCR2, CCR3) MIP-1, RANTES (CCR1, CCR5) IP-10(?) (CXCR3) Fractalkine

IgA nephropathy, MPGN**

MCP-1 IP-10(?) (CXCR3)

Diabetic nephropathy

MCP-1

Tubulointerstitial nephritis

MCP-1 Eotaxin

Transplanted kidney rejection

MCP-1 (CCR2) MCP-4 (CCR2, CCR3) IL-8, ENA-78

Underlined molecules were detected in renal resident cells. *JE is a mouse-specific molecule with substantially different properties of human MCP-1 in length. **MPGN, membranoproliferative glomerulonephritis.

Chemokines and Renal Fibrosis

75

anti-CCR1 antibodies significantly reduced the accumulation of inflammatory cells and collagen deposition, resulting in dramatic improvement of survival in bleomycin-induced lung fibrosis in mice [57]. The identification of organ- and phase-specific roles of CCR1 will be required. Similarly, deleting MCP-1 dramatically reduces macrophage and T-cell recruitment, protects kidney pathology, reduces proteinuria and prolongs survival in MCP-1-deficient MRLFaslpr mice [58]. MCP-1-deficient mice were not protected from glomerular injury (histopathology, proteinuria, and macrophage influx) in nephrotoxic serum nephritis; however, there was a marked reduction in tubular injury in MCP-1-deficient mice [41]. In contrast, the intensity of glomerulonephritis in CCR2 knockout mice was more severe induced by nephrotoxic serum. These results suggested that glomerulonephritis is not solely dependent on the presence of CCR2 for progression of disease and that the intriguing possibility that a CCR2 gene product ameliorates glomerulonephritis in this murine model [59]. Further studies, therefore, will be required to identify the detailed molecular mechanisms of chemokine and chemokine receptor actions in renal diseases in vivo. Establishment of Cell-Mediated Renal Injury via ex-vivo Gene Transfer of Chemokine Few studies revealed the direct evidence that locally or systematically produced chemokines in the kidneys induced the leukocyte infiltration and activation. We constructed an ex-vivo gene transfer system to deliver cytokines into the kidney and circulation using genetically modified renal tubular epithelial cells. TEC were infected with recombinant retroviruses expressing macrophage growth factors, cytokines or chemokines using a retroviral Moloney murine leukemia virus-based MFG vector [60]. RANTES may explain the cellular infiltration including Th1 T cells in the interstitium in human renal diseases via its cognate receptors, CCR1 and CCR5 [31]. In addition, RANTES and its receptors were upregulated in the kidneys of a murine lupus nephritis model, MRL-Faslpr mice prior to renal injury and increased with progressive injury [61, 62]. To explore whether locally produced RANTES incites renal injury with T-cell infiltration, tubular epithelial cells genetically modified to secrete RANTES infused under the renal capsule incites interstitial nephritis in MRLFaslpr mice [61]. RANTES fostered the accumulation of a distinct subset of T cells (e.g. CD4-positive T cells), which would be compatible to clinical findings. Supporting this notion, circulating components including CD4-positive T cells are required to incite renal injury in MRL-Faslpr mice via both cellular and humoral immune responses [63, 64]. The manipulation of T cells dependent on chemokines/chemokine receptors would be of therapeutic importance in renal diseases.

Yokoyama/Wada/Furuichi

76

Chemokines and Chemokine Cascades in Human Renal Diseases with Tubulointerstitial Lesions Chemokines in Human Proliferative Glomerulonephritis In inflammatory renal diseases, the types of leukocytes migrated to the diseased kidneys depend on the types of insults; neutrophils in acute inflammation, macrophages, lymphocytes and plasma cells in chronic inflammation, resulting in glomerulosclerosis and renal interstitial fibrosis. To clarify the mechanism of specific leukocyte infiltration into the diseased kidneys and the involvement of chemokines and their cognate receptors in renal diseases, both urinary and plasma levels of chemokines were measured in patients with various renal diseases at different phases. Firstly, urinary IL-8 levels elevated in the acute phase and acute exacerbation of inflammatory renal diseases including IgA nephropathy, acute glomerulonephritis, lupus nephritis and crescentic glomerulonephritis. Urinary levels of IL-8 were correlated with glomerular endocapillary proliferation and the degree of hematuria [26, 27]. Elevated urinary IL-8 levels during the acute phase or exacerbation decreased during spontaneous or glucocorticoid therapy-induced convalescence in all patients examined. Taken together, locally produced IL-8 in the diseased kidneys may reflect the acute inflammatory phase based on leukocyte infiltration in the diseased kidneys (table 3). Secondly, urinary MCP-1 levels were significantly higher in patients with advanced renal diseases, and were correlated with pathological progressive factors such as mesangial proliferation, crescentic formation and interstitial lesions associated with CD68-positive macrophages in patients with IgA nephropathy, lupus nephritis, crescentic glomerulonephritis and other chronic inflammatory renal diseases [27, 29, 30]. Urinary MCP-1 levels in lupus nephritis patients with active lesions were significantly higher than those with inactive lesions. Moreover, elevated urinary MCP-1 levels were dramatically decreased during steroid therapy-induced convalescence. MCP-1 was mainly detected in vascular endothelial cells, tubular epithelial cells and infiltrated mononuclear cells in the interstitial lesions via both immunohistochemical and in situ hybridization analyses [27, 29, 30]. The expression of MCP-1 receptor, CCR2B mRNA was observed in cells in infiltrating leukocytes in the interstitium, as well as in cells in glomeruli and crescents [65]. Collectively, these observations suggest that MCP-1 is probably involved in the pathogenesis of progressive inflammatory human renal diseases, especially tubulointerstitial lesions possibly through the recruitment and activation of macrophages. Moreover, the measurement of urinary MCP-1 levels may be a useful clinical tool for monitoring the disease activity of inflammatory renal diseases. Considering the presence of chemokine amplification that CXC chemokines induced CC chemokines and CXC chemokines as well in mesangial cells

Chemokines and Renal Fibrosis

77

in vitro [25], switches from acute inflammation to chronic inflammation in human renal diseases may be dependent on the subsequent expression of CC chemokines (e.g. MCP-1) followed by CXC chemokines (e.g. IL-8). In addition, MCP-1 mediates collagen deposition in experimental glomerulonephritis by TGF- [66], MCP-1 expression in chronic inflammation may result in renal sclerosis/fibrosis. Supporting this notion, the administration of anti-MCP-1 antibodies prevented glomerular sclerosis and interstitial fibrosis as well as the leukocyte infiltration [17]. Therefore, the possible positive amplification loop from CXC chemokines to CC chemokines ‘chemokine cascade’ may play a crucial role in the progression of human renal diseases resulting in glomerulosclerosis and interstitial fibrosis (table 3). Human Crescentic Glomerulonephritis: Th1-Type Immune Response and Chemokine/Chemokine Receptors In rapidly progressive nephritic syndrome, crescentic glomerulonephritis, associated with a comparable degree of TIN and anemia, is a prominent feature, which leads to eventual loss of renal function [30]. Therefore, specific molecule(s) involved in the pathogenesis and the development of clinical monitoring systems for disease activities are required for the better understanding of crescentic glomerulonephritis. Urinary MIP-1 was specifically detected in patients with crescentic glomerulonephritis, even though it was below detectable levels in healthy subjects and patients with renal diseases without crescentic formation [30]. MIP-1-positive cells were mainly detected in crescentic lesions. In addition, recent studies revealed that chemokine receptors, CXCR3 and CCR5 are preferentially expressed on Th1 T cells [67–71]. Infiltrating CCR5-positive leukocytes were found mainly in the interstitium, often clustering around Bowman’s capsules in biopsies from crescentic glomerulonephritis [23]. In addition, CCR1-positive cells as well as CCR5positive cells, cognate receptors for MIP-1, were detected in diseased glomeruli and interstitium [31]. Using a dual staining technique, these positive cells were mainly CD3-positive T cells and CD68-positive macrophages. Urinary MIP-1 levels in the patients with crescentic glomerulonephritis were well correlated with percentage of cellular crescent and the number of CD68positive infiltrating cells, CCR1- and CCR5-positive cells in the glomeruli [31]. Moreover, elevated urinary levels of MIP-1 and the increased number of CCR5-positive cells dramatically decreased during glucocorticoid therapyinduced convalescence. These data suggest that MIP-1 may be involved specifically in the pathogenesis of crescentic glomerulonephritis via recruiting and activating macrophages and T cells as Th1-type immune responses and the measurement of urinary MIP-1 may be the excellent system for monitoring the clinical activities of crescentic glomerulonephritis. In contrast, urinary

Yokoyama/Wada/Furuichi

78

MCP-1 levels were well correlated with the percentage of both total crescents and fibrocellular/fibrous crescents and the number of CD68-positive infiltrating cells in the interstitium. Moreover, MCP-1 was detected mainly in the interstitium [30]. Much the same, MCP-4 was primarily expressed at peritubular, periglomerular and perivascular sites, irrespective of the inflammatory condition, and was associated with infiltrating CD3-positive lymphocytes and CD68-positive monocyte/macrophages. CCR3-, CCR5- and CCR2-expressing leukocyte populations were identified at sites of MCP-4 expression [35]. These observations suggest that locally produced MIP-1 may be involved in the development of cellular crescents in acute phase via CCR1 and CCR5 and that MCP-1 and MCP-4 may be involved mainly in the development of interstitial lesions possibly through macrophages and T-cell recruitment and activation. MCP-1: A Common Regulatory Molecule of Chronic Inflammation in Immune- and Non-Immune-Mediated Renal Diseases Resulting in Renal Sclerosis/Fibrosis MCP-1 may be involved in progressive glomerular and interstitial damage resulting in renal sclerosis/fibrosis in inflammatory renal diseases. Recent studies revealed that MCP-1 also plays an important role in the pathogenesis of non-proliferative glomerular diseases such as membranous nephropathy and metabolic disorders such as diabetic nephropathy. Locally expressed MCP-1 in interstitium participates in human diabetic nephropathy, especially in interstitial lesions, via recruiting and activating macrophages [72]. Urinary MCP-1 levels were significantly elevated in patients with diabetic nephrotic syndrome and advanced tubulointerstitial lesions. Moreover, urinary levels of MCP-1 were well correlated with the number of CD68-positive infiltrating macrophages in the interstitium. In contrast, serum MCP-1 levels remained as similar as those of healthy volunteers. Furthermore, MCP-1-positive cells were detected in the interstitium of diabetic nephropathy via both immunohistochemical and in situ hybridization analyses [72]. These observations suggest that locally produced MCP-1 may be involved in the development of advanced diabetic nephropathy, especially in the formation of tubulointerstitial lesions possibly through macrophages recruitment and activation. In addition, overexpression of MCP-1 and other fibrogenic cytokines including PDGF and TGF- were associated with an interstitial accumulation of mononuclear cells and an increase in myofibroblastic activity in patients with membranous nephropathy, non-inflammatory nephrotic syndrome [73, 74]. In contrast, patients with minimal change nephrotic syndrome showed undetectable levels of urinary MCP-1, similarly as those of healthy volunteers [29, 30]. These results suggest that there might be no correlation between levels of urinary MCP-1 and proteinuria caused by

Chemokines and Renal Fibrosis

79

Pro-inflammatory cytokines Immune complexes Complement activation Oxidant stress Lipid

Hyperglycemia, AGEs Angiotensin Endothelin Proteinuria

Renal resident cells Mesangial cells Tubular epithelial cells

Endothelial cells MCP-1 chemokines IL-1 TNF-

Coagulation

TGF- Inflammatory cells

Inflammation

Extracellular matrix

Glomerular sclerosis Interstitial fibrosis

Fig. 2. MCP-1: the pivotal molecule of acute and chronic inflammation resulting in renal inflammation and fibrosis.

non-inflammatory glomerular capillary lesions, such as minimal change nephrotic syndrome. Therefore, some additional factors in patients with membranous nephropathy may be responsible for the upregulation of MCP-1 resulting in interstitial inflammation and fibrosis. Taking these together with the results of inflammatory renal diseases, upregulation of MCP-1 may be a common regulatory pathway involved in the progressive renal damage with any etiologies leading to interstitial fibrosis (fig. 2). Additional Chemokines in Human Renal Diseases In addition to MCP-1 and RANTES as mentioned above, other chemokines and chemokine receptors may be involved in renal interstitial diseases. IL-8, MCP-1 and other C-C chemokines were involved in the pathogenesis of allograft rejection [28, 75]. In addition, MCP-1 mRNA and protein expression was clearly upregulated in acute TIN. The gene and the protein expression were primarily localized in tubular and glomerular parietal epithelial cells, as well as in infiltrating mononuclear cells [76]. Little is known about chemokines/chemokine receptors toward Th2 immune responses in situ in human kidney diseases [71]. Locally produced eotaxin, the cognate ligand for CCR3, may contribute to renal eosinophil

Yokoyama/Wada/Furuichi

80

Chemokines

Neutralizing antibodies Antagonists

Effector cell

Antagonists

NF-B AP-1 SP-1

Chemokine genes

Nucleus

Chemokines

IFNs Cyclosporin Glucocorticoids FK506 Vitamin D Aspirin FR167653

Analogues

Signals

Chemokine receptor antagonists

Fig. 3. Strategies for ‘anti-chemokine therapy’.

infiltration in the interstitium and that the elevated urinary levels of eotaxin fell dramatically during the glucocorticoid-induced convalescence, suggesting that they may reflect disease activities [77]. Recently, fractalkine expressed in the interstitium was reported to be involved leukocyte influx including CD16-positive cells [32]. Although the intervention of fractalkine receptor CX3CR1 showed the prevention of glomerular damage with the decrease in leukocyte infiltration [42], the blockade of fractalkine/its receptor may be beneficial to the prevention of interstitial damage as well as glomerular diseases. Thus, the development of therapeutic approaches via chemokines and their cognate receptors will be anticipated for the prevention of progressive renal injury.

Anti-Chemokine Therapy: Novel Therapeutic Intervention to Renal Diseases

Based on in vitro and in vivo studies, selective intervention of chemokines/chemokine receptor systems and the adequate timing of antichemokine therapy may have the potential as the particular therapeutic strategies to combat with renal diseases culminating in renal sclerosis/fibrosis [78]. Several anti-chemokine therapies are under development (fig. 3).

Chemokines and Renal Fibrosis

81

Neutralizing Antibodies against Chemokines Chemokines have been verified as inflammatory mediators of experimental nephritis by using anti-chemokine antibodies. Immune Complex-Mediated Glomerulonephritis The pathophysiological role of IL-8 was explored in animal model of acute immune complex-mediated glomerulonephritis by administering a neutralizing antibody against IL-8. Anti-IL-8 treatment decreased neutrophil number in glomeruli by 40% and dramatically prevented the fusion of epithelial cell foot process. Furthermore, anti-IL-8 treatment completely reversed the urinary levels of protein and albumin to the normal levels [16]. Anti-GBM Antibody Nephritis The specific inhibition of MCP-1 may prevent long-term renal dysfunction in concomitant reduction of glomerulosclerosis and interstitial fibrosis in an animal model of crescentic glomerulonephritis. Administration of a small dose of nephrotoxic serum induced severe proliferative and necrotizing glomerulonephritis, with crescentic formation in the early phase and glomerulosclerosis and interstitial fibrosis in the later phase in WKY rats [17, 45, 46]. Anti-MCP-1 antibodies decreased the number of macrophages in glomeruli, proteinuria and crescentic formation [17, 46]. Furthermore, this treatment remarkably reduced glomerulosclerosis and interstitial fibrosis and improved renal function on day 56 [17]. In the accelerated form of this disease model of Lewis rats, in vivo administration of anti-CINC IgG selectively attenuated the influx of neutrophils into the glomerulus and commensurately diminished proteinuria. Moreover, neutralizing antibodies to MIP-1 attenuated the acute phase proteinuria, but not the accompanying influx of neutrophils. A combination of both antibodies was not significantly more effective than either alone. In addition, the expression of another CXC chemokine, MIP-2 correlated with glomerular neutrophil influx in rat model. A single dose of the anti-MIP-2 antibodies 30 min before antiGBM antibodies was effective in reducing neutrophil influx (40% at 4 h). Functional improvement in the glomerular damage was evidenced by a reduction of abnormal proteinuria [79–81]. Anti-Thy-1.1 Antibody Nephritis Wenzel et al. [82] analyzed rats received a neutralizing anti-MCP-1 antiserum following the induction of glomerulonephritis by an anti-thymocyte antibody. The glomerular mRNA levels of MCP-1 and IL-1 increased at 3 and 24 h following the induction of the injury. In animals that received anti-MCP-1

Yokoyama/Wada/Furuichi

82

antibody, glomerular MCP-1 mRNA expression increased. However, the antiMCP-1 antiserum significantly reduced the chemoattractant activity for monocytes released into supernatants of isolated glomeruli and the glomerular monocyte and macrophage infiltration at 24 h by 40%, but was without effect on glomerular polymorphonuclear granulocyte recruitment or growth of the resident glomerular cells. These results may suggest that (1) chemokines are essentially involved in the recruitment and activation of leukocytes in the diseased kidneys, (2) IL-8 and MCP-1 may be closely related to the function of epithelial foot processes and the excreted amounts of protein, and (3) MCP-1 may play an important role in the impairment of renal functions and renal sclerosis/fibrosis. Approaches via Chemokine/Chemokine Receptor Systems Accumulating data suggest that the blockade of select chemokines and/or their cognate receptors provide the key to the improvement of renal diseases. Antagonists against RANTES receptor using Met-RANTES [83, 84] or AOPRANTES [85] decrease the cell infiltration and improve the pathological findings in the models of renal transplant acute rejection, anti-GBM nephritis in WKY rats and anti-Thy-1.1 rat nephritis. In addition, recent studies revealed that the larger DNA viruses such as poxviruses and herpesviruses thwart or exploit the activities of chemokines and their receptors by these viral proteins: virus-encoded secreted chemokine ligand homologs, which function as antagonists or agonists; virusencoded cell-surface chemokine receptor homologs, and virus-encoded secreted chemokine-binding proteins. A natural viral chemokine receptor antagonist of human herpes 8 viral origin, vMIP-II, was shown to displace chemokines binding to the CC chemokine receptors, CCR1, CCR2, CCR5, CXCR4 and CX3CR1 [86]. vMIP-II can reduce proteinuria as well as cell infiltration in experimental crescentic glomerulonephritis [43]. In addition, blocking the CCR1 using the non-peptide antagonist BX471 substantially reduced cell accumulation and renal fibrosis after unilateral ureter obstruction [87]. Considering the importance of therapeutic approaches via chemokine/chemokine receptor systems in HIV infection, the development of these small peptides or small molecular antagonists would be of use for the application to the renal diseases. Inhibiting Chemokine Signals: p38 Mitogen-Activated Kinases (MAPK) and Chemokines Mitogen-activated protein kinase (MAPK) signal transduction pathway is thought to be involved in proliferation and apoptosis in various types of inflammatory diseases [88]. The activation of MAPK isoform p38, detected also in mesangial cells, is closely related to apoptosis, stress responses and inflammation [88, 89]. Moreover, the phosphorylation of p38 MAPK may contribute to

Chemokines and Renal Fibrosis

83

the activation of NF-B and activating protein (AP)-1, which may be essentially involved in inflammatory processes. FR167653 dramatically decreased the phosphorylation of p38 MAPK in cultured rat mesangial cells stimulated by IL-1 [90]. The administration of FR167653 reduced glomerular damage, including crescentic formation, proteinuria and interstitial fibrosis [90, 91]. In addition, FR167653 markedly decreased renal expression of MIP-1 and MCP-1. This study may provide evidence that p38 MAPK is a novel target for the therapy of glomerulonephritis. Other Approaches to Modulate Chemokine/Chemokine Receptor Systems: A New Insight of Corticosteroid as a Chemokine Regulator Agents that have impacts on cAMP [92] or NF-B such as antioxidants, glucocorticoids and aspirin can modulate chemokine expression, leading to improve renal pathology [93–96]. In nephrotoxic serum-induced glomerulonephritis, AP-1 DNA-binding activity was increased in nephritic glomeruli. Activation of both NF-B and AP-1 preceded proteinuria. Treatment with prednisolone prevented activation of both NF-B and AP-1 in glomeruli and subsequent mRNA expression of NF-B- and AP-1-regulated genes [97]. The enhanced gene expression of chemokines, MCP-1, MCP-3, TCA3, MIP-1, RANTES and lymphotactin was inhibited strongly by a low dose of methylprednisolone (5 mg/kg). The level of crescents and numbers of glomerular and interstitial monocytes/macrophages and T lymphocytes were reduced by 5 mg/kg/day, but maximal effect was obtained by 30 mg/kg/day of methylprednisolone in WKY rats. Thus, corticosteroid reduces the number of infiltrating mononuclear cells and crescents in a dose-dependent fashion despite the strong inhibition of chemokine expression at a lower dose [95, 96]. The development of humanized monoclonal antibodies, particular antagonists against chemokines/chemokine receptors or chemokine-specific signal transduction that would block intrarenal chemokines could well provide significant beneficial effects in treating acute renal inflammation, as well as renal fibrosis.

Conclusion

The essential involvement of chemokine/chemokine receptor systems in the pathogenesis of the early phase of renal fibrosis was established by the intervention of chemokine/chemokine receptor systems in animal models. In human renal diseases, despite their redundancy, chemokines and chemokine receptors were also involved in the progression of fibrosis in inflammatory and non-inflammatory renal diseases. Therefore, the selective blockade of

Yokoyama/Wada/Furuichi

84

chemokines/chemokine receptor systems may have the potential to become the particular therapeutic strategies in the specific phases of renal fibrosis in future.

References 1 2

3

4 5 6 7 8

9 10 11

12 13

14

15

16

17

18 19 20

Henle J, Pfeufer C, Bright M: Klinische Mitteilungen. Z Rationelle Med Zürich 1:1844. Ridson RA, Sloper JC, Wardener HE: Relationship between renal function and histological changes found in renal biopsy specimens from patients with persistent glomerular nephritis. Lancet 1968;ii:363–366. Striker GE, Schainuck LI, Culter RE, Benditt EP: Structural-functional correlations in renal disease. I. A method for analyzing and classifying histopathological changes in renal disease. Hum Pathol 1970;1:615–630. Schainuck LI, Striker GE, Culter RE, Benditt EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1970;1:631–641. Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon M: Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol 2001;22:199–204. Segerer S, Nelson PJ, Schlöndorff D: Chemokines, chemokine receptors and renal disease: From basic science to pathophysiologic and therapeutic studies. J Am Soc Nephrol 2000;11:152–176. Eddy AA: Molecular basis of renal fibrosis. Pediatr Nephrol 2000;15:290–301. Mukaida N, Harada A, Matsushima K: Interleukin-8 and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine Growth Factor Rev 1998;9:9–23. Zlotnik A, Yoshie O: Chemokines: A new classification system and their role in immunity. Immunity 2000;12:121–127. Baggiolini M: Chemokines and leukocyte traffic. Nature 1998;392:565–568. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA: International union of Pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 2000;52:145–176. Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ: A new class of membrane-bound chemokine with a CX3C motif. Nature 1997;385:640–644. Segawa C, Wada T, Takaeda M, Furuichi K, Matsuda I, Hisada Y, Ohta S, Takasawa K, Takeda S, Kobayashi K, Yokoyama H: In situ expression and soluble form of P-selectin in human glomerulonephritis. Kidney Int 1997;52:1054–1063. Yokoyama H, Takaeda M, Wada T, Ohta S, Hisada Y, Segawa C, Furuichi K, Kobayashi K: Glomerular ICAM-1 expression related to circulating TNF- in human glomerulonephritis. Nephron 1997;76:425–433. Romagnani P, Beltrame C, Annuziato F, Lasagni L, Luconi M, Galli G, Cosmi L, Maggi E, Salvadori M, Pupilli C, Serio M: Role for interactions between IP-10/MIG and CXCR3 in proliferative glomerulonephritis. J Am Soc Nephrol 1999;10:2518–2526. Wada T, Tomosugi N, Naito T, Yokoyama H, Kobayashi K, Mukaida N, Matsushima K: Prevention of proteinuria by the administration of anti-interleukin 8 antibody in experimental acute immune complex-induced glomerulonephritis. J Exp Med 1994;180:1135–1140. Wada T, Yokoyama H, Furuichi K, Kobayashi K, Harada K, Naruto M, Su SB, Akiyama M, Mukaida N, Matsushima K: Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1). FASEB J 1996;10:1418–1425. Garin EH, Blanchard DK, Matsushima K, Djeu JY: IL-8 production by peripheral blood mononuclear cells in nephrotic patients. Kidney Int 1994;45:1311–1317. Koch AE, Polverni PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner SG, Strieter RM: Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798–1801. Strieter RM, Polverni PJ, Kunkel SL, Arenberg DA, Burdick MD, Kasper J, Dzuiba J, Van Damme J, Walz A, Marriott D, Chan SY, Roczniak S, Schnafelt AB: The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 1995;270:27348–27357.

Chemokines and Renal Fibrosis

85

21

22

23

24

25 26

27

28

29

30

31

32

33

34

35

36

37

38

Dragic T, Litwin V, Allaway GP, Martin SR, Huang YX, Nagashima KA, Cayaran C, Maddon PJ, Koup RA, Moore JP, Paxton WA: HIV-1 entry into CD4 cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996;381:667–673. Hadley TJ, Lu ZH, Wasniowska K, Martin AW, Peiper SC, Hesselgesser J, Horuk R: Postcapillary venule endothelial cells in kidney express a multispecific chemokine receptor that is structurally and functionally identical to the erythroid isoform, which is the Duffy blood group antigen. J Clin Invest 1994;94:985–991. Segerer S, Regele H, MacK M, Kain R, Cartron JP, Colin Y, Kerjaschki D, Schlondorff D: The Duffy antigen receptor for chemokines is up-regulated during acute renal transplant rejection and crescentic glomerulonephritis. Kidney Int 2000;58:1546–1556. Banasa B, Luckow B, Moeller M, Holthoefer H, Nelson PJ, Schadde E, Brigl M, Halevy D, Reinhart B, Schlondorff D: Chemokine and chemokine receptor expression in a novel human mesangial cell line. J Am Soc Nephrol 1999;10:2314–2322. Luo Y, Lloyd C, Gutierrez-Ramos JC, Dorf ME: Chemokine amplification in mesangial cells. J Immunol 1999;163:3985–3992. Wada T, Yokoyama H, Tomosugi N, Hisada Y, Ohta S, Naito T, Kobayashi K, Mukaida N, Matsushima K: Detection of urinary interleukin-8 in glomerular diseases. Kidney Int 1994;46:455–460. Yokoyama H, Wada T, Furuichi K, Segawa C, Shimizu M, Kobayashi K, Su SB, Mukaida N, Matsushima K: Urinary levels chemokines (MCAF/MCP-1, IL-8) reflect distinct disease activities and phases of human IgA nephropathy. J Leukoc Biol 1998;63:493–499. Wada T, Tomosugi N, Kobayashi K, Ishikawa I, Shinoda A, Tsugawa R, Mukaida N, Matsushima K: Appearance of interleukin-8 in the urine predicts acute rejection of renal transplantation. Nephrol Dial Transplant 1994;9:874–875. Wada T, Yokoyama H, Su SB, Mukaida N, Iwano M, Dohi K, Takahashi Y, Sasaki T, Furuichi K, Segawa C, Hisada Y, Ohta S, Takasawa K, Kobayashi K, Matsushima K: Monitoring urinary levels of monocyte chemotactic and activating factor reflects disease activity of lupus nephritis. Kidney Int 1996;49:761–767. Wada T, Furuichi K, Segawa C, Shimizu M, Sakai N, Takeda S, Takasawa K, Kida H, Kobayashi K, Mukaida N, Ohmoto Y, Matsushima K, Yokoyama H: MIP-1 and MCP-1 contribute crescents and interstitial lesions in human crescentic glomerulonephritis. Kidney Int 1999;56:995–1003. Furuichi K, Wada T, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Kobayashi K, Takasawa K, Kida H, Takeda S, Mukaida N, Matsushima K, Yokoyama H: Distinct expression of CCR1 and CCR5 in glomerular and interstitial lesions of human glomerular diseases. Am J Nephrol 2000; 20:291–299. Furuichi K, Wada T, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda S, Takasawa K, Kida H, Kobayashi K, Mukaida N, Matsushima K, Yokoyama H: Upregulation of fractalkine in human crescentic glomerulonephritis. Nephron 2001;87:314–320. Hisada Y, Sugaya T, Yamanouchi M, Uchida H, Fujimura H, Sakurai H, Fukamizu A, Murakami K: Angiotensin II plays a pathogenic role in immune-mediated renal injury in mice. J Clin Invest 1999;103:627–635. Wolf G, Ziyadeh FN, Thaiss F, Tomaszewski J, Caron RJ, Wenzel U, Zahner G, Helmchen U, Stahl RA: Angiotensin II stimulates expression of the chemokine RANTES in rat glomerular endothelial cells: Role of the angiotensin type 2 receptor. J Clin Invest 1997;100:1047–1058. Chakravorty SJ, Howie AJ, Girdlestone J, Gentle D, Savage CO: Potential role for monocyte chemotactic protein-4 in monocyte/macrophage recruitment in acute renal inflammation. J Pathol 2001;194:239–246. Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DC: Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 1997;8:1537–1545. Zoja C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, Remuzzi G: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-B activation. Kidney Int 1998;53:1608–1615. Zoja C, Benigni A, Remuzzi G: Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators. Exp Nephrol 1999;7:420–428.

Yokoyama/Wada/Furuichi

86

39

40 41

42 43

44

45

46

47 48 49 50 51 52

53 54 55 56

57 58

59

60

Wang Y, Rangan GK, Tay YC, Wang Y, Harris DC: Induction of monocyte chemoattractant protein-1 by albumin is mediated by NF-B in proximal tubule cells. J Am Soc Nephrol 1999;10: 1204–1213. Wolf G: Angiotensin II is involved in the progression of renal disease: Importance of nonhemodynamic mechanisms. Nephrologie 1998;19:451–456. Tesch GH, Schwarting A, Kinoshita K, Lan HY, Rollins BJ, Kelley VR: Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis. J Clin Invest 1999;103:73–80. Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P: Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1. Kidney Int 1999;56:612–620. Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, Thompson DA, Siani MA, Yamamoto T, Harrison JK, Feng L: In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto rats by vMIP-II. J Exp Med 1998;188: 193–198. Schneider A, Harendza S, Zahner G, Jocks T, Wenzel U, Wolf G, Thaiss F, Helmchen U, Stahl RA: Cyclooxygenase metabolites mediate glomerular monocyte chemoattractant protein-1 formation and monocyte recruitment in experimental glomerulonephritis. Kidney Int 1999;55:430–444. Fujinaka H, Yamamoto T, Feng L, Kawasaki K, Yaoita E, Hirose S, Goto S, Wilson CB, Uchiyama M, Kihara I: Crucial role of CD8-positive lymphocytes in glomerular expression of ICAM-1 and cytokines in crescentic glomerulonephritis of WKY rats. J Immunol 1997;158:4978–4983. Fujinaka H, Yamamoto T, Takeya M, Feng L, Kawasaki K, Yaoita E, Kondo D, Wilson CB, Uchiyama M, Kihara I: Suppression of anti-glomerular basement membrane nephritis by administration of anti-monocyte chemoattractant protein-1 antibody in WKY rats. J Am Soc Nephrol. 1997;8:1174–1178. Tang WW, Yin S, Wittwer AJ, Qi M: Chemokine gene expression in anti-glomerular basement membrane antibody glomerulonephritis. Am J Physiol 1995;269:F323–F330. Natori Y, Sekiguchi M, Ou Z, Natori Y: Gene expression of CC chemokines in experimental crescentic glomerulonephritis. Clin Exp Immunol 1997;109:143–148. Natori Y, Ou ZL, Yamamoto-Shuda Y, Natori Y: Expression of lymphotactin mRNA in experimental crescentic glomerulonephritis. Clin Exp Immunol 1998;113:265–268. Tang WW, Qi M, Warren JS, Van GY: Chemokine expression in experimental tubulointerstitial nephritis. J Immunol 1997;159:870–876. Ou ZL, Natori Y, Natori Y: Gene expression of CC chemokines in experimental acute tubulointerstitial nephritis. J Lab Clin Med 1999;133:41–47. Tang WW, Feng L, Mathison JC, Wilson CB: Cytokine expression, upregulation of intercellular adhesion molecule-1, and leukocyte infiltration in experimental tubulointerstitial nephritis. Lab Invest 1994;70:631–638. Chandraker A, Takada M, Nadeau KC, Peach R, Tilney NL, Sayegh MH: CD28-B7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury. Kidney Int 1997;52:1678–1684. Jones EA, Shoskes DA: The effect of mycophenolate mofetil and polyphenolic bioflavonoids on renal ischemia reperfusion injury and repair. J Urol 2000;163:999–1004. Eddy AA, Giachelli CM: Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 1995;47:1546–1557. Topham PS, Csizmadia Y, Solder D, Hines D, Gerad CJ, Salant DJ, Hancock WW: Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis. J Clin Invest 1999;104:1549–1557. Tokuda A, Itakura M, Onai N, Kimura H, Kuriyama T, Matsushima K: Pivotal role of CCR1-positive leukocytes in bleomycin-induced lung fibrosis in mice. J Immunol 2000;164:2645–2751. Tesch GH, Maifert S, Schwarting A, Rollins BJ, Kelley VR: Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Faslpr mice. J Exp Med 1999;190:1813–1824. Bird JE, Giancarli MR, Kurihara T, Kowala MC, Valentine MT, Gitlitz PH, Pandya DG, French MH, Durham SK: Increased severity of glomerulonephritis in C-C chemokine receptor 2 knockout mice. Kidney Int 2000;57:129–136. Yokoyama H, Naito T, Wada T, Kelley VR: Application of genetically engineered tubular epithelial cells in kidney disease. Exp Nephrol 1999;7:267–272.

Chemokines and Renal Fibrosis

87

61 62

63 64 65

66

67 68 69

70 71 72

73

74

75 76

77

78 79

80

81

Moore KJ, Wada T, Barbee SD, Kelley VR: Gene transfer of RANTES elicits autoimmune renal injury in MRL-Faslpr mice. Kidney Int 1998;53:1631–1641. Lema GP, Maier H, Nieto E, Vielhauer V, Luckow B, Mampaso F, Schlöndorff D: Chemokine expression precedes inflammatory cell infiltration and chemokine receptor and cytokine expression during the initiation of murine lupus nephritis. J Am Soc Nephrol 2001;12:1369–1382. Wada T, Naito T, Griffiths RC, Coffman TM, Kelley VR: Systemic autoimmune nephrogenic components induce CSF-1 and TNF- in MRL mice. Kidney Int 1997;52:934–941. Wada T, Schwarting A, Chesnutt MS, Wofsy D, Kelley VR: Nephritogenic cytokines and disease in MRL-Faslpr kidneys are dependent on multiple T-cell subsets. Kidney Int 2001;59:565–578. Segerer S, Cui Y, Hudkins KL, Goodpaster T, Eitner F, Mack M, Schlöndorff D, Alpers CE: Expression of the chemokine monocyte chemoattractant protein-1 and its receptor chemokine receptor-2 in human crescentic glomerulonephritis. J Am Soc Nephrol 2000;11:2231–2242. Schneider A, Panzer U, Zahner G, Wenzel U, Wolf G, Thaiss F, Helmchen U, Stahl RA: Monocyte chemoattractant protein-1 mediates collagen deposition in experimental glomerulonephritis by transforming growth factor-. Kidney Int 1999;56:135–144. Sallusto F, Lanzavecchia A, Mackay CR: Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today 1998;19:568–574. Loetscher P, Uguccioni M, Bordoli L, Baggiolini M, Moser B, Chizzolini C, Dayer JM: CCR5 is characteristic of Th1 lymphocytes. Nature 1998;391:344–345. Yoneyama H, Harada A, Imai T, Baba M, Yoshie O, Zhang Y, Higashi H, Murai M, Asakura H, Matsushima K: Pivotal role of TARC, a CC chemokine, in bacteria-induced fulminant hepatic failure in mice. J Clin Invest 1998;102:1933–1941. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A: Flexible programs of chemokine receptor expression on human polarized T-helper 1 and 2 lymphocytes. J Exp Med 1998;187:875–883. Holdsworth SR, Kitching AR, Tipping PG: Th1 and Th2 T-helper cell subsets affect patterns of injury and outcomes in glomerulonephritis. Kidney Int 1999;55:1198–1216. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda S, Takasawa K, Yoshimura M, Kida H, Kobayashi K, Mukaida N, Naito T, Matsushima K, Yokoyama H: Up-regulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney Int 2000;58:1492–1499. Mezzano SA, Droguett MA, Burgos ME, Ardiles LG, Aros CA, Caosrsi I, Egido J: Overexertion of chemokines, fibrogenic cytokines and myofibroblasts in human membranous nephropathy. Kidney Int 2000;57:147–158. Wada T, Yokoyama H, Sakai N, Izumiya Y, Shimizu M, Misaki T, Furuichi K, Segawa C, Kobayashi K: Cytokine up-regulation in tubulointerstitial nephritis associated with membranous nephropathy. Clin Exp Nephrol 1999;3:130–132. Robertson H, Morley AR, Talbot D, Callanan K, Kirby JA: Renal allograft rejection: -Chemokine involvement in the development of tubulitis. Transplantation 2000;69:684–687. Grandaliano G, Gesualdo L, Ranieri E, Monno R, Montinaro V, Marra F, Schena FP: Monocyte chemotactic peptide-1 expression in acute and chronic human nephritides: A pathogenetic role in interstitial monocytes recruitment. J Am Soc Nephrol 1996;7:906–913. Wada T, Furuichi K, Sakai N, Shimizu M, Segawa C, Kobayashi K, Mukaida N, Kasahara T, Matsushima K, Yokoyama H: Eotaxin contributes to renal interstitial eosinophilia. Nephrol Dial Transplant 1999;14:76–80. Rovin BH: Chemokine blockade as a therapy for renal diseases. Curr Opin Nephrol Hypertens 2000;13:225–232. Wu X, Dolecki GJ, Sherry B, Zagorski J, Lefkowith JB: Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat. J Immunol 1997;158:3917–3924. Wu X, Wittwer AJ, Carr LS, Crippes BA, DeLarco JE, Lefkowith JB: Cytokine-induced neutrophil chemoattractant mediates neutrophil influx in immune complex glomerulonephritis in rat. J Clin Invest 1994;94:337–344. Feng L, Xia Y, Yoshimura T, Wilson CB: Modulation of neutrophil influx in glomerulonephritis in the rat with anti-macrophage inflammatory protein-2 antibody. J Clin Invest 1995;95:1009–1017.

Yokoyama/Wada/Furuichi

88

82

83

84

85

86 87

88 89

90

91

92

93 94

95 96 97

Wenzel U, Schneider A, Valente AJ, Abboud HE, Thaiss F, Helmchen UM, Stahl RA: Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in anti-thymocyte antibodyinduced glomerulonephritis. Kidney Int 1997;51:770–776. Grone HJ, Weber C, Weber KS, Grone EF, Rabelink T, Kiler CM, Wells TN, Proudfoot AE, Schlondorff D, Nelson PJ: Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: Blocking monocyte arrest and recruitment. FASEB J 1999;13:1371–1383. Lloyd CM, Minto AW, Dorf ME, Proudfoot A, Wells TN, Salant DJ, Gutierrez-Ramos JC: RANTES and monocyte chemoattractant protein-1 play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis. J Exp Med 1997;185:1371–1380. Panzer U, Schneider A, Wilken J, Thompson DA, Kent SB, Stahl RA: The chemokine receptor antagonist AOP-RANTES reduces monocyte infiltration in experimental glomerulonephritis. Kidney Int 1999;56:2107–2115. Lalani AS, Barrett JW, McFadden G: Modulating chemokines: More lessons from viruses. Immunol Today 2000;21:100–106. Anders HJ, Vielhauer V, Frink M, Linde Y, Cohen CD, Blattner SM, Kretzler M, Strutz F, Mack M, Grone HJ, Onuffer J, Horuk R, Nelson PJ, Schlöndorff D: A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation. J Clin Invest 2002;109:251–259. Herlaar E, Brown Z: p38 MAPK signaling cascades in inflammatory disease. Mol Med Today 1999;5:439–447. Rovin BH, Wilmer WA, Danne M, Dickerso JA, Dixon CL, Lu L: The mitogen-activated protein kinase p38 is necessary for interleukin 1-induced monocyte chemoattractant protein-1 expression by human mesangial cells. Cytokine 1999;11:118–126. Wada T, Furuichi K, Sakai N, Hisada Y, Kobayashi K, Mukaida N, Tomosugi N, Matsushima K, Yokoyama H: Phosphorylation of p38 mitogen-activated protein kinase followed by NF-B activation and chemokine expression in crescentic glomerulonephritis. Am J Kidney Dis 2001;36:1169–1177. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Kobayashi K, Mukaida N, Matsushima K, Yokoyama H: A new anti-inflammatory compound, FR167653 ameliorates crescentic glomerulonephritis in WKY rats. J Am Soc Nephrol 2000;11:1534–1541. Satriano JA, Hora K, Shan Z, Stanley ER, Mori T, Schlöndorff D: Regulation of monocyte chemoattractant protein- and macrophage colony-stimulating factor-1 by IFN-, tumor necrosis factor-, IgG aggregates and cAMP in mouse mesangial cells. J Immunol 1993;150:1971–1978. Rangan GK, Wang Y, Tay YC, Harris DC: Inhibition of NF-B activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int 1999;56:118–134. Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto S, Kasahara T, Matsushima K: Novel mechanism of glucocorticoid-mediated gene repression. NF-B is target for glucocorticoid-mediated interleukin-8 gene repression. J Biol Chem 1994;269:13289–13295. Yamamoto-Shuda Y, Nakayama K, Saito T, Natori Y: Therapeutic effect of glucocorticoid on experimental crescentic glomerulonephritis. J Lab Clin Med 1999;134:410–418. Ou ZL, Nakayama K, Natori Y, Doi N, Saito T, Natori Y: Effective methylprednisolone dose in experimental crescentic glomerulonephritis. Am J Kidney Dis 2001;37:411–417. Sakurai H, Shigemori N, Hisada Y, Ishizuka T, Kawashima K, Sugita T: Activation of transcription factor NF-B in experimental glomerulonephritis in rats. Biochim Biophys Acta 1996;1316: 132–138.

Hitoshi Yokoyama, MD, DMSc, Department of Gastroenterology and Nephrology and Division of Blood Purification, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa 920-8641 (Japan) Tel. 81 76 2652030, Fax 81 76 2344250, E-Mail [email protected]

Chemokines and Renal Fibrosis

89

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 90–101

Pathological Significance of Renal Expression of NF-␬B Mehmet S. Inana, Mohammed S. Razzaqueb,c, Takashi Taguchic a

Genomic Center, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Mass., USA; bDepartment of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Mass., USA and c Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Introduction

Nuclear factor-␬B (NF-␬B) is a transcription factor that is responsive to various pathological processes including immunoinflammatory disorders. It also plays critical roles in cell growth, survival and apoptosis. Although transient activation of NF-␬B is a normal physiological response, the abnormal, constitutive activation of NF-␬B has been associated with a number of human diseases, including chronic inflammatory renal diseases, by regulating the expression of genes encoding for cytokines, inflammatory enzymes, growth factors, chemokines and adhesion molecules. NF-␬B Structure and Functions

NF-␬B was first discovered by Sen and Baltimore [1] in 1986 in mature B cells as a nuclear factor that binds to ␬-immunoglobin light chain enhancer. Shortly after its discovery, it became clear that NF-␬B exerts its biological activities in a wide variety of cell types. Activation of NF-␬B by numerous inducers has been the focus of investigation in a number of disease processes that involve cellular inflammation and proliferation, and apoptosis. In mammalian cells, NF-␬B, which belongs to Rel family proteins, consists of a homodimer or heterodimer of 5 possible different subunits [2].

Protein

Mass

p65 (RelA)

65 kDa

c-Rel

68 kDa

RelB

66 kDa

Structure

NH2 terminal domain Rel homology domain Nuclear localization signal Trans-activation domain Glysine-rich domain Proteolytic processing site

p50/p105 (NF-␬B1) 50/105 kDa

Ankyrin repeat domain PEST domain

p52/p100 (NF-␬B2) 52/100 kDa

I␬B-␣

37 kDa

I␬B-␤

41 kDa

Bcl-3

45 kDa

I␬B-␧

45 kDa

Fig. 1. Schematic illustration of mammalian NF-␬B and I␬B protein families.

These subunits are RelA (p65), c-Rel, NF-␬B1 (p50), NF-␬B2 (p52) and RelB (fig. 1). Although all possible combinations can form, the most abundant and studied form in the cells is p50/p65 heterodimer. All Rel family proteins have a highly conserved domain, the so-called Rel homology domain (RHD), which contains amino acid sequences essential for the formation of dimers, nuclear localization, recognition and binding DNA consensus sequence (␬ site), and association with I␬B molecules (fig. 1). In addition to RHDs, RelA, c-Rel and RelB carry a trans-activation domain which plays an important role in NF-␬Bactivated gene expression. Since p50 and p52 subunits lack a trans-activation domain that exists at the C-terminus of other members, they act as a weak activator as well as a suppressor. In non-stimulated cells, NF-␬B sequestered in the cytoplasm in its inactive state by association with inhibitory molecules, the so-called inhibitory ␬B (I␬B). The I␬B family of proteins includes I␬B-␣, I␬B-␤, I␬B-␥, I␬B-␧ and Bcl-3, which carry 5–7 ankyrin repeat units, interact directly with NF-␬B. The N-terminal regions of the I␬Bs often serve as a signal responsive domain, and the C-terminal region usually contains a proline (P), glutamate (E), serine (S) and threonine (T) (PEST) sequence, which is essential for the inhibition of NF-␬B DNA binding [3, 4] (fig. 1). The activity of NF-␬B can also be inhibited by the p105 and p100 proteins. These proteins are derived from the same polypeptides that give rise to p50 and p52, but unlike the DNA-binding factors, p105 and p100 do not undergo proteolytic processing.

NF-␬B and Kidney

91

Table 1. Partial list of genes regulated by NF-␬B Cytokines and growth factors IL-1 IL-2 IL-3 IL-6 IL-12 TNF-␣ TNF-␤ INF-␤ LT-␣ LT-␤ GM-CSF G-CSF TGF-␤1 Chemokines IL-8 MIP-1␣ MCP-1 RANTES IP10 Eotaxin GRO-1

Inflammatory enzymes iNOS COX-2 Phospholipase A2 Lipoxygenase Tissue factor Transglutaminase Apoptotic genes Fas and Fas ligand c-IAP-1 c-IAP-2 MnSOD A20 Bcl-X Adhesion molecules E-selectin ICAM VCAM

Transcription factors and inhibitors c-myc cyclin D1 I␬B-␣ Bcl-3 ESE-1 ESE-3 Receptors involves in immune functions IL-2 receptor Ig␬ light chain TCR ␤ chain ␤2-Microglobulin MHC-I, II Acute phase proteins SAA CRP C3, C4 Metalloproteinase Ferritin

IL ⫽ Interleukin; TNF-␣ ⫽ tumor necrosis factor-␣; INF-␤ ⫽ interferon-␤, LT ⫽ lymphotoxin; GM-CSF ⫽ granulocyte-macrophage colony-stimulating factor; G-CSF ⫽ granulocyte colony-stimulating factor; TGF-␤1 ⫽ transforming growth factor-␤1; MIP-1␣ ⫽ macrophage inflammatory protein-1␣; MCP-1 ⫽ monocyte chemoattractant protein-1; RANTES ⫽ regulated on activation normal T-cell expression and secreted; IP10 ⫽ IFN-␥-inducible protein 10 kDa; GRO-1 ⫽ growth-regulated oncogene 1; iNOS ⫽ inducible nitric oxide synthase; COX-2 ⫽ cyclooxygenase-2; c-IAP ⫽ inhibitor of apoptosis protein; MnSOD ⫽ Mn superoxide dismutase; A20 ⫽ anti-apoptotic protein; ICAM ⫽ intercellular adhesion molecules; VCAM ⫽ vascular cell adhesion molecules; ESE ⫽ epithelium-specific Ets transcription factor; MHC ⫽ major histocompatibility complex; TCR ⫽ T-cell receptor; SAA ⫽ serum amyloid A; CRP ⫽ C-reactive protein; C ⫽ complement.

I␬B molecules differ in their affinity for Rel proteins. For example, I␬B-␣ has a high affinity for complexes that have RelA and c-Rel, whereas p105 is the primary inhibitor of p50 homodimers. In addition, I␬B molecules can show different sensitivities to different stimuli. Depending on the cell type and on the stimulus, I␬Bs react divergently to NF-␬B-inducing signals. Generally, I␬B-␣ is rapidly degraded, whereas in other I␬Bs degradation occurs with slower

Inan/Razzaque/Taguchi

92

Table 2. Partial list of inducers of NF-␬B Cytokines and growth factors TNF-␣ and ␤ IL-1 and 2 INF-␥ GM-CSF PDGF Membrane protein ligands CD40 CD40 ligand CD28 CD3 CD4 ␣␤TCR Adhesion molecules

Bacterial and viral agents LPS EB virus HIV CMV Hepatitis B Stress ROS Hypoxia Dehydration Ischemia Hyperglycemia Acetaldehyde Lipoproteins UV light

Mitogens Phorbol esters PAF dsRNA PKC stimuli Hyaluronic acid Sphingomyelinase Ceramide

PDGF ⫽ Platelet-derived growth factor; TCR ⫽ T-cell receptor; LPS ⫽ lipopolysaccharide; EB ⫽ Epstein-Barr; HIV ⫽ human immunodeficiency virus; CMV ⫽ cytomegalovirus; ROS ⫽ reactive oxygen species; UV ⫽ ultraviolet; PAF ⫽ platelet-activating factor; PKC ⫽ protein kinase C.

kinetics [3–5]. Furthermore, different I␬Bs inhibit NF-␬B with different efficiencies. For example, I␬B-␣ is a stronger inhibitor of NF-␬B than other I␬Bs [6]. Activation of NF-␬B is a paradigm for signal transduction through the ubiquitin-proteosome pathway. Some of the genes regulated by NF-␬B are listed in table 1. Tumor necrosis factor-␣ (TNF-␣) is one of the most studied and potent inducers of NF-␬B. Following stimulation by various stimuli (table 2), I␬B-␣ is phosphorylated by a specific kinase, targeting it for an ubiquitin-ligase enzyme, which targets I␬B-␣ for subsequent degradation by the proteasome. Degradation of I␬B-␣ allows NF-␬B to translocate into the nucleus, binds the ␬B enhancer, and stimulates a number of transcriptions including pro-inflammatory, pro- and anti-apoptotic genes (fig. 2). However, NF-␬B-regulated transcription of I␬B-␣ ensues rapid re-synthesis of the I␬B-␣ protein, and this newly synthesized I␬B exports NF-␬B out of the nucleus back to cytoplasm by masking nuclear localization and DNA-binding region of NF-␬B [7, 8]. NF-␬B activation has been linked to apoptosis, acting either in an anti- or pro-apoptotic role, depending on cell type and stimuli. The relationship between apoptosis and NF-␬B was first described in RelA knockout mice studied by Beg and Baltimore [9] in 1996. These mice die embryonically because

NF-␬B and Kidney

93

Cytokines, stress agents, bacterial and viral agents, mitogens, membrane ligands Ub

I␬B on

ti

iva

I␬B

I␬

B

de gr ad

at

io

Pr

ot

n

ea

m

NF-␬B ␬B promoter elements

Basal transcription factors

r

e

NF␬B n u tran sloc clear atio n

om

ibi

to

NF-␬B

B I␬

nh

as

ei

e ot

so

Pr

NF

␬B

I␬B phosphorylation

NF-␬B

t

ac

Proteasome

Gen transc e ription

Fig. 2. A schematic pathway for NF-␬B signaling. Upon exposure of cells to stimuli, such as TNF-␣, LPS, or FasL, a kinase cascade is initiated which results in the activation of the I␬B kinase (IKK). The IKK phosphorylates I␬Bs, which then targets these proteins for degradation by the ubiquitin-dependent proteasome. Degradation of I␬B results NF-␬B to translocate into the nucleus and binds promotor of target genes.

of extensive apoptosis in the developing liver. It was also demonstrated that embryonic fibroblasts from RelA-deficient mice treated with TNF-␣ die within 8 h, whereas wild-type cells survive the treatment. The simple conclusion from this study is that RelA-activated genes induced by TNF-␣ suppress apoptosis. The mechanism whereby NF-␬B protects cells from apoptosis includes the activation of anti-apoptotic genes like A20 [9, 10] and MnSOD [11]. More recent reports have implicated NF-␬B in the induction of apoptosis. Glutamate cytotoxicity in neurons [12] and T-cell apoptosis are two situations in which NF-␬B promotes cell death. Pro-apoptotic genes that are regulated by NF-␬B include c-myc, Fas, Fas ligand and p53 [13, 14]. Identifying the factors that determine whether NF-␬B promotes or inhibits apoptosis is a major challenge. High expression of c-Rel results in a cell cycle arrest at the G1/S-phase transition [15]. In addition, NF-␬B is a positive regulator of c-myc [16], cyclin D1 [17] and other genes involved in the cell cycle. Given the role of NF-␬B in regulating cell proliferation and death, NF-␬B is also one of the crucial transcription factors that regulate the expression of

Inan/Razzaque/Taguchi

94

pro-inflammatory genes. NF-␬B has been found to be extremely active at the site of inflammation in a number of diseases. Activation of NF-␬B increases the expression of adhesion molecules, cytokines, chemokines, etc. Moreover, prevention of NF-␬B activity reduces adhesions and therefore prevents transmigration of leukocyte [18]. In experimental nephritis, activated NF-␬B enhances the production of pro-inflammatory cytokines [19, 21]. Recent studies have suggested a possible role of NF-␬B in the pathogenesis of various renal diseases, some of which eventually develop renal fibrosis. To understand the possible role of NF-␬B in the pathogenesis of various renal diseases, we need to recognize the involvement of pro-inflammatory cytokines in the initial phase of the disease process. We have chosen interleukin-1 (IL-1) and TNF-␣, which are known components of early stages of various renal diseases, as prototype molecules, and discuss their relevance to NF-␬B.

Inflammatory Cytokines and Kidney

Inflammatory responses, as a result of known and unknown factors, are encountered in the initial phase in most of the fibrotic renal diseases. The synthesis and release of pro-inflammatory cytokines and growth factors initiate a downstream cascade, which eventually helps in inducing fibrogenic molecules, and resulting in matrix remodeling in progressive renal diseases. We will present here the role of IL-1 and TNF-␣ in the inflammatory phase of various renal diseases. IL-1 and TNF-␣ are pro-inflammatory cytokines, both of which play essential roles in mediating early renal injuries in human and experimental nephritis [22–24]. IL-1 and TNF-␣ are structurally distinct and exert their bioactivities through different receptors. However, these two cytokines share many similar biological activities, such as induction of various cytokines, chemokines, adhesion molecules, metalloproteinases and tissue factor. In addition to the infiltrating leukocytes, and macrophages, which are known sources of IL-1 and TNF-␣, intrinsic renal cells, including glomerular cells and tubular epithelial cells, have shown to produce these cytokines in human and experimental nephritis [25, 26]. In vivo studies have convincingly demonstrated that blocking the bioactivities of IL-1 and TNF-␣ modulate the progression of nephritis to a certain extent, both in terms of renal functions and morphological changes [27–29]. For instance, neutralizing antibodies for IL-1, a soluble form of the IL-1 receptor and the IL-1 receptor antagonist (IL-1ra) treatment, resulted in the suppression of the progression of nephritis in experimental models; in vivo blocking the bioactivities of IL-1 in the rat model of crescentic nephritis leads to decrease proteinuria, less deterioration of renal function, and reduced renal fibrosis [27]. Similarly, blocking the bioactivities of TNF-␣ with a neutralizing anti-TNF-␣

NF-␬B and Kidney

95

antibody or a soluble form of the TNF-␣ receptor, inhibited the induction of glomerular injury in rat models of anti-GBM nephritis and acute aminonucleoside nephrosis [28]. Furthermore, when an experimental model of rats with crescentic nephritis were treated with a soluble form of the TNF-␣ receptor, a marked suppression of proteinuria, maintenance of normal renal function, and less renal fibrosis was detected [29]. These in vivo studies have clearly demonstrated that both IL-1 and TNF-␣ contribute significantly to the earlier stages of renal injury in a number of diseases involved in the kidney. More interestingly, the combined blockade of IL-1 and TNF-␣ did not provide any additional suppression of renal injury, in comparison to individual blocking of IL-1 and TNF-␣, suggesting a common downstream cascade for both of these molecules in mediating early stages of renal injuries. The introduction and relative success of anti-cytokine therapy in the treatment of certain chronic inflammatory diseases has generated considerable interest among clinicians in clinical trials. Selective inhibitors of TNF-␣, such as neutralizing antibodies or soluble TNF-␣ receptors, showed good results in clinical trials in rheumatoid arthritis and Crohn’s disease [30, 31]. Despite the impressive results in certain diseases, whether blocking a single pro-inflammatory molecule will suffice for an adequate treatment is still questionable. Hypothetically, anti-NF-␬B therapy, which provides the means to simultaneously inhibit the expression of numerous inflammatory molecules, might be a better choice for long-term treatment in chronic progressive diseases. It has been convincingly demonstrated that both IL-1 and TNF-␣ could exert their bioactivities by activating transcription factor NF-␬B.

NF-␬B and Kidney

NF-␬B is a ubiquitous transcription factor that is activated by a variety of cytokines and mitogens, and assumed to be a key regulator of genes involved in inflammation. The promoter regions of both IL-1 and TNF-␣ genes contain NF-␬B-binding sites, which act to induce transcription of a number of genes, known to mediate both inflammatory and fibrotic events in the kidney. For instance, activation of NF-␬B leads to transcription of such genes as IL-1 and TNF-␣, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor, inducible nitric oxide synthase and tissue factor. All these molecules have shown to play an important role in the induction and propagation of various renal diseases [32–34]. In vivo and in vitro studies have shown the activation of NF-␬B in a number of renal cells. For example, a high concentration of albumin has been shown

Inan/Razzaque/Taguchi

96

to induce NF-␬B activation by proximal tubular epithelial cells [35, 36]. In adriamycin-induced proteinuric rats, renal activation of NF-␬B has been documented in cortical tissues, while antioxidant pyrrolidine dithiocarbamate (PDTC) treatment resulted in improvement of interstitial damage [37]. Similar to numerous experimental animal models, activation of NF-␬B was also detected in renal biopsy sections, obtained from patients with minimal change disease, idiopathic membranous nephropathy, and IgA nephropathy [38]. By using Southwestern histochemistry, NF-␬B was detected mainly in the tubular epithelial cells of the above-mentioned proteinuric patients [38]. Activation of NF-␬B has also been documented in mesangial cells in LPS-treated animals [39]. Some of the main inducers of NF-␬B are listed in table 2. Activation of NF-␬B in the glomeruli has been demonstrated in immune complex-mediated experimental nephritis, and this activation could be suppressed, in vivo, by using PDTC (inhibitor of NF-␬B) [40]; PDTC treatment resulted in the decreased production of IL-1, MCP-1 and ICAM-1 in the glomeruli of nephritic rats. Importantly, a reduced urinary excretion of protein has been documented in PDTC-treated nephritic rats [40]. Activation of NF-␬B has also been detected during the inflammatory phase of several other renal diseases, which eventually develop renal fibrosis, including unilateral ureteral obstruction model. Removal of one kidney is a very common surgical procedure that is performed in a substantial number of cases with unavoidable circumstances, such as renal tumor. Unilateral nephrectomy results in a compensatory increase in the size and function of the remaining kidney. Hypertension is one of the longterm risks associated with unilateral nephrectomy, and usually requires longterm medical therapy to control resultant hypertension. In an animal model of unilateral ureteral obstruction, activation of NF-␬B was documented in the occluded kidneys, while no such activation was noted in the contralateral kidney, or sham-operated animals [41, 42]. Moreover, animals with unilateral ureteral obstruction, treated with an oral angiotensin-converting enzyme (ACE) inhibitor, resulted in decreased activation of NF-␬B with relatively less renal injuries [41, 42]. The beneficial effects of using ACE inhibitors are not surprising, as both angiotensin II and endothelin-1 (ET-1) are able to activate NF-␬B, possibly through AT1 and AT2 receptors, and ETA and ETB receptors, respectively [43]. In contrast to the ACE inhibitors, certain widely used drugs including Tacrolimus exert their nephrotoxic effects through the activation of NF-␬B. Tacrolimus (FK506) is a macrolide immunosuppressant which possesses slightly more potent immunosuppressant properties than cyclosporine. Tacrolimus (FK506) is a widely prescribed immunosuppressive agent which is effectively used in organ transplantation. However, nephrotoxicity is somewhat

NF-␬B and Kidney

97

limited in its clinical use. Acute nephrotoxicity is associated with reversible hemodynamic changes due to renal vasoconstriction, while chronic FK506-induced nephrotoxicity is progressive in nature and mostly irreversible. In FK506-induced nephropathy, inflammatory responses such as monocyte/macrophage influx are relatively early features that gradually lead to the development of tubulointerstitial fibrosis. In a recent study, renal activation of NF-␬B has been shown to mediate monocyte/macrophage infiltration in kidneys obtained from chronic FK506treated rats by regulating the expression of macrophage chemoattractant MCP-1 [44]. It is well known that NF-␬B plays an important role in the transcriptional regulation of MCP-1 expression [45]. Furthermore, PDTC treatment not only blocked FK506-induced renal infiltration of macrophages, but also suppressed tubular injury and interstitial fibrosis [44]. It is convincingly demonstrated that monocytes/macrophages are an important source of several fibrogenic molecules including TGF-␤1, which actively contributes to the renal fibrogenesis. Therefore, modulating the early inflammatory events by selectively blocking NF-␬B activation could play an important role in delaying and/or suppressing the progression of renal fibrosis. NF-␬B also acts as a positive regulator of fibroblast and myofibroblast growth by inducing expression of c-myc and cyclin D1 [46–48]. An increased proliferation of matrix-producing fibroblasts and myofibroblasts is a known phenomenon during renal fibrogenesis. Although c-myc is required for cell proliferation, it could also induce cell death unless certain survival factors are available to prevent it. PDGF is one such factor that helps in suppressing the pro-apoptotic activity of c-myc. The expression of PDGF and activation of NF-␬B in renal cells are not surprising. In fact, the production of PDGF and the activation of NF-␬B have been reported in various human and experimental renal diseases [49–51]. PDGF, a potent mitogen, has been shown to help in the proliferation of various matrix-producing cells, including mesangial cells, fibroblasts and myofibroblasts, in various fibrotic renal diseases. It appears likely that activation of NF-␬B and its effects on matrix-generating cells are important in subsequent renal fibrosis; it could exert mitogenic effects on matrixproducing fibroblasts and myofibroblasts during fibrogenesis. Recent studies have documented that therapeutic modulation of the activation of NF-␬B could suppress the progression of the fibrotic process. For example, experimental cholestasis by ligating bile duct causes focal necrosis, apoptosis and fibrotic changes in the liver. Using this experimental model, Zhong et al. [52] demonstrated that rats treated with adenovirus carrying mitochondrial Mn-SOD gene could reduce hepatic injury caused by experimental cholestasis, possibly by reducing the formation of oxygen radicals (4-hydroxynonenal, a product of lipid peroxidation), suppressing the activation of NF-␬B and by decreasing the synthesis of proinflammatory (TNF-␣) and profibrogenic (TGF-␤) molecules.

Inan/Razzaque/Taguchi

98

Conclusion

Further studies are required to determine the pathologic role of NF-␬B in the kidney, however a limited number of studies have suggested involvement of NF-␬B in the early phase of injury in various renal diseases. It is likely that initial renal insult due to known and unknown causes might lead to the activation of NF-␬B, which in turn may enhance the expression of pro-inflammatory cytokines, chemokines and adhesion molecules, and thereby contribute to the induction and propagation of renal injuries. Although blocking individual cytokines, chemokines and adhesion molecules have shown to have some beneficial effect on the progression of certain experimental renal diseases; however, targeting their common inducers, such as NF-␬B, might be a more useful therapeutic choice. NF-␬B not only exerts pro-inflammatory effects during various renal diseases, but also has other essential biological functions, including host defense. Because of its multifaceted role, inhibiting NF-␬B would be likely to have an adverse impact on its numerous essential cellular functions. Therefore, any such blockage of the activation of NF-␬B needs to be selective and specific to the particular disease process of the kidney, in order to avoid disruption of its other essential cellular functions. Further studies will determine whether complex renal diseases could be molecularly modulated in the early inflammatory stages, with the aim to reduce, and possibly inhibit the progression of renal scarring, by therapeutically modulating the bioactivities of NF-␬B.

References 1 2 3 4 5 6 7 8 9 10

Sen R, Baltimore D: Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986;46:705–716. Beg AA, Baldwin AS Jr: The I␬B proteins: Multifunctional regulators of Rel/NF-␬B transcription factors. Genes Dev 1993;7:2064–2070. Ghosh S, Karin M: Missing pieces in the NF-␬B puzzle. Cell 2002;109:S81–S96. Baldwin AS Jr: The NF-␬B and I␬B proteins: New discoveries and insights. Annu Rev Immunol 1996;14:649–683. Ghosh S, May MJ, Kopp EB: NF-␬BB and Rel proteins: Evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225–260. Thanos D, Maniatis T: NF-␬B: A lesson in family values. Cell 1995;80:529–532. Simeonidis S, Stauber D, Chen G, Hendrickson WA, Thanos D: Mechanisms by which I␬B proteins control NF-␬B activity. Proc Natl Acad Sci USA 1999;96:49–54. Malek S, Huxford T, Ghosh G: I␬B␣ functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-␬B. J Biol Chem 1998;273:25427–25435. Beg AA, Baltimore D: An essential role for NF-␬B in preventing TNF-␣-induced cell death. Science 1996;274:782–784. Ferran C, Stroka DM, Badrichani AZ, Cooper JT, Wrighton CJ, Soares M, Grey ST, Bach FH: A20 inhibits NF-␬B activation in endothelial cells without sensitizing to tumor necrosis factormediated apoptosis. Blood 1998;91:2249–2258.

NF-␬B and Kidney

99

11

12

13 14

15 16 17 18

19 20

21 22

23 24

25 26 27

28

29

30 31 32

Sarma V, Lin Z, Clark L, Rust BM, Tewari M, Noelle RJ, Dixit VM: Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J Biol Chem 1995;270:12343–12346. Xu Y, Kiningham KK, Devalaraja MN, Yeh CC, Majima H, Kasarskis EJ, St Clair DK: An intronic NF-␬B element is essential for induction of the human manganese superoxide dismutase gene by tumor necrosis factor-␣ and interleukin-1␤. DNA Cell Biol 1999;18:709–722. Coyle JT, Puttfarcken P: Oxidative stress, glutamate and neurodegenerative disorders. Science 1993;262:689–695. Giardina C, Boulares H, Inan MS: NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-␣ and Fas ligation: The role of reactive oxygen species. Biochim Biophys Acta 1999;1448:425–438. Grilli M, Memo M: Possible role of NF-␬B and p53 in the glutamate-induced pro-apoptotic neuronal pathway. Cell Death Differ 1999;6:22–27. Bash J, Zong WX, Gelinas C: c-Rel arrests the proliferation of HeLa cells and affects critical regulators of the G1/S-phase transition. Mol Cell Biol 1997;17:6526–6536. La Rosa FA, Pierce JW, Sonenshein GE: Differential regulation of the c-myc oncogene promoter by the NF-␬B Rel family of transcription factors. Mol Cell Biol 1994;14:1039–1044. Chen CC, Rosenbloom CL, Anderson DC, Manning AM: Selective inhibition of E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression by inhibitors of I␬B␣ phosphorylation. J Immunol 1995;155:3538–3545. Sakurai H, Hisada Y, Ueno M, Sugiura M, Kawashima K, Sugita T: Activation of transcription factor NF-␬B in experimental glomerulonephritis in rats. Biochim Biophys Acta 1996;1316:132–138. Sakurai H, Shigemori N, Hisada Y, Ishizuka T, Kawashima K, Sugita T: Suppression of NF-␬B and AP-1 activation by glucocorticoids in experimental glomerulonephritis in rats: Molecular mechanisms of anti-nephritic action. Biochim Biophys Acta 1997;1362:252–262. Mudge SJ, Paizis K, Auwardt RB, Thomas RJ, Power DA: Activation of nuclear factor-␬B by podocytes in the autologous phase of passive Heymann nephritis. Kidney Int 2001;59:923–931. Akai Y, Iwano M, Kitamura Y, Shiiki H, Dohi Y, Dohi K, Moriyama T, Yonemasu K: Intraglomerular expressions of IL-1␣ and platelet-derived growth factor-B mRNA in experimental immune complex-mediated glomerulonephritis. Clin Exp Immunol 1994;95:29–34. Waldherr R, Noronha IL, Niemir Z, Kruger C, Stein H, Stumm G: Expression of cytokines and growth factors in human glomerulonephritides. Pediatr Nephrol 1993;7:471–478. Onbe T, Kashihara N, Yamasaki Y, Makino H, Ota Z: Expression of mRNAs of cytokines and growth factors in experimental glomerulonephritis. Res Commun Mol Pathol Pharmacol 1994;86: 131–138. Noronha IL, Kruger C, Andrassy K, Ritz E, Waldherr R: In situ production of TNF-␣, IL-1␤ and IL-2R in ANCA-positive glomerulonephritis. Kidney Int 1993;43:682–692. Matsumoto K, Dowling J, Atkins RC: Production of interleukin-1 in glomerular cell cultures from patients with rapidly progressive crescentic glomerulonephritis. Am J Nephrol 1988;8:463–470. Karkar AM, Koshino Y, Cashman SJ, Dash AC, Bonnefoy J, Meager A, Rees AJ: Passive immunization against tumour necrosis factor-␣ and IL-1␤ protects from LPS enhancing glomerular injury in nephrotoxic nephritis in rats. Clin Exp Immunol 1992;90:312–318. Tang WW, Feng L, Vannice JL, Wilson CB: Interleukin-1 receptor antagonist ameliorates experimental anti-glomerular basement membrane antibody-associated glomerulonephritis. J Clin Invest 1994;93:273–279. Martin A, Molina A, Bricio T, Mampaso F: Passive dual immunization against tumour necrosis factor-␣ and IL-1␤ maximally ameliorates acute aminonucleoside nephrosis. Clin Exp Immunol 1995;99:283–288. Bickston SJ, Cominelli F: Treatment of Crohn’s disease at the turn of the century. N Engl J Med 1998;339:401–402. O’Dell JR: Anticytokine therapy – A new era in the treatment of rheumatoid arthritis? N Engl J Med 1999;340:310–312. Taal MW, Zandi-Nejad K, Weening B, Shahsafaei A, Kato S, Lee KW, Ziai F, Jiang T, Brenner BM, MacKenzie HS: Proinflammatory gene expression and macrophage recruitment in the rat remnant kidney. Kidney Int 2000;58:1664–1676.

Inan/Razzaque/Taguchi

100

33 34

35

36

37

38 39 40 41 42 43

44

45

46 47 48

49 50 51 52

Razzaque MS, Taguchi T: Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc 2002;35:68–80. Tomita N, Morishita R, Tomita S, Gibbons GH, Zhang L, Horiuchi M, Kaneda Y, Higaki J, Ogihara T, Dzau VJ: Transcription factor decoy for NF-␬B inhibits TNF-␣-induced cytokine and adhesion molecule expression in vivo. Gene Ther 2000;7:1326–1332. Zoja C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, Remuzzi G: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-␬B activation. Kidney Int 1998;53:1608–1615. Drumm K, Gassner B, Silbernagl S, Gekle M: Inhibition of Na superset⫹/H superset⫹ exchange decreases albumin-induced NF-␬B activation in renal proximal tubular cell lines (OK and LLC-PK1cells). Eur J Med Res 2001;6:422–432. Rangan GK, Wang Y, Tay YC, Harris DC: Early administration of PDTC in adriamycin nephropathy: Effect on proteinuria, cortical tubulointerstitial injury and NF-␬B activation. Ren Fail 2001; 23:773–780. Mezzano SA, Barria M, Droguett MA, Burgos ME, Ardiles LG, Flores C, Egido J: Tubular NF-␬B and AP-1 activation in human proteinuric renal disease. Kidney Int 2001;60:1366–1377. Rangan GK, Wang Y, Tay YC, Harris DC: Inhibition of NF-␬B activation with antioxidants is correlated with reduced cytokine transcription in PTC. Am J Physiol 1999;277:F779–F789. Khachigian LM, Collins T, Fries JW: N-acetylcysteine blocks mesangial VCAM-1 and NF-␬B expression in vivo. Am J Pathol 1997;151:1225–1229. Morrissey JJ, Klahr S: Enalapril decreases nuclear factor-␬B activation in the kidney with ureteral obstruction. Kidney Int 1997;52:926–933. Klahr S, Morrissey JJ: The role of growth factors, cytokines and vasoactive compounds in obstructive nephropathy. Semin Nephrol 1998;18:622–632. Asai T, Nakatani T, Yamanaka S, Tamada S, Kishimoto T, Tashiro K, Nakao T, Okamura M, Kim S, Iwao H, Miura K: Magnesium supplementation prevents experimental chronic cyclosporine A nephrotoxicity via a renin-angiotensin system independent mechanism. Transplantation 2002;74: 784–791. Tamada S, Nakatani T, Asai T, Tashiro K, Komiya T, Sumi T, Okamura M, Kim S, Iwao H, Kishimoto T, Yamanaka S, Miura K: Inhibition of nuclear factor-␬B activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy. Kidney Int 2003;63:306–314. Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T: NF-␬B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 1994;153:2052–2063. Romashkova JA, Makarov SS: NF-␬B is a target of AKT in antiapoptotic PDGF signalling. Nature 1999;401:86–90. Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS Jr: NF-␬B controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999;19:5785–5799. Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M: NF-␬B function in growth control: Regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol 1999;19:2690–2698. Nath KA: Reshaping the interstitium by platelet-derived growth factor. Implications for progressive renal disease. Am J Pathol 1996;148:1031–1036. Abboud HE: Role of platelet-derived growth factor in renal injury. Annu Rev Physiol 1995; 57:297–309. Guijarro C, Egido J: Transcription factor-␬B and renal disease. Kidney Int 2001;59:415–424. Zhong Z, Froh M, Wheeler MD, Smutney O, Lehmann TG, Thurman RG: Viral gene delivery of superoxide dismutase attenuates experimental cholestasis-induced liver fibrosis in the rat. Gene Ther 2002;9:183–191. Mohammed S. Razzaque, MBBS, PhD Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (USA) Fax ⫹1 425 732 1965, E-Mail [email protected]

NF-␬B and Kidney

101

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 102–119

Oxidant Stress and Regulation of Chemokines in the Development of Renal Interstitial Fibrosis Anita L. Cochrane, Sharon D. Ricardo Monash University, Clayton, Vic., Australia

Introduction

Progressive interstitial fibrosis is currently regarded as the most important determinant of chronic renal failure [1]. There is a strong correlation between the extent of tubulointerstitial fibrosis and the decline in renal function and long-term renal outcome in chronic glomerular injury. A histological correlate has been observed between tubular abnormalities and declining renal filtration rate in renal biopsies from patients with a variety of nephropathies. The early migration of immune cells into the renal interstitium is observed in a number of immune and non-immune renal diseases [2]. The recruitment of macrophages into the renal interstitium plays a central role in the downstream pathway of tubulointerstitial injury culminating in interstitial fibrosis. Infiltrating macrophages are thought to contribute to the fibrogenic process through production of reactive oxygen species (ROS) and many pro-fibrogenic and inflammatory cytokines. Although knowledge about the mechanisms contributing to fibrosis in progressive renal disease is expanding, the exact pathways leading to the development of tubulointerstitial injury and fibrosis remain to be elucidated. This article will review the cellular and molecular mechanisms involved in macrophage infiltration, the release of pro-inflammatory cytokines and the accumulation of extracellular matrix (ECM) proteins. Increasing evidence supports the notion that oxidant stress by an overproduction of ROS, generation of reactive nitrogen species (RNS), and/or altered capacity of the cellular

antioxidant enzymes, plays a central role stimulation of fibrogenic cytokines and increased transcription and synthesis of ECM proteins. We will examine the molecular biological studies implicating intrarenal oxidant stress, owing to an overproduction of ROS and reduced capacity of cellular antioxidant enzymes, in the induction of fibrogenic cytokines and chemoattractants and increased transcription and synthesis of collagen. Evidence implicating a nonhemodynamic role of angiotensin II (AII) leading to tubulointerstitial injury will be discussed. In addition, AII-induced oxidant stress through generation of ROS and RNS, and the role of this vasoactive protein in chemoattractant stimulation and ECM accumulation will be reviewed.

Macrophage Infiltration and Progressive Renal Disease

Macrophage infiltration into the glomerular and tubulointerstitial compartments of the kidney plays a key role in the pathogenesis of progressive renal diseases of diverse etiology. The degree of macrophage infiltration and proliferation within the tubulointerstitial compartment is widely recognized to correlate with the severity of renal failure [3]. Macrophage infiltration into the glomerular and tubular interstitium is associated with a number of clinical and experimental models of progressive renal disease [4, 5]. Macrophage infiltration is seen in biopsy specimens from patients with glomerulonephritis [6] and diabetic nephropathy [7]. Experimental evidence demonstrates that macrophage infiltration plays a key role in the pathogenesis of a variety of renal disorders, including passive Heymann nephritis [8], ischemia [9, 10], polycystic kidney disease [11], aminonucleoside and adriamycin nephrosis [12, 13], nephrotic serum nephritis [14], renal ablations [15, 16], diabetic nephropathy [17, 18], and unilateral ureteral obstruction (UUO) [3, 19]. Therapeutic intervention of macrophage infiltration has been found to reduce the severity of a range of immune cell-mediated renal diseases [20–22], reinforcing the importance of the macrophage in the pathogenesis of progressive renal disease. Macrophage influx into the renal interstitium is one of the earliest responses of the kidney to ureteral ligation. A rat model of UUO originally described by Klahr [23] serves as a useful experimental model of renal hydronephrosis and interstitial fibrosis as it is non-proteinuric and nonlipidemic, with no direct immune or toxic renal insult. An initial macrophage infiltrate has been within observed within 4–12 h of the onset of obstruction, with marked serial increments in cortical interstitial macrophage number continuing to increase through to 96 h [3, 19]. If unrelieved, this florid macrophage infiltration of the kidney contributes to the cascade of events that ultimately leads to the development of tubulointerstitial fibrosis.

Oxidant Stress and Chemokine Regulation

103

In diabetic nephropathy, macrophage infiltration has been shown to precede the development of tubulointerstitial injury. Macrophage infiltration in the glomerular and tubular compartments of the renal cortex has been demonstrated in human diabetic nephropathy [22]. A prominent glomerular macrophage infiltrate has also been observed in rats with streptozotocin (STZ)induced diabetic nephropathy [18]. Kato et al. [24] suggested that macrophage recruitment and activation may play a key role in the pathogenesis of early diabetic glomerular injury mediated via AII. In the transgenic (mRen-2)27 rat with STZ-induced diabetic nephropathy, macrophage infiltration has been found to occur in close proximity to cortical tubules expressing the chemokine osteopontin [25]. Infiltrating macrophages may release pro-inflammatory cytokines and cytotoxic mediators to destroy invading pathogens and initiate tissue repair. The pro-inflammatory cytokine transforming growth factor-1 (TGF-1) acts to stimulate the synthesis of ECM proteins and inhibits matrix degradation, events that culminate in the development of tubulointerstitial fibrosis. Other examples of pro-inflammatory mediators that may affect ECM accumulation include platelet-derived growth factor (PDGF), interleukin-1, insulin-like growth factor, and epidermal growth factor, tumor necrosis factor- (TNF-), fibroblast growth factor, proteases and eicosanoids. In addition to releasing pro-inflammatory agents, macrophages are a potent source of ROS, suggested to be one of the primary mediators of the pathogenesis of renal injury. Macrophage infiltration into the cortical interstitium is associated with not only the release of ROS and ECM accumulation, but also with cytotoxicity. A number of studies have demonstrated that macrophage cytotoxicity is associated with increased nitric oxide (NO) production within the kidney [26, 27]. Macrophage cytotoxicity is enhanced by the depletion of the NO precursor L-arginine. Under these circumstances, inducible NO synthase (iNOS)mediated superoxide anion and peroxynitrite could be particularly important in the cytotoxic actions of macrophages [28]. Furthermore, monocytes and activated macrophages produce ROS under many of the same circumstances in which they make NO [29].

Chemoattractants and Macrophage Infiltration

Chemoattractants play an important role in the recruitment and facilitation of macrophages into the renal interstitium. A number of chemoattractant molecules, such as osteopontin and monocyte chemoattractant protein (MCP)-1, have been identified to be important in infiltration of inflammatory cells in renal disease. Other chemoattractants of the small cytokine

Cochrane/Ricardo

104

(Scy superfamily/intercrine) family, including intercellular adhesion molecule (ICAM-1) and RANTES, are also known to have pro-inflammatory or reparative properties. Osteopontin, a glycosylated phosphoprotein, was originally isolated in bone and is normally present in the renal medulla in the loop of Henle and distal nephrons. After renal injury, osteopontin is up-regulated in the renal cortex, specifically in the proximal tubules and glomeruli [30]. An important structural feature of osteopontin thought to promote adhesion and cell attachment is the presence of an Arg-Gly-Asp cell-binding sequence and a thrombin-cleavage site [31]. Osteopontin is primarily involved in inflammation as a consequence of its ability to function as a regulator of inflammation [32]. Osteopontin has a variety of diverse functions apart from acting as a chemoattractant for macrophages. These include mineralization, immunity, cell adhesion and regeneration. Increased production of osteopontin has been observed in response to mitogens, growth factors, hormones, calcium, phosphate and drugs [33]. Osteopontin is overexpressed in a variety of clinical and experimental models of chronic renal disease including experimental hydronephrosis, autosomal-dominant polycystic kidney disease (ADPKD), glomerulonephritis, ischemia/reperfusion injury, diabetic nephropathy, hypertension, renal ablation, and AII-induced tubulointerstitial disease [for review, see 33]. In addition to up-regulated expression in renal injury, osteopontin is associated with cell proliferation and regeneration [33]. Giachelli et al. [34] observed a dramatic increase in osteopontin expression in kidneys from rats with focal tubulointerstitial injury associated with AII infusion. Our group has found a role for a local renin-angiotensin system (RAS) in the secretion of osteopontin from rat proximal tubular cells following cell stretch [35]. Antisense oligonucleotide transfection, targeted at angiotensinogen and the AII type 1 receptor (AT1), was found to ameliorate stretch-induced chemokine release from cultured proximal tubular cells. A mechanical/stretch-induced insult to the proximal tubular cell following ureteral ligation may increase AII synthesis and AT1 stimulation leading to the production of ROS and dysfunction of the cellular antioxidant defense enzymes [35]. Further insight into AII-mediated chemoattractant release and macrophage infiltration into the renal interstitium is demonstrated in our studies on osteopontin in the transgenic diabetic (mRen-2)27 rat [25]. Treatment of diabetic rats with the angiotensin-converting enzyme (ACE) inhibitor, perindopril, significantly ameliorated the overexpression of osteopontin and decreased macrophage accumulation [25]. The chemoattractant MCP-1, a member of a family of small cytokines (Scy superfamily/intercrines), is produced by a variety of immune and nonimmune cells and is a chemokine that mediates the recruitment of monocytes and lymphocytes. MCP-1 has an important role in macrophage influx leading

Oxidant Stress and Chemokine Regulation

105

to subsequent ECM deposition in the development of glomerular and tubulointerstitial injury. The induction of MCP-1 and RANTES expression may be due to stimulation of AII as a result of renal injury [36]. Many recent clinical and experimental studies have shown a role for MCP-1 in a number of renal disorders including glomerulosclerosis [37], ADPKD [38], cyclosporin nephrotoxicity [39], anti-glomerular basement membrane nephritis [40], minimal change disease [41], and 5/6 nephrectomy [42]. In patients with obstructive nephropathy, MCP-1 mRNA was markedly increased in the tubulointerstitium and directly correlated with the extent of macrophage infiltration and levels of urine excretion [43]. AII may play a pivotal role in the up-regulation of MCP-1 in UUO following the initial tubular injury [for review, see 44]. In a mouse model of UUO, mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to sites of tubulointerstitial damage and macrophage infiltration [45]. Pro-inflammatory cytokines, such as TNF-, plasminogen activator inhibitor type 1 and interferon (IFN)- have been found to stimulate MCP-1 activity [46]. Schwarz et al. [47] demonstrated that IFN--stimulated cultured human mesangial cells increased expression of MCP-1. Moreover, MCP-1 enhanced expression of its own mRNA and transcription factors nuclear factor kappa beta (NF-) and AP-1, suggesting autocrine feedback mechanisms in MCP-1 regulation. Wang et al. [48] have demonstrated that MCP-1 expression in proximal tubular cells is dependent on NF- activation. ROS and other free radical intermediates may be involved in the activation of genes for MCP-1 that play a central role in mediation of inflammatory cell infiltration following renal injury [49]. Sato et al. [50] demonstrated that peroxynitrite, an oxidant formed by the interaction of superoxide anion and NO, may play a regulatory role in inflammation by regulating monocyte chemotactic activity.

Oxidant Stress, an Important Forerunner for Renal Fibrosis

Oxidative Stress and Antioxidants ROS (superoxide anion, hydrogen peroxide, hydroxyl radical), including the production of RNS derived from NO (peroxynitrite, nitrogen dioxide), may play an important role in tubulointerstitial inflammation and the development of oxidant stress observed in the pathogenesis of renal injury. ROS are essential for the normal physiological process of respiration and for host defense of many immune cells. The intracellular antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) act to metabolize and detoxify ROS. Oxidant stress occurs as a result of an overproduction of ROS and/or a reduced capacity of antioxidant enzymes (fig. 1).

Cochrane/Ricardo

106

O2

•

O2

SOD H2O2

Fe3 Fe2

•

OH

Catalase GPx

H2O Overproduction of ROS

Down-regulated antioxidants

Oxidant stress

Fig. 1. The overproduction of ROS and reduced antioxidant enzyme capacity leads to the development of oxidant stress. The reduction of oxygen to water generates ROS inter• mediates comprising the free radicals, O• 2 and OH, and the powerful oxidant H2O2. Under • normal conditions, O2 is dismutased by SOD to form H2O2. The oxidant H2O2 is then reduced to form the highly reactive •OH through the actions of the ferrous ion (Fe2 to Fe3). The cellular antioxidants catalase and GPx reduce H2O2 and •OH to water. Oxidant stress results from an overproduction of ROS and/or a down-regulated expression of the cellular • antioxidant enzymes. O• 2 , superoxide anion; H2O2, hydrogen peroxide; OH, hydroxyl radical; SOD, superoxide dismutase; GPx, glutathione peroxidase.

An overproduction of ROS and down-regulated expression of cellular antioxidant enzymes may play a pivotal role in mediation of glomerular, tubular, vascular and interstitial damage [for reviews, see 51, 52]. Numerous experimental and clinical studies have shown the protective effects of antioxidants, including vitamin E and -lipoic acid, in the pathogenesis of glomerulosclerosis [53, 54], diabetic nephropathy [55, 56], cardiovascular renal disease [57], cyclosporine nephrotoxcitiy [58], and ischemic acute renal failure [59]. Increasing numbers of clinical studies confirm the pro-oxidant nature of chronic renal insufficiency irrespective of the type of disease or age of the patient [for reviews, see 52, 60]. Dietary and therapeutic intervention studies using antioxidants are an attempt to slow the rate of progression of renal disease. Nath et al. [61] demonstrated that feeding rats a pro-oxidant diet low in antioxidants, selenium and vitamin E induced renal enlargement, proteinuria, tubulointerstitial disease and reduced glomerular filtration rate (GFR) [61]. An up-regulated expression of TGF-1 in conjunction with increased collagen

Oxidant Stress and Chemokine Regulation

107

type I, III and IV mRNA expression was observed in the kidneys of rats maintained on the deficient diet. In UUO, a down-regulated expression of antioxidant enzymes may contribute to progressive tubular injury and atrophy and the development of tubulointerstitial fibrosis [for review, see 62]. The reduced activities of SOD, catalase and GPx have been observed to a greater extent following sodium depletion in a rat model of UUO [63]. We have demonstrated an increased generation of ROS in obstructed kidneys, in concert with renal macrophage influx, and impairment of normal antioxidant defense mechanisms as evidenced by down-regulated Cu-ZnSOD and catalase mRNA and protein expression [64]. Modi et al. [65] have observed that pre-treatment of probucol, a synthetic antioxidant, to rats with a 24-hour release of bilateral ureteral obstruction reduced levels of malondialdehye, a measure of lipid peroxidation, decreased macrophage infiltration, and decreased levels of oxidized glutathione, compared to rats with bilateral ureteral obstruction not receiving probucol [65]. ROS generated as a result of hyperglycemia may play a key role in the development of glomerular microvascular and pathological changes in diabetic nephropathy. Experimental and clinical evidence indicates that oxidative stress is involved in both the pathogenesis and the complications of diabetes [66–68]. Renal Cu-ZnSOD and GPx activity is decreased in patients with established type 1 diabetes mellitus and coincides with markers of tubular dysfunction [68]. Decreased Cu-ZnSOD and catalase activity have been observed in the kidneys of rats with STZ-induced diabetic nephropathy [69]. Furthermore, the administration of glutathione to rats with STZ-induced diabetic nephropathy restores renal cortical Cu-ZnSOD activity levels [69]. Long-term administration of antioxidants inhibits the development of early diabetic retinopathy in rats [70]. Nicotinamide adenine dinucleotide phosphate (NADPH) may play a central role in advanced glycation end-product (AGE)-mediated generation of ROS [71]. Recent reports suggest that AGE inhibitors, previously shown to have beneficial effects in diabetes, may act to inhibit the chemical modification of proteins during lipid peroxidation and may act directly as an antioxidant [72]. Hyperglycemia is thought to lead to glucose-induced activation of protein kinase C (PKC), increased formation of AGEs, and increased glucose flux through the aldose reductase pathway. Increased glucose-induced generation of ROS may serve as a unifying mechanism of induction between elevated glucose and each of the three major pathways responsible for diabetic damage [73]. Nishikawa et al. [74] recently demonstrated that ROS activates aldose reductase, induces diacylglycerol (DAG), activates PKC and AGE formation, and activates transcription factors. These studies demonstrated that hyperglycemia increases the production of ROS in cultured aortic endothelial cells. Normalizing levels of mitochondrial ROS with an inhibitor of electron transport chain complex II, an

Cochrane/Ricardo

108

uncoupler of oxidative phosphorylation and the addition of SOD, was found to prevent glucose-induced activation of PKC, formation of AGEs, sorbitol accumulation and NF- activation [74]. ROS may stimulate signaling pathways and transcription factors including NF- and AP-1 [75, 76]. An antioxidant-sensitive pathway involving the redoxsensitive NF-, may play a role in the activation of macrophages and transcriptional up-regulation of inflammatory and fibrogenic cytokines including TGF-1, PDGF and TNF-. NF- modulates ROS generation and the genes encoding the transcription of inflammatory genes and adhesion molecules. NF- has been suggested to play a role in diabetes and its complications. Hyperglycemia activates NF-, in part by activation of PKC via overproduction of ROS [74]. Activation of the DAG-PKC pathway plays a key role in the development of diabetic vascular complications via ROS and glycation products. Hyperglycemia may activate PKC isoforms indirectly via both ligation of AGE receptors and increased activity of the polyol pathway via ROS generation [73]. Oxidative Stress and the Development of Interstitial Fibrosis In addition to direct cytotoxic effects of ROS leading to tubulointerstitial inflammation, activation of ROS may directly stimulate collagen synthesis leading to ECM accumulation and fibrosis [77]. Antioxidant enzymes and ROS play an integral role in the activation of chemoattractant gene expression, the overexpression of fibrogenic cytokines, and increased transcription and synthesis of ECM proteins. Oxidative stress may be involved in the activation of genes for MCP-1 and ICAM-1, cell adhesion molecules that play a central role in the mediation of inflammatory cell infiltration. ROS may also stimulate cell proliferation and collagen synthesis, important events leading to the development of interstitial fibrosis. Furthermore, the phenotypic transformation of fibroblasts to myofibroblasts may involve ROS [77]. Oxidative stress has previously been found to stimulate collagen 1(1) gene expression in cultured human fibroblasts [78]. The addition of -tocopherol to the fibroblast cultures was found to decrease collagen synthesis and gene transcription. ROS may act directly with the lipid bilayers of cell membranes resulting in toxic aldehydic end-products of lipid peroxidation. Lipid aldehydes can modulate genetic overexpression of fibrogenic cytokines [75, 76]. This disruption of the focal contacts between oxidatively modified cells and surrounding matrix may play a role in the development of tubulointerstitial disease. More recently, Park et al. [79] demonstrated that hydrogen peroxide is a direct inducer of connective tissue growth factor (CTGF), a stimulator of collagen type I and fibronectin synthesis via JAK-2/-3 activation. It has recently been demonstrated [80] that some of the pro-fibrotic effects of TGF-1 may be mediated via the formation of hydrogen peroxide. In these studies, TGF-1-induced up-regulation

Oxidant Stress and Chemokine Regulation

109

SOD activity (U/mg protein)

350 300 250 200

*

*

*

150

* 100

24 hour

48 hour

96 hour

UUO CUK Cand UUO Cand CUK

UUO CUK Cand UUO Cand CUK

UUO CUK Cand UUO Cand CUK

0

UUO CUK Cand UUO Cand CUK

50

1 week

Fig. 2. Represents a graph of Cu-ZnSOD activity, a superoxide anion scavenging enzyme, in rats following 24, 48, 96 hours and 1 week of ureteral ligation. At all time points, the renal cortical homogenates of obstructed kidneys from UUO rats receiving candesartan (1 mg/kg body weight) showed significantly elevated Cu-ZnSOD activity, compared to the obstructed kidneys from UUO rats not receiving candesartan (24 h, 130.67 58.66 vs. 262.49 74.93, p 0.027; 48 h, 144.28 48.55 vs. 212.46 69.40, p 0.020; 96 h, 98.3 17.6 vs. 139.64 17.01, p 0.013, and 1 week, 150.45 25.62 vs. 197.28 25.81, p 0.021; n 5 rats per group/time point). UUO, unilateral ureteral obstruction; CUK, contralateral unobstructed kidney; Cand UUO, the obstructed kidney from UUO rats receiving candesartan; Cand CUK, the contralateral unobstructed kidney from candesartan UUO rats.

of (1) procollagen gene was associated with production of hydrogen peroxide in cultured hepatic stellate cells [80]. Angiotensin II and ROS Production Changes in renal hemodynamics and GFR occur as a result of complex interactions between vasoconstrictor and vasodilatory agents. Recently, a nonhemodynamic action of AII has been described in renal injury, namely, the induction of oxidant stress [81–83]. In a rat model of UUO, we have demonstrated that AII acts as an initial mediator of oxidant stress-induced renal injury. Cu-ZnSOD activity in the obstructed kidney was decreased following UUO in comparison to the contralateral unobstructed kidneys. Administration of the AT1 inhibitor, candesartan, was found to restore antioxidant activity towards control values (fig. 2). Hannken et al. [81] have demonstrated that AII induces ROS,

Cochrane/Ricardo

110

subsequently leading to induction of p27(Kip1) and hypertrophy of proximal tubular cells. Recent evidence suggests that AII may stimulate production of superoxide anion in LLC-PK1 cells generated through activation of membranebound NADPH oxidase [83]. A flavoprotein inhibitor as well as p22phox antisense oligonucleotide treatment abolished the AII-induced generation of ROS, concluding that the effect was transduced through the AT1 receptor and depended on activation of membrane NADPH oxidase [83]. In addition, Haugen et al. [82] reported that AII infusion to rats in vivo induced oxidative stress and increased the renal expression of the oxidant-sensitive gene, hemeoxygenase (HO-1). In studies using cultured LLC-PK1 cells, increased HO-1 mRNA expression was observed in response to AII [82].

Reactive Nitrogen Species in Renal Fibrosis

Nitric Oxide: A Double-Edged Sword NO production has been implicated in the development of progressive renal disease, with studies demonstrating that macrophage cytotoxicity is associated with increased NO production within the kidney [84]. In contrast to the beneficial vasomodulatory actions of NO, the diverse actions of this molecule are also implicated in a variety of inflammatory renal diseases. When produced in normal physiological concentrations, macrophage-derived NO, acting as a free radical, plays an important role in immunological host defense. However, when NO is generated in large concentrations, tissue inflammation and cell destruction can result from the formation of RNS [85, 86]. NO is a free radical formed from L-arginine in a reaction catalyzed by NOS enzymes that requires O2 and NADPH. Neuronal NOS (nNOS, type I) and endothelial NOS (eNOS, type III) are constitutively expressed and calcium dependent. eNOS and nNOS produce low levels of NO for only a short period of time per response. Conversely, inducible NOS (iNOS, type II) can produce NO for long periods of time, is cytokine inducible and calcium independent. Sustained action may account for the large magnitude of NO produced by the iNOS isoform, a factor that may be important in determining whether NO plays a beneficial or damaging role during the development of tubulointerstitial fibrosis. Renal NO production has been linked with apoptosis contributing to renal cell injury. Using in situ hybridization, iNOS has been localized to the renal medullary thick ascending limb, proximal tubules and collecting ducts [87]. In addition, iNOS has been found in the arcuate arteries and vasa recta bundles [88]. Glomerular mesangial cells, endothelial cells and epithelial cells exposed to NO undergo cell death, displaying both the morphological and biochemical

Oxidant Stress and Chemokine Regulation

111

changes characteristic of apoptosis [89]. Amore et al. [90] demonstrated that cyclosporine A induces apoptosis in cultured mesangial, cortical and endothelial cells via the induction of iNOS, leading to increased expression of p53. NO-dependent apoptosis has also been associated with ischemia/ reperfusion renal cell injury in transgenic sickle cell mouse kidneys where iNOS activation and superoxide anion formation contributed to the formation of RNS [91].

Peroxynitrite and the Development of Oxidant Stress The production of highly RNS derived from NO may contribute to oxidative stress leading to tubulointerstitial fibrosis. RNS are formed when NO and superoxide anion act together to initiate oxidant injury via the formation of peroxynitrite (ONOO): NO O• 2 → ONOO

Peroxynitrite is a potent and versatile oxidant that can react with lipids, proteins and DNA, inducing tissue injury and dysfunction [92, 93]. The production of iNOS has been implicated as the source of NO contributing to peroxynitrite formation in renal disease [94, 95]. During the development of interstitial fibrosis, the reaction between superoxide anion and NO is favored as a result of oxidant stress. NO is generated by cells that are also capable of producing superoxide anion. Indeed, NOS enzymatic activity may be responsible for superoxide anion generation. Traylor and Mayeux [96] found that NO and superoxide anion are both produced by proximal tubule NOS. This study used NOS inhibition and the addition of L-arginine to demonstrate that blocking the formation of either NO or superoxide anion ameliorates cytotoxicity. Pathological factors that enhance the local release of superoxide anion are likely to induce peroxynitrite formation and oxidant injury. Heeringa et al. [97] demonstrated nitrotyrosine localization in the renal interstitium of rats with anti-myeloperoxidase-associated cresentic glomerulonephritis. Furthermore, double-staining experiments revealed that most nitrotyrosine-expressing cells also produced both superoxide anion and iNOS, indicating that iNOS-derived NO may contribute to peroxynitrite formation during glomerulonephritis. In contrast, it is suggested that eNOS-induced NO has a renoprotective role during the development of glomerulonephritis [98]. Increased production of nitrotyrosine and ROS, associated with an up-regulation of iNOS and eNOS, has been attributed to the development of proteinuria, tubular atrophy, glomerulosclerosis and interstitial fibrosis in chronic iron overload [99]. Nitrotyrosine has previously been found in proximal tubule cells of patients with diabetic nephropathy where excessive peroxynitrite generation is

Cochrane/Ricardo

112

suggested to be an important mediator of tubulointerstitial injury [100]. Interestingly, rats with early proteinuric STZ-induced diabetes mellitus display oxidant stress associated with enhanced renal expression of eNOS and nitrotyrosine, changes that were prevented via interruption of the RAS [101]. Macrophages from spontaneously diabetic db/db mice exhibit increased levels of NO and altered cytokine secretion [102]. Interestingly, iNOS-derived NO has previously been found to contribute to peroxynitrite-dependent platelet damage in diabetes [103]. Up-regulation of iNOS mRNA and increased iNOS activity has been observed in murine mesangial cells and macrophages exposed to high glucose, conditions that mimic the diabetic cellular environment [104]. Ishii et al. [105] recently concluded that increased renal cortical synthesis of NO occurred during STZ-induced diabetes mellitus. This NO formation occurred in the setting of accelerated superoxide anion production and subsequently resulted in the rapid formation of the potent oxidant peroxynitrite.

Conclusions

Figure 3 represents the putative pathway of events occurring in the development of tubulointerstitial injury centered on the UUO model as a prototype disorder. The central theme drawn from data in this model is that the mechanical disturbance as a result of ureteral obstruction may lead to up-regulation of a local RAS in the proximal tubular epithelium. Proximal tubular ACE may facilitate the epithelial generation of AII that then stimulates, in an autocrine manner, the production of macrophage recruitment factors, such as osteopontin. These chemoattractants mediate macrophage influx into the renal interstitium. Proximal tubule-derived AII has been found to act via the AT1 receptor to up-regulate chemoattractant expression and induce iNOS transcription. Chemoattractants and adhesion molecules facilitate macrophage infiltration into the cortical interstitium within hours of ureteral ligation. Macrophage and tubular-derived iNOS generate NO and ROS leading to the formation of peroxynitrite, a powerful oxidant species intermediate. We have also observed that coincident with tubular injury after UUO, there is a dramatic early reduced capacity of cellular antioxidant enzymes. An overproduction of ROS and RNS, together with down-regulated expression of cellular antioxidants, may lead to increased intrarenal oxidant stress. In addition to direct toxic effects on renal cells, ROS and toxic end-products of lipid peroxidation may modulate the expression of chemoattractants and play an integral role in the overexpression of fibrogenic cytokines. Oxidative stress may also directly stimulate increased synthesis of collagen, an important forerunner for the events leading to interstitial fibrosis.

Oxidant Stress and Chemokine Regulation

113

PT

Ureteral obstruction

ANG II AT1 iNOS

Osteopontin ICAM-1 MCP-1 RANTES

iNOS Antioxidant down-regulation Tubular injury Tubular loss

Macrophage infiltration

NO & ROS

TGF-1

ONOO Oxidant stress

↑TIMP-1 Myofibroblast ↓MMP transformation

Extracellular matrix accumulation

Interstitial fibrosis

Fig. 3. Schematic diagram showing the putative molecular and cellular events mediating the pro-inflammatory response to ureteral obstruction, leading to the development of oxidant stress, culminating in ECM accumulation and ultimately the development of interstitial fibrosis. ANG II, angiotensin II; AT1, angiotensin II type 1 receptor; ICAM-1, intracellular adhesion molecule-1; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinases; MCP-1, monocyte chemoattractant peptide-1; NO, nitric oxide; ONOO, peroxynitrite; ROS, reactive oxygen species; TGF-1, transforming growth factor-1; TIMP-1, tissue inhibitor of metalloproteinases-1.

Finally, activated macrophages infiltrating into the tubulointerstitium as a result of UUO release an array of pro-inflammatory and fibrogenic cytokines. One of the many macrophage-derived products is TGF-1. Macrophagederived TGF-1 promotes fibrogenesis by stimulating the synthesis of ECM proteins in parallel with the down-regulation of matrix metalloproteinases and the up-regulation of matrix metalloproteinase inhibitors. In addition, TGF-1 and generation of ROS can lead to modulation of fibroblasts to a more contractile phenotype leading to increased transcription and synthesis

Cochrane/Ricardo

114

of collagen and production of pro-inflammatory cytokines. A net effect of these events is tubulointerstitial inflammation and the development of interstitial fibrosis.

References 1 2 3 4 5 6 7

8

9

10 11

12 13 14 15 16

17 18 19

Eddy AA: Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. J Am Soc Nephrol 1994;5:1273–1287. Nikolic-Paterson DJ, Lan HY, Hill PA, Atkins RC: Macrophages in renal injury. Kidney Int Suppl 1994;45:S79–S82. Schreiner GF, Harris KP, Purkerson ML, Klahr S: Immunological aspects of acute ureteral obstruction: Immune cell infiltrate in the kidney. Kidney Int 1988;34:487–493. Nikolic-Paterson DJ, Lan HY, Hill PA, Atkins RC: Macrophages in renal injury. Kidney Int Suppl 1994;45:S79–S82. Schreiner GF: The role of the macrophages in glomerular injury. Semin Nephrol 1991;11: 268–275. Yang N, Isbel NM, Nikolic-Paterson DJ, Li Y, Ye R, Atkins RC, Lan HY: Local macrophage proliferation in human glomerulonephritis. Kidney Int 1998;54:143–151. Uesugi N, Sakata N, Horiuchi S, Nagai R, Takeya M, Meng J, Saito T, Takebayashi S: Glycoxidation-modified macrophages and lipid peroxidation products are associated with the progression of human diabetic nephropathy. Am J Kidney Dis 2001;38:1016–1025. Eddy AA, Ho GC, Thorner PS: The contribution of antibody-mediated cytotoxicity and immunecomplex formation to tubulointerstitial disease in passive Heymann nephritis. Clin Immunol Immunopathol 1992;62:42–55. Goes N, Urmson J, Ramassar V, Halloran PF: Ischemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-, transforming growth factor-1, granulocyte-macrophage colony-stimulating factor, interleukin-2 and interleukin-10. Transplantation 1995;59:565–572. Truong LD, Farhood A, Tasby J, Gillum D: Experimental chronic renal ischemia: Morphologic and immunologic studies. Kidney Int 1992;41:1676–1689. Takahashi H, Calvet JP, Dittemore-Hoover D, Yoshida K, Grantham JJ, Gattone VH 2nd: A hereditary model of slowly progressive polycystic kidney disease in the mouse. J Am Soc Nephrol 1991;1:980–989. Jones CL, Buch S, Post M, McCulloch L, Liu E, Eddy AA: Renal extracellular matrix accumulation in acute puromycin aminonucleoside nephrosis in rats. Am J Pathol 1992;141:1381–1396. Saito T, Atkins RC: Contribution of mononuclear leucocytes to the progression of experimental focal glomerular sclerosis. Kidney Int 1990;37:1076–1083. Eddy AA: Tubulointerstitial nephritis during the heterologous phase of nephrotoxic serum nephritis. Nephron 1991;59:304–313. Van Goor H, van der Horst ML, Fidler V, Grond J: Glomerular macrophage modulation affects mesangial expansion in the rat after renal ablation. Lab Invest 1992;66:564–571. Wu LL, Yang N, Roe CJ, Cooper ME, Gilbert RE, Atkins RC, Lan HY: Macrophage and myofibroblast proliferation in remnant kidney: Role of angiotensin II. Kidney Int Suppl 1997;63: S221–S225. Ziyadeh FN, Goldfarb S: The renal tubulointerstitium in diabetes mellitus. Kidney Int 1991;39:464–475. Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, Couser WG, Seidel K: Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int 1995;47:935–944. Sato N, Shiraiwa K, Kai K, Watanabe A, Ogawa S, Kobayashi Y, Yamagishi-Imai H, Utsunomiya Y, Mitarai T: Mizoribine ameliorates the tubulointerstitial fibrosis of obstructive nephropathy. Nephron 2001;89:177–185.

Oxidant Stress and Chemokine Regulation

115

20

21

22 23

24

25

26 27 28 29 30

31 32 33 34

35

36 37

38 39

40

41

Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P, Wilson CB, Harrison JK, Bacon KB: Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication. Kidney Int 1999;56:612–620. Romero F, Rodriguez-Iturbe B, Parra G, Gonzalez L, Herrera-Acosta J, Tapia E: Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats. Kidney Int 1999;55:945–955. Yokoyama H, Wada T, Furuichi K: Chemokines in renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 66–89. Klahr S: New insights into the consequences and mechanisms of renal impairment in obstructive nephropathy. The modulation of macrophage migration and eicosanoid metabolism. Am J Kidney Dis 1991;18:689–699. Kato S, Luyckx VA, Ots M, Lee KW, Ziai F, Troy JL, Brenner BM, MacKenzie HS: Renin-angiotensin blockade lowers MCP-1 expression in diabetic rats. Kidney Int 1999;56: 1037–1048. Kelly DJ, Wilkinson-Berka JL, Ricardo SD, Cox AJ, Gilbert RE: Progression of tubulointerstitial injury by osteopontin-induced macrophage recruitment in advanced diabetic nephropathy of transgenic (mRen-2)27 rats. Nephrol Dial Transplant 2002;17:985–991. Ketteler M, Border WA, Noble NA: Cytokines and L-arginine in renal injury and repair. Am J Physiol 1994;267:F197–F207. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM: Nitric oxide: A cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988;157:87–94. Xia Y, Zweier JL: Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 1997;94:6954–6958. Klebanoff SJ: Oxygen metabolites from phagocytes; in Gallin J, Goldstein I, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates. New York, Raven Press, 1992, pp 541–588. Diamond JR, Kees-Folts D, Ricardo SD, Pruznak A, Eufemio M: Early and persistent up-regulated expression of renal cortical osteopontin in experimental hydronephrosis. Am J Pathol 1995;146:1455–1466. Denhardt DT, Guo X: Osteopontin: A protein with diverse functions. FASEB J 1993;7:1475–1482. Giachelli CM, Steitz S: Osteopontin: A versatile regulator of inflammation and biomineralization. Matrix Biol 2000;19:615–622. Xie Y, Sakatsume M, Nishi S, Narita I, Arakawa M, Gejyo F: Expression, roles, receptors and regulation of osteopontin in the kidney. Kidney Int 2001;10:1645–1657. Giachelli CM, Pichler R, Lombardi D, Denhardt DT, Alpers CE, Schwartz SM, Johnson RJ: Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis. Kidney Int 1994;45:515–524. Ricardo SD, Franzoni DF, Roesener CD, Crisman JM, Diamond JR: Angiotensinogen and AT1 antisense inhibition of osteopontin translation in rat proximal tubular cells. Am J Physiol 2000;278:F708–F716. Wolf G: Angiotensin II is involved in the progression of renal disease: Importance of nonhemodynamic mechanisms. Nephrologie 1998;19:451–456. Park YS, Guijarro C, Kim Y, Massy ZA, Kasiske BL, Keane WF, O’Donnell MP: Lovastatin reduces glomerular macrophage influx and expression of monocyte chemoattractant protein-1 mRNA in nephrotic rats. Am J Kidney Dis 1998;31:190–194. Cowley BD Jr, Ricardo SD, Nagao S, Diamond JR: Increased renal expression of monocyte chemoattractant protein-1 and osteopontin in ADPKD. Kidney Int 2001;60:2087–2096. Di Paolo S, Grandaliano G, Gesualdo L, Ranieri E, Schena FP: Low-density lipoproteins enhance transforming growth factor-1 and monocyte chemotactic protein-1 expression induced by cyclosporin in human mesangial cells. Clin Exp Immunol 1999;117:355–360. Suganami T, Mukoyama M, Sugawara A, Mori K, Nagae T, Kasahara M, Yahata M, Makino H, Fujinaga Y, Ogawa Y, Tanaka I, Nakao K: Overexpression of brain natriuretic peptide in mice ameliorates immune-mediated renal injury. J Am Soc Nephrol 2001;12:2652–2663. Mezzano SA, Barria M, Droguett MA, Burgos ME, Ardiles LG, Flores C, Egido J: Tubular NF-B and AP-1 activation in human proteinuric renal disease. Kidney Int 2001;60: 1366–1377.

Cochrane/Ricardo

116

42

43

44 45

46

47 48

49 50

51 52 53

54 55 56

57 58

59 60 61 62 63

Taal MW, Chertow GM, Rennke HG, Gurani A, Jiang T, Shahsafaei A, Troy JL, Brenner BM: Mechanisms underlying renoprotection during renin-angiotensin system blockade. Am J Physiol 2001;280:F343–F355. Grandaliano G, Gesualdo L, Bartoli F, Ranieri E, Monno R, Leggio A, Paradies G, Caldarulo E, Infante B, Schena FP: MCP-1 and EGF renal expression ad urine excretion in human congenital obstructive nephropathy. Kidney Int 2000;58:182–192. Klahr S: Urinary tract obstruction. Semin Nephrol 2001;21:133–145. Vielhauer V, Anders HJ, Mack M, Cihak J, Strutz F, Stangassinger M, Luckow B, Grone HJ, Schlondorff D: Obstructive nephropathy in the mouse: Progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes. J Am Soc Nephrol 2001;12:1173–1187. Ghanim H, Garg R, Aljada A, Mohanty P, Kumbkarni Y, Assian E, Hamouda W, Dandona P: Suppression of nuclear factor and stimulation of inhibitor by troglitazone: Evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese. J Clin Endocrinol Metab 2001;86:1306–1312. Schwarz M, Wahl M, Resch K, Radeke HH: IFN- induces functional chemokine receptor expression in human mesangial cells. Clin Exp Immunol 2002;128:285–294. Wang Y, Rangan GK, Tay YC, Wang Y, Harris DC: Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor B in proximal tubular cells. J Am Soc Nephrol 1999;10:1204–1213. Schlondorff D: The role of chemokines in the initiation and progression of renal disease. Kidney Int 1995;47:S44–S47. Sato E, Simpson KL, Grisham MB, Koyama S, Robbins RA: Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro. Am J Physiol 1999;277:L543–L549. Rohrmoser MM, Mayer G: Reactive oxygen species and glomerular injury. Kidney Blood Press Res 1996;19:263–269. Mathur S, Devaraj S, Jialal I: Accelerated atherosclerosis, dyslipidemia and oxidant stress in endstage renal disease. Curr Opin Nephrol Hypertens 2002;11:141–147. Trachtman H, Chan JC, Chan W, Valderrama E, Brandt R, Wakely P, Futterweit S, Maesaka J, Ma C: Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy. Pediatr Res 1996;40:620–626. Scheinman JI, Trachtman H, Lin CY, Langman CB, Chan JC: IgA nephropathy: To treat or not to treat? Nephron 1997;75:251–258. Imaeda A, Kaneko T, Aoki T, Kondo Y, Nagase H: DNA damage and the effect of antioxidants in streptozotocin-treated mice. Food Chem Toxicol 2002;40:979–987. Evans JL, Heymann CJ, Goldfine ID, Gavin LA: Pharmokinetics, tolerability and fructosaminelowering effect of a novel, controlled-release formulation of -lipoic acid. Endocr Pract 2002;8:29–35. Schackenberg CG: Oxygen radicals in cardiovascular-renal disease. Curr Opin Pharmacol 2002;2:121–125. Satyanarayana PS, Singh D, Chopra K: Quercetin, a bioflavanoid, protects against oxidative stressrelated renal dysfunction by cyclosporine in rats. Methods Findings Exp Clin Pharmacol 2001;23:175–181. Takaoka M, Ohkita M, Kobayashi Y, Yuba M, Matsumura Y: Protective effect of -lipoic acid against ischaemic acute renal failure in rats. Clin Exp Pharmacol Physiol 2002;29:189–194. Davis ID, MacRae Dell K, Sweeney WE, Avner ED: Can progression of autosomal dominant or autosomal recessive polycystic kidney disease be prevented? Semin Nephrol 2001;21:430–440. Nath KA, Grande J, Croatt A, Haugen J, Kim Y, Rosenberg ME: Redox regulation of renal DNA synthesis, transforming growth factor-1 and collagen gene expression. Kidney Int 1998;53: 367–381. Ricardo SD, Diamond R: The role of macrophages and reactive oxygen species in experimental hydronephrosis. Semin Nephrol 1998;18:612–621. Kinter M, Wolstenholme JT, Thornhill BA, Newton EA, McCormick ML, Chevalier RL: Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion. Kidney Int 1999;55:1327–1334.

Oxidant Stress and Chemokine Regulation

117

64 65 66

67 68 69

70 71

72 73 74

75 76 77 78

79

80 81

82 83

84

85 86

Ricardo SD, Ding G, Eufemio M, Diamond J: Antioxidant expression in experimental hydronephrosis: Role of mechanical stretch and growth factors. Am J Physiol 1997;272:F789–F798. Modi KS, Morrissey J, Shah SV, Schreiner GF, Klahr S: Effects of probucol on renal function in rats with bilateral ureteral obstruction. Kidney Int 1990;38:843–850. Wautier MP, Chappey O, Lorda S, Stern DM, Schmidt AM, Wautier SL: Activation of NADPH oxidation by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol 2001;280:E685–E694. Onorato JM, Jenkins AJ, Thorpe SR, Baynes JW: Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. J Biol Chem 2000;275:21177–21184. Yaqoob M, McClelland P, Patrick AW, Stevenson A, Mason H, White MC, Bell GM: Evidence of oxidant injury and tubular damage in early diabetic nephropathy. Q J Med 1994;87:601–607. Loven D, Schedl H, Wilson H, Daabees TT, Stegink LD, Diekus M, Oberley L: Effect of insulin and oral glutathione on glutathione levels and superoxide dismutase activities in organs of rats with streptozotocin-induced diabetes. Diabetes 1986;35:503–507. Kowluru RA, Tang J, Kern TS: Abnormalities of retinal metabolism in diabetes and experimental alactosemia. Diabetes 2001;50:1938–1949. Wautier MP, Chappey O, Lorda S, Stern DM, Schmidt AM, Wautier SL: Activation of NADPH oxidation by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol 2001;280:E685–E694. Onorato JM, Jenkins AJ, Thorpe SR, Baynes JW: Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. J Biol Chem 2000;275:21177–21184. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414:813–820. Nishikawa T, Edelstein D, Liang XD, Yamagishi SI, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Browlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemic damage. Nature 2000;404:787–790. Palmer HJ, Paulson KE: Reactive oxygen species and antioxidants in signal transduction and gene expression. Nutr Rev 1997;55:353–361. Lander HM: An essential role for free radicals in signal transduction. FASEB J 1997;11:118–124. Sorescu D, Griendling KK: Reactive oxygen species, mitochondria and NAD(P)H oxidases in the development and progression of heart failure. Congest Heart Fail 2002;8:132–140. Houglam K, Brenner DA, Chojkier M: d--Tocopherol inhibits collagen 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest 1991;87:2230–2235. Park SK, Kim J, Seomun Y, Choi J, Kim DH, Han IO, Lee EH, Chung SK, Joo CK: Hydrogen peroxide is a novel inducer of connective tissue growth factor. Biochem Biophys Res Commun 2001;284:966–971. Dominguez-Rosales JA, Mavi G, Levenson SM, Rojkind M: H2O2 is an important mediator of physiological and pathological healing processes. Arch Med Res 2000;31:15–20. Hannken T, Schroeder R, Stahl RAK, Wolf G: Angiotensin II-mediated expression of p27kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. Kidney Int 2002;54:1923–1933. Haugen EN, Croatt AJ, Nath KA: Angiotensin II induced renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int 2000;58:144–152. Hannken T, Schroeder R, Zahner G, Stahl RAK, Wolf G: Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27kip1:role in angiotensin II-mediated hypertrophy of proximal tubular cells. J Am Soc Nephrol 2000;11:1387–1397. Hoerrner, LA, Kowalski AJ, Diamond JR, Wilson PA, Denhardt DT: Homeostatic and pathological action of nitric oxide in the kidney; in Laskin JD, Laskin DL (eds): Cellular and Molecular Biology of Nitric Oxide. Basel, Karger, 1999, pp 137–169. Radi R, Peluffo G, Alvarez MN, Naviliat M, Cayota A: Unravelling peroxynitrite formation in biological systems. Free Radic Biol Med 2001;30:463–488. Beckman JS, Koppenol WH: Nitric oxide, superoxide and peroxynitrite: The good, the bad, and ugly. Am J Physiol 1996;271:C1424–C1437.

Cochrane/Ricardo

118

87 Ahn KY, Mohaupt MG, Madsen KM, Kone BC: In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney. Am J Physiol 1994;267:F748–F757. 88 Morrissey JJ, McCracken R, Kaneto H, Vehaskari M, Montani D, Klahr S: Location of an inducible nitric oxide synthase mRNA in the normal kidney. Kidney Int 1994;45:998–1005. 89 Muhl H, Sandau K, Brune B, Briner VA, Pfeilschifter J: Nitric oxide donors induce apoptosis in glomerular mesangial cells, epithelial cells and endothelial cells. Eur J Pharmacol 1996;317: 137–149. 90 Amore A, Emancipator SN, Cirina P, Conti G, Ricotti E, Bagheri N, Coppo R: Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells. Kidney Int 2000;57:1549–1559. 91 Bank N, Kiroycheva M, Ahmed F, Anthony GM, Fabry ME, Nagel RL, Singhal PC: Peroxynitrite formation and apoptosis in transgenic sickle cell mouse kidneys. Kidney Int 1998;54:1520–1528. 92 Pryor WA, Squadrito GL: The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am J Physiol 1995;268:L699–L722. 93 Ischiropoulos H: Biological tyrosine nitration: A pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;356:1–11. 94 Zhang C, Walker LM, Mayeux PR: Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney. Biochem Pharmacol 2000;59:203–209. 95 Goligorsky MS, Brodsky SV, Noiri E: Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int 2002;61:855–861. 96 Traylor LA, Mayeux PR: Superoxide generation by renal proximal tubule nitric oxide synthase. Nitric Oxide 1997;1:432–438. 97 Heeringa P, van Goor H, Moshage H, Klok PA, Huitema MG, de Jager A, Schep AJ, Kallenberg CG: Expression of iNOS, eNOS and peroxynitrite-modified proteins in experimental anti-myeloperoxidase-associated crescentic glomerulonephritis. Kidney Int 1998;53:382–393. 98 Heeringa P, Steenbergen E, van Goor H: A protective role for endothelial nitric oxide synthase in glomerulonephritis. Kidney Int 2002;61:822–825. 99 Zhou XJ, Laszik Z, Wang XQ, Silva FG, Vaziri ND: Association of renal injury with increased oxygen free radical activity and altered nitric oxide metabolism in chronic experimental hemosidersosis. Lab Invest 2000;80:1905–1914. 100 Thuraisingham RC, Nott CA, Dodd SM, Yaqoob MM: Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy. Kidney Int 2000;57:1968–1972. 101 Onozato ML, Tojo A, Goto A, Fujita T, Wilcox CS: Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 2002;61:186–194. 102 Zykova SN, Jenssen TG, Berdal M, Olsen R, Myklebust R, Seljelid R: Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice. Diabetes 2000;49:1451–1458. 103 Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, Mazzanti L, Mutus B: Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia 1999; 42:539–544. 104 Sharma K, Danoff TM, DePiero A, Ziyadeh FN: Enhanced expression of inducible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: Possible mediation via protein kinase C. Biochem Biophys Res Commun 1995;207:80–88. 105 Ishii N, Patel KP, Lane PH, Taylor T, Bian K, Murad F, Pollock JS, Carmines PK: Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus. J Am Soc Nephrol 2001;12:1630–1639.

A.L. Cochrane, Department of Anatomy and Cell Biology Monash University, Clayton, Vic 3800 (Australia) Tel. 61 3 9905 9096, Fax 61 3 9905 2766, E-Mail [email protected]

Oxidant Stress and Chemokine Regulation

119

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 120–140

Role of Myofibroblasts in Progressive Renal Diseases Toshiki Moriyama a,b, Enyu Imai b a

b

Department of Health Science II, School of Health and Sport Sciences, Osaka University, Toyonaka, Osaka, and Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine (A8), Yamadaoka, Osaka, Japan

Introduction

The progression of chronic renal disease to end-stage renal failure is still one of the biggest challenges in modern nephrology and is an enormous public health burden. The number of patients on maintenance chronic dialysis in Japan has continued to increase and is now more than 200,000, and the number of patients who were newly introduced to renal replacement therapy exceeded 30,000/year in 2000. Current concepts of the therapies of progressive renal diseases are primarily focused on the manipulations to retard the progression of renal failure. These include blood pressure control, reduction of proteinuria, correction of metabolic disorders such as hyperglycemia, hyperuricemia and hyperlipidemia, achieved by the combination of medications and dietary modifications. No therapy has been established to cure or reverse the organic changes of the kidneys. It is therefore important to seek the therapeutic maneuvers to intervene against the underlying mechanisms of the progressive renal diseases and to cure or restore the renal function loss in the process of disease progression. Towards this therapeutic goal, it is a powerful approach to gain insights into the common underlying mechanisms as therapeutic targets of progressive renal diseases of diverse etiology, including glomerulonephritis, diabetic nephropathy, nephrosclerosis, interstitial nephritis, etc. The appearance of myofibroblasts has been demonstrated to be a common pathological finding

in a wide variety of progressive renal diseases. Myofibroblasts are assumed to play important roles in the progression of renal morphological deterioration accompanied by progressive renal function loss in both glomerular and tubular interstitial lesions. In this review, we will highlight the ‘myofibroblasts’ and discuss their nature, origin, role and mechanisms in glomerular and tubulointerstitial lesions in progressive renal diseases.

Myofibroblasts: An Overview

Myofibroblasts are a unique mesenchymal cell population with ultrastructural properties of both muscle and non-muscle cells. These are originally described as ‘modified’ fibroblasts located in granulation tissue [1], and these cells have been subsequently described in many types of tissue during tissue repair and the fibrocontractive disease process [2, 3]. Numerous cell types have been characterized as myofibroblasts, including pericytes, stellate cells, mesangial cells, interstitial cells and granulation tissue fibroblasts. Although 30 years have passed since being first recognized, their molecular, cellular and developmental properties have not been well documented. Myofibroblasts may be defined morphologically and immunologically through identification of expressed cytoskeletal proteins [4]. The simplest definition of myofibroblasts is that they are smooth muscle-like fibroblasts. Morphologically they display prominent cytoplasmic actin microfilaments (stress fiber), and they are connected to each other by adherens and gap junctions. Assembly of stress fiber is regulated by Rho, a newly described member of small guanosine trisphosphatases (GTPases), Rho A, Rho B and Rho C [5]. These GTP-binding proteins are also known to regulate myofibroblast morphology [6]. Stress fiber assembly is inhibited by the pharmacological blockade of Rho-ROCK signaling pathway using Y-27632, a specific ROCK inhibitor in liver stellate cells [7] and in cultured mesangial cells and renal interstitial fibroblasts [Nagatoya and Moriyama, unpubl. observations]. Immunohistological characterization to classify myofibroblasts is performed by combination of the expression of vimentin, desmin and smooth muscle ␣-actin (SM␣A). Myofibroblasts that express only vimentin are termed V-type myofibroblasts, those that express vimentin and desmin are called VD-type, those that express vimentin, SM␣A and desmin are called VAD-type, and those that express vimentin and SM␣A are called VA-type [8]. In kidney, SM␣A-positive cell populations (i.e. VAD and VA-type) are commonly recognized as myofibroblasts, however, the presence and significance of SM␣A-negative fibroblast-like cells has recently been suggested in a rat Goodpasture model [9], and further investigation is required to confirm the observation.

Myofibroblasts in Renal Diseases

121

Role of Myofibroblasts in Health and Tissue Repair

In normal situations, myofibroblasts are involved in organogenesis or morphogenesis – the growth and differentiation of the tissue or organ through the secretion of soluble mediators of inflammation and growth factors and expression of their receptors and through secretion and formation of extracellular matrix (ECM). Myofibroblasts also play fundamental roles in many disease states, either through activation and proliferation or through apoptosis. They play a central role in wound healing, presumably as an extension or accentuation of their role in normal growth and differentiation. After injury, three main events take place to repair the damaged tissues. These are the inflammatory phase, granulation tissue development, and scar formation. The inflammatory phase starts at the time of injury. A disruption of blood vessels occurs and bleeding followed by clot formation takes place. Aggregated platelets release various growth factors and cytokines, which act as chemotactic factors and/or mitogens on various cells including leukocytes, smooth muscle cells, endothelial cells and fibroblasts. Consequently, neutrophills followed by monocytes/ macrophages are recruited to the injured area. These infiltrated cells release various growth factors, cytokines and reactive oxygen species that may act as an activator of resident cells. Then granulation tissue formation starts in parallel with the leukocyte infiltration, and this phase is characterized by fibroblast proliferation and migration, ECM deposition, and angiogenesis. Proliferating fibroblasts in granulation tissues acquire smooth muscle cell-like phenotypes, and are recognized as myofibroblasts [2]. They appear to be involved in the formation and repair of the ECM and proliferation and differentiation. Their contractile property is also thought to play a role in the wound healing process, presumably by reducing the amount of denuded (injured) surface area of wounded tissue and by regulating the blood flow [10]. Then, afterwards, scar formation occurs involving tissue remodeling with ECM degradation and decrease of cellularity by apoptosis. In the situations with non-healing or excessive scarring, pathological conditions develop leading to tissue fibrosis and eventual function loss.

Origin of Myofibroblasts in Kidney

It remains unclear whether myofibroblasts originate from progenitor stem cells or simply transdifferentiate from epithelial cells (epithelial mesenchymal transdifferentiation), or differentiate and proliferate from resident cells (interstitial fibroblasts or mesangial cells), or originate from tissue cells of smooth muscle origin (smooth muscle cells or pericytes). It has recently been suggested that

Moriyama/Imai

122

renal tubular epithelial cells differentiate into fibroblasts/myofibroblasts in progressive renal diseases [11]. This phenomenon is also designated as epithelial mesenchymal transdifferentiation in various cell types [12–14], and its significance in kidney diseases will be discussed in more detail below. However, under such a circumstance, many resident renal fibroblasts proliferate and probably undergo phenotypic changes and acquire myofibroblast characteristics.

Factors Affecting the Differentiation of Myofibroblasts

Numerous factors have been investigated and described that are involved in the differentiation/activation of myofibroblasts. Among many fibrogenic cytokines (IL-1␤, tumor necrosis factor-␣ (TNF-␣), platelet-derived growth factor (PDGF), fibroblast growth factor and transforming growth factor-␤ (TGF-␤)) investigated so far, TGF-␤ appears to be the most important factor inducing the expression of SM␣A and causing an activated phenotype of exaggerated collagen secretion [15–17]. It is very interesting that the 5⬘-flanking region of the SM␣A gene has responsiveness to TGF-␤, providing molecular evidence for the importance of TGF-␤ in the differentiation of myofibroblasts [18]. Vasoactive peptides such as angiotensin II and endothelin-1 are also assumed to be important ‘players’ in myofibroblast activation [19, 20].

Cytokines, Growth Factors and Inflammatory Mediators Produced by Renal Myofibroblasts

One of the prominent characteristics of myofibroblasts related to the pathogenesis of fibrosis is their capability of soluble factor production. There is a list of bioactive polypeptides secreted by myofibroblasts. Very recently, macrophage colony-stimulating factor (M-CSF) was shown to be up-regulated in human glomerulonephritis in tubules and in SM␣A-positive interstitial myofibroblasts where heavy infiltration of macrophages is present. Some mesangial cells also showed an increased expression of M-CSF [21]. A cytokine that may act downstream of TGF-␤ to regulate matrix metabolism is connective tissue growth factor (CTGF). Human CTGF was originally identified as a product of human umbilical vein endothelial cells that was chemotactic and mitogenic for fibroblasts. The biologic activities of CTGF have not been fully understood. However, an important emerging role for CTGF in tissue fibrosis including renal fibrosis is attracting increasing attention [22]. CTGF mRNA is expressed by fibroblasts in the lesions of patients with progressive systemic sclerosis, keloids and localized scleroderma, while there is no

Myofibroblasts in Renal Diseases

123

expression in adjacent normal skin [23]. It was shown that CTGF mRNA was expressed at 50- to 100-fold higher levels in atherosclerotic blood vessels compared with normal arteries. CTGF-positive cells were found predominantly in areas with ECM accumulation and fibrosis. In contrast, in normal arteries, neither CTGF mRNA nor immunoreactive CTGF protein was detected [24]. In human kidneys, Ito et al. [25] first described the enhanced expression of CTGF mRNA in human progressive renal diseases including crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis and diabetic nephropathy in severe mesangial proliferative lesions. They also found that CTGF mRNA-positive cells were increased at the site of chronic tubulointerstitial damage. These cells were found to be SM␣A-positive and negative for macrophage markers, indicating that interstitial myofibroblasts are the cells expressing CTGF and contributing to interstitial fibrosis. CTGF was identified as one of 15 high glucose-induced genes in cultured human mesangial cells, and elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy [26]. Very recently, up-regulation of CTGF mRNA in renal cortex of streptozotocin-induced diabetic rats was demonstrated. Immunohistology indicated that CTGF was expressed particularly in dilated-appearing proximal tubules [27]. It was also demonstrated in rat UUO kidneys that TGF-␤ and CTGF mRNA expression was coordinately up-regulated from the early stage of interstitial fibrosis, followed by marked induction of fibronectin and type I collagen mRNA expression [28]. They also demonstrated in rat kidney fibroblast cell line (NRK-49F) that blockade of endogenous CTGF by transfecting antisense ODN significantly attenuated TGF-␤1-induced fibronectin and type I collagen expression.

Myofibroblasts in the Kidney

The glomerular mesangial cells were first recognized as a subset of distinct cells about 70 years ago [29]. Mesangial cells constitute 30–40% of the glomerular cell population consisting of three major resident renal cells (mesangial cells, endothelial cells and epithelial cells). Mesangial cells have cellular processes that attach to the glomerular basement membrane in the paramesangial areas. Mesangial cells in in vitro cell culture show a contraction in response to various kind of stimuli such as angiotensin II, endothelin-1, arginine vasopressin and PDGF. Although no direct experimental evidence is available about the contractility of mesangial cells in vivo, it is assumed that mesangial cell contraction in vivo contributes to produce a tension and counteract the high capillary pressure and maintain the proper structure of glomerular capillary network. Increased contractility under a pathological condition with mesangial

Moriyama/Imai

124

hypercellularity may contribute to the decrease of glomerular filtration rate by reducing the capillary surface area [30]. Our knowledge on mesangial cell function has been obtained largely from an in vitro cell culture study [31]. Mesangial cells in culture can secrete a variety of soluble factors including growth factors, cytokines and inflammatory mediators. Mesangial cells in vitro also secrete an ECM such as types I, III, IV, V, fibronectin, and laminin. In contrast, in vivo mesangial cells do not appear to express interstitial collagens, i.e. type I and III collagens. Mesangial cells in culture express high levels of contractile related proteins such as SM␣A, myosin heavy chain [32] and caldesmon [33]. Recently it was demonstrated that calponin, an actin-binding protein, was up-regulated in glomerular lesion [34]. Interestingly, they report that the increased calponin plays a suppressive role in glomerulonephritis. This is the first suggestion about the functional role of smooth muscle proteins in myofibroblasts in the pathophysiology of glomerulonephritis. The role of SM␣A, a well-established marker for myofibroblasts, in the disease condition has not been elucidated yet. These proteins are not expressed in vivo in normal glomerular mesangial cells but are up-regulated in disease state. Another prominent feature of mesangial cells in culture is their high proliferative activity. These phenotypic characteristics of cultured mesangial cells are closely related to those of in vivo mesangial cells in a pathological condition. Activation of mesangial cells in vivo and in vitro is discussed below.

Myofibroblasts in Glomerular Lesion

Activation of mesangial cells in vivo has been demonstrated to play a central role in glomerular injury leading to glomerulosclerosis. Up-regulation of SM␣A in glomerular mesangial cells has been widely accepted as a phenotypic marker of mesangial activation in vivo. This was first described in 1991 in experimental animals [35], and in 1992 in human glomerulonephritis [36]. Since then, glomerular up-regulation of other contractile proteins in progressive renal diseases has been documented. Increased expression of non-muscle type (brain/embryonic form) myosin heavy chain (SMemb) was demonstrated in various forms of mesangioproliferative glomerulonephritis [37]. Glomerular SMemb expression showed a correlation with glomerulosclerosis and prognosis in IgA nephropathy [38]. We found that caldesmon, an actin-binding protein, was also up-regulated in glomeruli of IgA nephropathy patients [39]. Quantitative analysis of immunohistochemical staining of caldesmon revealed a significant correlation with mesangial cell proliferation, matrix expansion and SM␣A expression. We concluded in this study that caldesmon is an additional marker of myofibroblasts in glomerular disease. Four- to six-week

Myofibroblasts in Renal Diseases

125

treatment consisted of oral administration of glucocorticoid and continuous infusion of heparin significantly reduced daily urinary protein excretion, the degree of glomerular myofibroblast expansion, mesangial hypercellularity and matrix expansion. A randomized prospective treatment regimen of glucocorticoid treatment therapy in early IgA nephropathy patients also demonstrated a clinical improvement along with histological evidence of myofibroblast regression 1 year after the start of the therapy [40]. The appearance of SM␣Apositive myofibroblasts has been documented focusing its prognostic value in IgA nephropathy [41], crescentic glomerulonephritis [42] and other types of glomerulonephritis [43, 44], diabetic nephropathy [45] and in chronic allograft nephropathy in post-transplant patients [46, 47]. These observations in human renal diseases warrant the significance of myofibroblasts in glomerular diseases, and numerous experimental investigations utilize myofibroblast expansion as a marker of progression, and its suppression as a marker of therapeutic efficacy. For this purpose, SM␣A is the most widely accepted myofibroblast marker, partly because of the sensitivity, reproducibility and simplicity of its detection by immunohistochemical staining using anti-SM␣A monoclonal antibody (clone 1A4). The mechanism of mesangial cell activation has been a matter of interest from the viewpoint of seeking therapeutic target. It is, however, practically difficult to identify the factors involved in myofibroblast formation in glomerulonephritis patients in vivo. In an experimental study using whole animals in combination with a direct in vivo gene transfer and overexpression method [48], we proved the direct involvement of PDGF for mesangial proliferative lesion, and TGF-␤ for matrix expansion in the mesangial area [49]. We also found that glomerular overexpression of renin and angiotensinogen genes resulted in myofibroblast expansion in the mesangial area, indicating that local activation of the renin-angiotensin system can cause mesangial transdifferentiation to myofibroblasts [50]. Experimental gene therapies to intervene against the up-regulated TGF-␤ [51, 52] or PDGF [53] in rat anti-Thy-1 glomerulonephritis model have been conducted in our laboratory with satisfactory therapeutic efficacy on mesangial cell proliferation, ECM expansion and SM␣A-positive myofibroblast expansion. These results also support the central role of PDGF and TGF-␤ in glomerular mesangial cell transdifferentiation in vivo. In vitro mesangial cell culture has been utilized to study the physiological, biochemical and cell biological nature of mesangial cells. It should be noted, however, that the mesangial cells in culture are already ‘activated’ and mimic myofibroblast phenotypes seen in vivo disease states. Elger et al. [54] clearly demonstrated the time course of expression of SM␣A in the process of mesangial cell primary culture that SM␣A-positive mesangial cells start outgrowth 3 days after explantation of isolated glomeruli to the culture dish. Since mesangial

Moriyama/Imai

126

cells in vitro are already showing phenotypes resembling myofibroblasts in vivo, this is not a good system to investigate the molecular mechanisms involved in myofibroblast transdifferentiation.

Molecular Mechanisms of Mesangial Cell Activation

As has been discussed above, up-regulated expression of SM␣A gene is a prominent peculiarity of activated mesangial cells observed in in vivo disease states or under an in vitro cell culture condition. We have been seeking the molecular mechanisms of mesangial cell transdifferentiation to activated ‘myofibroblasts’ by investigating the transcriptional mechanisms of SM␣A in myofibroblasts [55]. Actin is not a unique protein but exists as six isoforms: two cytoplasmic or non-muscle actins (␤ and ␥), and four muscle-type actins (two striated muscle actins, ␣-skeletal and ␣-cardiac actins, and two smooth muscle actins, ␣ and ␥). These six isoforms are encoded by a separate gene and their temporal and spatial pattern of expression is distinct and specifically regulated during development. The primary structure of actins is highly homologous among isoforms (93.5–99.5% homology) and well conserved over species in humans, rodent and chicken. Our working hypotheses in this investigation are as follows: (1) transcription factor(s) regulating the gene expression of SM␣A in myofibroblasts are not merely specific to SM␣A but also involved in the regulation of other phenotypes of myofibroblasts, i.e., production of cytokines, ECM production and proliferative activity, etc., and (2) the molecular mechanisms of SM␣A gene regulation in myofibroblasts are a potential target for pharmacological intervention against myofibroblast formation and the resultant fibrotic lesion. We performed in vivo promoter analyses of human SM␣A gene utilizing transgenic mice harboring a different region of SM␣A promoter regions fused to the reporter gene. We clearly demonstrated the essential role of gene region around the CArG motif element in intron 1 of SM␣A gene for expression of SM␣A in smooth muscle cells and in activated mesangial cells in vivo glomerulonephritis and in vitro cell culture [55]. Interestingly, in vitro transient transfection analysis using the same promoter construct failed to show the positive role of the intronic CArG element in SM␣A transcription, suggesting the importance of chromosomal integration of the SM␣A promoter for active transcription of the gene. The CArG (CC(A/T)6GG) motif is a binding site for serum response factor (SRF) originally identified in the c-fos gene promoter region [56]. SRF regulates many muscle and growth factorinducible genes via CArG motif. Since SRF is a ubiquitous factor and not specific for muscle, it has been postulated to activate muscle genes by recruiting transcriptional co-factor(s) which confer tissue specificity.

Myofibroblasts in Renal Diseases

127

Myofibroblasts in Tubulointerstitial Fibrosis

Progressive interstitial fibrosis accompanied by a loss of renal tubules and interstitial peritubular capillaries is a final common histopathological feature of all progressive renal diseases. Severity of chronic tubulointerstitial fibrosis has been shown to be a single best histologic correlate of the decline in renal function and long-term prognosis [57–60]. Many morphometric studies indicate that tubulointerstitial changes correlate better with renal function loss than glomerular changes [61, 62]. In the process of interstitial fibrosis, renal tubules, fibroblasts and infiltrated macrophages are important cell populations involved in the pathogenesis. The former two types of cells are considered to be origins of interstitial myofibroblasts. Interstitial myofibroblasts are assumed to play important roles in the pathogenesis of tubulointerstitial fibrosis. In normal human kidney, SM␣Apositive cells are essentially confined to smooth muscle cells in arterial/arteriolar walls and pericytes around peritubular capillaries. The appearance of interstitial SM␣A-positive myofibroblasts in human progressive renal diseases has been well demonstrated in a wide variety of diseases including IgA nephropathy [63], membranous nephropathy [64], crescentic glomerulonephritis [63], diabetic nephropathy [65], lupus nephritis [66], chronic allograft nephropathy [46, 67] and obstructive nephropathy. The prognostic value of interstitial myofibroblast expansion in membranous nephropathy was demonstrated [68]. A correlation between the amount of myofibroblast in renal biopsy specimens and renal function at the time of biopsy was shown in IgA nephropathy [41, 69] and in diabetic nephropathy [70]. We observed in IgA nephropathy patients that the interstitial expression of caldesmon and SM␣A increased where interstitial cell infiltration and fibrosis were found. Higher interstitial expression of caldesmon was related to the higher urinary protein excretion. Glucocorticoid and heparin therapy reduced urinary protein excretion along with suppression of interstitial cell infiltration and myofibroblast expansion [71]. Expansion of interstitial SM␣A-positive myofibroblasts has been described in a wide variety of experimental renal injury models. The appearance of SM␣A-positive cells is used as a sensitive marker of early pathological changes of interstitium, and a useful indicator of therapeutic efficacy. Obstructive uropathy is a form of tubulointerstitial injury leading to progressive loss of normal structure and renal function of affected kidneys [72]. Unilateral ureteral obstruction (UUO) is an experimental renal injury model of obstructive uropathy pioneered by Klahr and Pukerson [73]. Myofibroblast expansion in UUO kidney was described in detail [74], and SM␣A-positive myofibroblast has been used so far for quantitative evaluation of pre-fibrotic lesion in the interstitium. We quantitated the SM␣A-positive interstitial area in interstitium

Moriyama/Imai

128

by a computer-aided manipulator and evaluated the therapeutic efficacy of various drugs [75–77], and experimental gene therapy using antisense oligodeoxynucleotides (ODN) for TGF-␤ [78]. UUO is a simple and excellent model of tubulointerstitial injury because it is non-proteinuric, normotensive and non-hyperlipidemic without any apparent immunologic or toxic renal insult. Pathogenesis of interstitial fibrosis in UUO is a complex sequel of initial acute occlusion of ureter including mechanical tension stress to tubules, hypoxia induced by a marked decline in renal plasma flow, up-regulation of monocyte chemoattractant peptide-1, osteopontin, intercellular adhesion molecule-1, macrophage influx into interstitium, up-regulation of cytokines and growth factors (IL-1␤, TNF-␣, IL-6, PDGF, TGF-␤), increased oxidative stress in the interstitium. Enhanced activity of the renin-angiotensin system has also been documented [79]. Almost all kinds of local factors (except for proteinuria) postulated in the pathogenesis of progressive renal diseases are reported to be involved in the progression of tubulointerstitial fibrosis in UUO. Therefore, UUO is a good model to evaluate the usefulness of new therapeutic approach targeting a single molecule/signaling pathway. Successful therapy by angiotensin-converting enzyme inhibitors or angiotensin type-1 receptor antagonist in UUO kidneys has been reported [80]. The renin-angiotensin system was shown to be enhanced in the obstructed kidneys. Interestingly, interstitial influx of monocytes/macrophages into obstructed kidneys was significantly attenuated by angiotensin-converting enzyme inhibitor administration. We have recently demonstrated that specific inhibition of ROCK, a major downstream effector molecule of Rho-GTPase, by a new compound, Y-27632, suppressed SM␣Apositive myofibroblast expansion and interstitial fibrosis in the kidney of mouse with UUO [77]. In UUO kidneys, up-regulation of Rho B and Rho C mRNAs was found. Smooth muscle-specific gene regulation through a CArG (CC(A/T)6GG) motif element such as SM␣A and SM22 transcription is regulated by the Rho A signaling pathway, and promoter activity of SM␣A and SM22 in smooth muscle cell is inhibited by Y-27632 [81]. We also found that Y-27632 inhibited SM␣A expression in cultured renal fibroblasts. Our observation on the therapeutic efficacy of Y-27632 in UUO is in good agreement with the in vitro effect of Y-27632 on SM␣A gene regulation and myofibroblast formation. We also found that interstitial macrophage infiltration was significantly suppressed by Y-27632 treatment. There was no inhibitory effect of Y-27632 on renal MCP-1, osteopontin and ICAM-1 mRNA levels that are supposed to facilitate macrophage migration. Instead, we found that Y-27632 directly inhibited the migration of the macrophage cell line under MCP-1, angiotensin II, or PDGF stimulation in a dose-dependent manner. This inhibitory effect on macrophage migration may partly contribute to the anti-myofibroblast effect of Y-27632 in addition to its direct suppression of SM␣A gene regulation.

Myofibroblasts in Renal Diseases

129

Although TGF-␤ is the most investigated and a well-established culprit for interstitial fibrosis, there has been no direct evidence that specific inhibition of TGF-␤ expression or activity in tubulointerstitium is of therapeutic value towards interstitial fibrosis. We have established an efficient gene transfer method via ureter targeting tubulointerstitium [82], and applied it to gene therapy using antisense ODN for TGF-␤. A drastic reduction of interstitial fibrosis, type I collagen mRNA expression and SM␣A-positive myofibroblasts was observed in TGF-␤ antisense ODN-treated obstructed kidneys [78]. This observation strongly suggests the pivotal role of TGF-␤ for in vivo formation of interstitial myofibroblasts.

Proteinuria

A body of evidence has accumulated to show that there is a close correlation between the degree of proteinuria and the rate of progression of chronic renal failure [83, 84]. In the Modification of Diet in Renal Diseases Study [85], those patients with proteinuria ⬎3 g protein/24 h had a decrease in glomerular filtration rate of 10 ml/min/year compared with a decrease in glomerular filtration rate of 3 ml/min/year in those patients with proteinuria ⬍1 g/24 h. In the recently reported analysis of AIPRD (Angiotensin-Converting Enzyme Inhibition and Progression of Renal Disease) study, a higher level of urine protein excretion was associated with a greater risk of progression (relative risk 5.56 for each 1.0 g/day higher protein excretion) [86]. For many years, the degree of proteinuria has been assumed to be merely an indicator of severity of glomerular damage. However, in the past two decades, proteinuria has been recognized as an independent mediator of progression of interstitial injury rather than simply a marker of glomerular dysfunction (reviewed by Remuzzi [87]). This concept arose from clinical observations, and is now being further pursued by experimental research both in vivo and in vitro. The mechanisms of proteinuria-induced tubulointerstitial injury include the following steps: (1) activation of tubular epithelial cells; (2) release of inflammatory mediators from the activated tubular cells; (3) recruitment of neutrophills, T cells, mast cells, and macrophages into interstitial space; (4) activation of myofibroblasts, and (5) progression of interstitial fibrosis. Interstitial myofibroblasts are a primary source of the ECM protein that accumulates in the interstitium to cause fibrosis. Therefore, myofibroblast activation undoubtedly plays a central role in tubulointerstitial damage induced by heavy proteinuria. Activation of proximal tubular cells is the primary event under high urinary protein excretion. In proximal tubular cells in culture, increasing the concentration of albumin, immunoglobulin G, or transferrin in culture medium causes

Moriyama/Imai

130

activation of nuclear factor-␬B (NF-␬B) [88] and consequent up-regulation of NF-␬B-dependent gene transcription such as MCP-1 [89] and RANTES [88]. Interestingly, potent vasoactive peptide, endothelin-1, was shown to be induced by a high concentration of albumin in culture [90]. A similar up-regulation of inflammatory mediators was shown in vivo proteinuric glomerulonephritis models [91–93]. Pharmacological intervention of NF-␬B activity by PDTC ameliorated the interstitial fibrosis in adriamycin-induced proteinuric rats [94]. Clinically, non-selective proteinuria is considered to be more toxic than albuminuria and iron-containing holotransferrin but not albumin was demonstrated to be toxic in cultured proximal tubule cells [95]. The infiltrated macrophages have been demonstrated to play key roles in the pathogenesis and myofibroblast activation. Macrophages have an ability to synthesize and produce fibrosisrelated growth factors (TGF-␤), cytokines (TNF-␣, IL-1␤), vasoactive substances, and reactive oxygen species that contribute to activation of myofibroblasts, or transdifferentiation of epithelial cells to myofibroblasts. Another important aspect of proteinuria in the tubulointerstitial injury is complement activation [96, 97]. It was suggested in human renal diseases by Morita et al. [98] that the degree of intratubular complement activation correlates with the level of proteinuria, type of glomerular disease, impairment of renal function, and metabolic acidosis. Interestingly, urinary complement activation was reduced by alkalization of the urine by sodium-bicarbonate administration. There are many experimental investigations regarding the involvement of complement activation in the pathogenesis of tubulointerstitial injury [97, 99–102].

Oxidative Stress

Oxidative stress has been implicated as a pathogenesis of tubulointerstitial injury for many years [103]. Nath and Salahudeen [104] demonstrated that long-term dietary deficiency of antioxidants (vitamin E and selenium) causes interstitial injury, and this manipulation was also shown to exacerbate ischemic injury in rat kidney [105]. In this model of long-term dietary deficiency of antioxidants, a chronic pro-oxidant state, with features that mimic those of clinical uremia, increased the DNA synthesis of renal tubular epithelium, and induction of mRNA expression for collagens I, III and IV in conjunction with the mRNA for the fibrogenic cytokine, TGF-␤1, was clearly demonstrated. They also found that hydrogen peroxide induces collagen III, collagen IV and TGF-␤1 mRNA in vitro. Kidneys from antioxidant-deficient rats exhibit increased mitochondrial generation of hydrogen peroxide in conjunction with suppression of glutathione peroxidase mRNA and activity. Lipid peroxidation was increased twofold in the cortex and medulla of the antioxidant-deficient

Myofibroblasts in Renal Diseases

131

kidneys [106]. In a chronic puromycin nephrosis model, chronic administration of taurine, an amino acid with potent antioxidant activity, significantly ameliorated the interstitial injury along with reduction in urinary protein excretion and improvement of renal function [107]. A lower amount of lipid peroxidation was observed in taurine-treated rat kidneys. Taurine administration in streptozotocin-induced diabetic rats was effective in decreasing urinary protein excretion, progression of renal failure and ameliorated the sclerotic change in glomeruli, and fibrosis in tubulointerstitium [108]. These studies suggest the importance of oxidative stress as a pathogenesis of tubulointerstitial injury. In the setting of tubulointerstitial injury, oxidative stress may originate from many pathways including infiltrated macrophages, activated proximal tubules, and proteins in glomerular filtrate. Macrophages in the interstitium undoubtedly play a central role in the pathogenesis of renal damages. In clinical and experimental studies, the number of infiltrating macrophages correlates well with renal function decline [109–112]. Macrophages release superoxide anion [113] and also produce proinflammatory mediators such as IL-1␤ and TNF-␣. TNF-␣ is well known to induce superoxide production in various cell types. Therefore, interstitial influx of macrophages is an important source of oxidative stress in the setting of tubulointerstitial injury. Glomerular injury of any type causes impaired permselectivity of glomerular basement membrane leading to leakage of proteins including transferrin into glomerular filtrate. Transferrin provides a source of iron that has a potential to catalyze the generation of oxidants/free radicals [114]. The binding of iron to transferrin is pH-sensitive, as pH decreases iron that is liberated from transferrin. Ionized iron is capable of catalyzing radical production in tubular epithelial cells. Toxicity of holotransferrin on proximal tubule cells in vitro culture was augmented in acidic medium (pH 6.0) [95]. Iron-transferrin complex has been shown to accumulate in proximal tubular lysozomes in diseased kidneys [115–117]. It is also of interest to investigate the oxidative stress in the interstitial injury model without proteinuria to explore the possible involvement of other factors than proteinuria. In mouse UUO kidneys, we investigated the increased oxidative stress by immunohistological determination of carboxylmethyl lysine (CML), one of the major components of advanced glycation end-products formed by the process of both glycation and oxidation of proteins, and found that CML is accumulated in the interstitium of UUO kidneys 10 days after ureteral obstruction [118]. Heme oxygenase-1 mRNA was up-regulated as early as 12 h after ureteral obstruction. This is the first demonstration of increased oxidative stress in the interstitial fibrotic lesion of UUO kidneys. The mechanisms involved in the increased oxidative stress in UUO kidneys are not elucidated yet, however these are of importance to understand the pathogenesis of tubulointerstitial damages since the fibrotic lesion is not related to urinary

Moriyama/Imai

132

protein excretion in the setting of UUO. Next we investigated the possibility of the increased oxidative stress as a therapeutic target towards interstitial fibrosis in UUO kidneys [76]. We chose fluvastatin, one of the HMG-CoA reductase inhibitors (statins), as a candidate antioxidant. We also used pravastatin, another statin without antioxidant activity, as a control compound. Interstitial CML accumulation was significantly suppressed in fluvastatin-treated mouse kidneys but not in pravastatin-treated mice. Interstitial fibrosis and the appearance of SM␣A-positive myofibroblasts was also significantly suppressed in fluvastatintreated group but not in pravastatin-treated group. These observations strongly suggest the causal relationship between increased oxidative stress and tubulointerstitial fibrosis in UUO kidneys. The direct involvement of oxidative stress in myofibroblast formation in the interstitium was also suggested, however this hypothesis requires further investigation to be established.

Epithelial-Mesenchymal Transdifferentiation

Renal interstitial fibrosis, as the common pathway in progressive renal disease leading to functional deterioration and eventual loss of renal function, is classically characterized by an expansion of interstitial fibroblast population that mediates excessive deposition of interstitial matrix components resulting in interstitial fibrosis and atrophy of surrounding tubules. The fibroblasts may be resident renal fibroblasts, fibroblasts that migrate into the interstitium from an external source, or a specialized population of fibroblasts known as myofibroblasts. Recent observations stress a pivotal role of tubular epithelial cells as mediators of renal scarring [119]. Tubular epithelial cells function as a source of fibrogenic growth factors and chemokines in the initiation of fibrogenesis, contribute to tubular atrophy by undergoing apoptosis, and potentially contribute to increased numbers of interstitial fibroblasts by epithelial-mesenchymal transdifferentiation (EMT). EMT is defined as the acquisition of phenotypic as well as functional properties of mesenchymal fibroblasts by epithelial cells [120]. It occurs in development, carcinogenesis, and chronic diseases in different organs. Increasing evidence mainly in experimental renal injury and in some clinical settings suggests that EMT plays important roles in renal fibrogenesis. The current concept of EMT postulates a mechanism in which tubular epithelial cells become activated by exogenous stimuli, followed by a loss of contact with neighboring cells and basement membrane. After initiation of EMT, cells move through their basement membrane into the interstitial space where they become detectable as fibroblasts/myofibroblasts. The concept of renal transdifferentiation of tubular epithelial cells can be defined by the de novo expression of an otherwise fibroblast-specific protein

Myofibroblasts in Renal Diseases

133

(ex-FSP-1) or myofibroblast marker (ex-SM␣A) in tubular epithelial cells during fibrogenesis. It was described in human end-stage kidney with marked fibrosis that the widened interstitium contained single cells or loosely organized small cell clusters positive with both cytokeratin and the epithelial membrane antigen. These cells, though of fibroblast appearance and localization, still expressed several epithelial marker proteins, indicating that they may represent transitional cells between an epithelial and mesenchymal state [121]. The report described above was an early human study suggesting the presence of interstitial fibroblasts with some feature of tubular epithelial cells. Very recently, tubular phenotypic changes were demonstrated in progressive tubulointerstitial fibrosis in various forms of human glomerulonephritis [122]. De novo expression of SM␣A, a myofibroblast marker and loss of epithelial marker cytokeratin, and collagen production were used as the criteria of tubular phenotypic change and they found tubular phenotypic change in 36 of 90 cases of IgA nephropathy patients, in 9 of 18 RPGN. The incidence of SM␣Apositive tubules was 0.4% in IgA nephropathy and 3.8% in RPGN in cortical tubules. Interestingly, the same group described glomerular epithelial-myofibroblast transdifferentiation in the process of glomerular crescent formation [123]. This was the first demonstration of phenotypic change of glomerular epithelial cells. These observations were corroborated in an experimental study by Ng et al. [124] in the rat model of 5/6 nephrectomy. Using immunohistochemistry and in situ hybridizations, they noted de novo expression of the myofibroblast marker SM␣A in some tubular epithelial cells. By electron microscopy, these cells lost apical-basal polarity and tight junctions and acquired a fusiform cell shape characteristic of fibroblasts. Moreover, these cells separated from neighboring cells, lost contact with the tubular basement membrane and seemed to migrate to the interstitium. Interestingly, the process of transdifferentiation was restricted to areas where the tubular basement membrane was disrupted, underlining the importance of an intact anchoring of tubular epithelial cells. The number of smooth-muscle actin-positive tubular epithelial cells correlated with the number of cells expressing the activation marker within the interstitium and also with the degree of interstitial fibrosis. More recently, Yang and Liu [125] reported the key events during the process of EMT induced by TGF-␤1. Incubation of human tubular epithelial cells with TGF-␤1 induced de novo expression of SM␣A, loss of epithelial marker E-cadherin, acquisition of myofibroblastic morphology, and production of interstitial matrix. The transformed cells secreted a large amount of matrix metalloproteinase-2 that specifically degraded tubular basement membrane. They also exhibited an enhanced motility and invasive capacity. These alterations in epithelial phenotypes in vitro were also observed in a mouse model of renal fibrosis induced by UUO.

Moriyama/Imai

134

They concluded that tubular epithelial to myofibroblast transition requires four steps including: (1) loss of epithelial cell adhesion; (2) de novo ␣-smooth muscle actin expression and actin reorganization; (3) disruption of tubular basement membrane, and (4) enhanced cell migration and invasion.

Conclusion

Elucidation of nature and mechanisms of fibrogenesis in progressive renal diseases is one of the most important tasks of modern nephrology. A body of evidence has accumulated showing the important roles of myofibroblasts in progressive renal diseases. The concept of myofibroblasts and their importance in nephrology was overviewed in this chapter. In addition, recent advances in the novel pathway of myofibroblast formation from tubular epithelial cells, i.e. ‘epithelial-mesenchymal transdifferentiation’, were reviewed in detail. Further progress in myofibroblast research is expected to contribute to a better understanding of fibrogenesis of the kidney and to the development of a new therapeutic approach towards progressive renal diseases.

References 1 2 3 4 5 6 7

8 9 10 11 12

Gabbiani G, Ryan GB, Majne G: Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971;27:549–550. Schurch W, Seemayer TA, Gabbiani G: The myofibroblast: A quarter century after its discovery. Am J Surg Pathol 1998;22:141–147. Powell DW, Miffilin RC, Valentich JD, Crowe SE, Saada JI, West AB: Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 1999;277:C1–C19. Sappino AP, Schurch W, Gabbiani G: Differentiation repertoire of fibroblastic cells: Expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest 1990;63:144–161. Hall A: Rho GTPases and the actin cytoskeleton. Science 1998;279:509–514. Yee HFJ: Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton. Hepatology 1998;28:843–850. Kawada N, Seki S, Kuroki T, Kaneda K: ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1. Biochem Biophys Res Commun 1999; 266:296–300. Schmitt-Graff A, Desmouliere A, Gabbiani G: Heterogeneity of myofibroblast phenotypic features: An example of fibroblastic cell plasticity. Virchows Arch 1994;425:3–24. Okada H, Inoue T, Kanno Y, et al: Renal fibroblast-like cells in Goodpasture syndrome rats. Kidney Int 2001;60:597–606. Nedelec B, Ghahary A, Scott PG, Tredget EE: Control of wound contraction. Basic and clinical features. Hand Clin 2000;16:289–302. Okada H, Danoff TM, Kalluri R, Neilson EG: Early role of Fsp1 in epithelial-mesenchymal transformation. Am J Physiol 1997;273:F563–F574. Miettinen PJ, Ebner R, Lopez AR, Derynck R: TGF-␤ induced transdifferentiation of mammary epithelial cells to mesenchymal cells: Involvement of type I receptors. J Cell Biol 1994;127: 2021–2036.

Myofibroblasts in Renal Diseases

135

13

14 15

16 17 18

19 20 21

22 23

24 25 26

27 28

29 30 31 32

33 34 35

Caulin C, Scholl FG, Frontelo P, Gamallo C, Quintanilla M: Chronic exposure of cultured transformed mouse epidermal cells to transforming growth factor-␤1 induces an epithelial-mesenchymal transdifferentiation and a spindle tumoral phenotype. Cell Growth Differ 1995;6:1027–1035. Stahl PJ, Felsen D: Transforming growth factor-␤, basement membrane and epithelial-mesenchymal transdifferentiation: Implications for fibrosis in kidney disease. Am J Pathol 2001;159:1187–1192. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G: Transforming growth factor-␤1 induces ␣-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993;122:103–111. Border WA, Noble NA: Transforming growth factor-␤ in tissue fibrosis. N Engl J Med 1994;331: 1286–1292. Gressner AM: Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: A key event in hepatic fibrogenesis. Kidney Int 1996;54(suppl):39–45. Hautmann MB, Madsen CS, Owens GK: A transforming growth factor (TGF) control element drives TGF-induced stimulation of smooth muscle-actin gene expression in concert with two CArG elements. J Biol Chem 1997;272:10948–10956. Rockey D, Chung JJ: Endothelin antagonism in experimental hepatic fibrosis: Implications for endothelin in the pathogenesis of wound healing. J Clin Invest 1996;98:1381–1388. Weber KT: Metabolic responses of extracellular matrix in tissue repair. Ann Med 1997;29:333–338. Isbel NM, Nikolic-Paterson DJ, Hill PA, Dowling J, Atkins RC: Local macrophage proliferation correlates with increased renal M-CSF expression in human glomerulonephritis. Nephrol Dial Transplant 2001;16:1638–1647. Gupta S, Clarkson MR, Duggan J, Brady HR: Connective tissue growth factor: Potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 2000;58:1389–1399. Igarashi A, Nashiro K, Kikuchi K, et al: Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid and other fibrotic skin disorders. J Invest Dermatol 1996;106:729–733. Oemar BS, Werner A, Garnier JM, et al: Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation 1997;95:831–839. Ito Y, Aten J, Bende RJ, et al: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998;53:853–861. Murphy M, Godson C, Cannon S, et al: Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 1999;274:5830–5834. Wang S, Denichilo M, Brubaker C, Hirschberg R: Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy. Kidney Int 2001;60:96–105. Yokoi H, Sugawara A, Mukoyama M, et al: Role of connective tissue growth factor in profibrotic action of transforming growth factor-␤: A potential target for preventing renal fibrosis. Am J Kidney Dis 2001;38(suppl 1):134–138. Zimmermann KW: Über den Bau des Glomerulus der Säugerniere: weitere Mitteilungen. Z Mikrosk Anat Forsch 1933;32:176–278. Fujiwara Y, Orita Y, Sasaki E, Abe H, Takamitsu Y: Influence of mesangial proliferation on posturally induced change in glomerular filtration rate. Nephron 1983;35:35–38. Mene P, Simonson MS, Dunn MJ: Physiology of the mesangial cell. Physiol Rev 1989;69: 1347–1427. Hiraoka-Yoshimoto M, Higashida K, Takeda M, Kawamoto S, Ichikawa I, Hoover RL: Characterization of myosin heavy and light chains in cultured mesangial cells. Kidney Int 1991; 40:1013–1019. Ishino T, Kobayashi R, Wakui H, Fukushima Y, Nakamoto Y, Miura AB: Biochemical characterization of contractile proteins of rat cultured mesangial cells. Kidney Int 1991;39:1118–1124. Sugenoya Y, Yoshimura A, Yamamura H, et al: Smooth muscle calponin in mesangial cells: Regulation of expression and a role in suppressing glomerulonephritis. J Am Soc Nephrol 2002;13:322–331. Johnson RJ, Iida H, Alpers CE, et al: Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. ␣-Smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest 1991;87:847–858.

Moriyama/Imai

136

36 37

38

39

40

41 42 43

44

45 46 47 48 49

50

51

52

53 54 55 56 57

Alpers CE, Hudkins KL, Gown AM, Johnson RJ: Enhanced expression of ‘muscle-specific’ actin in glomerulonephritis. Kidney Int 1992;41:1134–1142. Kishino M, Kimura A, Yamaguchi K, et al: Increased expression of a brain/embryo-type myosin heavy chain isoform (MIIB2) in mesangial proliferative glomerulonephritis. Kidney Int 1996; 49:1350–1359. Mise N, Kimura K, Nagai R, et al: Mesangial expression of a non-muscle myosin heavy chain, SMemb, is associated with glomerular sclerosis and renal prognosis in IgA nephropathy. Nephron 1998;78:284–289. Ando Y, Moriyama T, Miyazaki M, et al: Enhanced glomerular expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy. Nephrol Dial Transplant 1998;13:1168–1175. Shoji T, Nakanishi I, Suzuki A, et al: Early treatment with corticosteroids ameliorates proteinuria, proliferative lesions and mesangial phenotypic modulation in adult diffuse proliferative IgA nephropathy. Am J Kidney Dis 2000;35:194–201. Goumenos DS, Brown CB, Shortland J, El Nahas AM: Myofibroblasts, predictors of progression of mesangial IgA nephropathy? Nephrol Dial Transplant 1994;9:1418–1425. Goumenos D, Tsomi K, Iatrou C, et al: Myofibroblasts and the progression of crescentic glomerulonephritis. Nephrol Dial Transplant 1998;13:1652–1661. MacPherson BR, Leslie KO, Lizaso KV, Schwarz JE: Contractile cells of the kidney in primary glomerular disorders: An immunohistochemical study using an anti-␣-smooth muscle actin monoclonal antibody. Hum Pathol 1993;24:710–716. Groma V, Marcussen N, Olsen S: A quantitative immunohistochemical study of the expression of mesangial ␣-smooth muscle actin and the proliferation marker Ki-67 in glomerulonephritis in man. Virchows Arch 1997;431:345–350. Makino H, Kashihara N, Sugiyama H, et al: Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes. Diabetes 1996;45:488–495. Pedagogos E, Hewitson TD, Walker RG, Nicholis KM, Becker GJ: Myofibroblast involvement in chronic transplant rejection. Transplantation 1997;64:1192–1197. Oka K, Moriyama T, Kawada N, et al: Significance of interstitial expression of ␣-smooth muscle actin in chronic allograft nephropaty. J Am Soc Nephrol 2000;11:700A. Imai E, Isaka Y: Strategies of gene transfer to the kidney. Kidney Int 1998;53:264–272. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: Glomerulosclerosis induced by in vivo transfection of transforming growth factor-␤ or platelet-derived growth factor gene into the rat kidney. J Clin Invest 1993;92:2597–2601. Arai M, Wada A, Isaka Y, et al: In vivo transfection of genes for renin and angiotensinogen into the glomerular cells induced phenotypic change of the mesangial cells and glomerular sclerosis. Biochem Biophys Res Commun 1995;206:525–532. Akagi Y, Isaka Y, Arai M, et al: Inhibition of TGF-␤1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1996;50:148–155. Isaka Y, Akagi Y, Ando Y, et al: Gene therapy by transforming growth factor-␤ receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1999;55:465–475. Nakamura H, Isaka Y, Tsujie M, et al: Electroporation-mediated PDGF receptor-IgG chimera gene transfer ameliorates experimental glomerulonephritis. Kidney Int 2001;59:2134–2145. Elger M, Drenckhahn D, Nobiling R, Mundel P, Kriz W: Cultured rat mesangial cells contain smooth muscle ␣-actin not found in vivo. Am J Pathol 1993;142:497–509. Kawada N, Moriyama T, Ando A, et al: Role of intron-1 in smooth muscle ␣-actin transcriptional regulation in activated mesangial cells in vivo. Kidney Int 1999;55:2338–2348. Shore P, Sharrocks AD: The MADS-box family of transcription factors. Eur J Biochem 1995; 229:1–13. Risdon RA, Sloper JC, De Wardener HE: Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 1968;ii:363–366.

Myofibroblasts in Renal Diseases

137

58 59 60 61

62 63

64 65 66 67

68 69 70

71

72 73

74 75 76

77 78 79 80 81

Schainuck LI, Striker GE, Cutler RE, Benditt EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1970;1:631–641. Nath KA: Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 1992;20:1–17. D’Amico G, Ferrario F, Rastaldi MP: Tubulointerstitial damage in glomerular diseases: Its role in the progression of renal damage. Am J Kidney Dis 1995;26:124–132. Bohle A, Muller GA, Wehrmann M, Mackensen-Haen S, Xiao JC: Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies and chronic interstitial nephritides. Kidney Int 1996;54(suppl):2–9. Ziyadeh FN: Significance of tubulointerstitial changes in diabetic renal disease. Kidney Int 1996;54(suppl):10–13. Alpers CE, Hudkins KL, Floege J, Johnson RJ: Human renal cortical interstitial cells with some features of smooth muscle cells participate in tubulointerstitial and crescentic glomerular injury. J Am Soc Nephrol 1994;5:201–209. Roberts IS, Burrows C, Shanks JH, Venning M, McWilliam LJ: Interstitial myofibroblasts: Predictors of progression in membranous nephropathy. J Clin Pathol 1997;50:123–127. Pedagogos E, Hewitson T, Fraser I, Nicholls K, Becker G: Myofibroblasts and arteriolar sclerosis in human diabetic nephropathy. Am J Kidney Dis 1997;29:912–918. Hurtado A, Asato C, Escudero E, et al: Clinicopathologic correlations in lupus nephritis in Lima, Peru. Nephron 1999;83:323–330. Pilmore HL, Painter DM, Bishop GA, McCaughan GW, Eris JM: Early up-regulation of macrophages and myofibroblasts: A new marker for development of chronic renal allograft rejection. Transplantation 2000;69:2658–2662. Badid C, Desmouliere A, McGregor B, et al: Interstitial ␣-smooth muscle actin: A prognostic marker in membranous nephropathy. Clin Nephrol 1999;52:210–217. Hewitson TD, Becker GJ: Interstitial myofibroblasts in IgA glomerulonephritis. Am J Nephrol 1995;15:111–117. Essawy M, Soylemezoglu O, Muchaneta-Kubara EC, Shortland J, Brown CB, el Nahas AM: Myofibroblasts and the progression of diabetic nephropathy. Nephrol Dial Transplant 1997;12:43–50. Ando Y, Moriyama T, Oka K, et al: Enhanced interstitial expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy. Nephrol Dial Transplant 1999;14:1408–1417. Klahr S: Obstructive nephropathy. Kidney Int 1998;54:286–300. Klahr S, Pukerson ML: The pathophysiology of obstructive nephropathy: The role of vasoactive compounds in the hemodynamic and structural abnormalities of the obstructed kidney. Am J Kidney Dis 1994;23:219–223. Diamond JR, van Goor H, Ding G, Engelmyer E: Myofibroblasts in experimental hydronephrosis. Am J Pathol 1995;146:121–129. Moriyama T, Kawada N, Ando A, et al: Up-regulation of HSP47 in the mouse kidneys with unilateral ureteral obstruction. Kidney Int 1998;54:110–119. Moriyama T, Kawada N, Nagatoy K, et al: Fluvastatin suppressed the oxidative stress and fibrosis in the interstitium of mouse kidneys with unilateral ureteral obstruction. Kidney Int 2001;59:2095–2103. Nagatoya K, Moriyama T, Kawada N, et al: Y-27632, a specific ROCK inhibitor, suppressed interstitial fibrosis of mouse kidneys with unilateral ureteral obstruction. Kidney Int 2002;61:1684–1695. Isaka Y, Tsujie M, Ando Y, et al: Transforming growth factor-␤1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int 2000;58:1885–1892. Klahr S, Ishidoya S, Morrissey J: Role of angiotensin II in the tubulointerstitial fibrosis of obstructive nephropathy. Am J Kidney Dis 1995;26:141–146. Klahr S, Morrissey JJ: Comparative study of ACE inhibitors and angiotensin II receptor antagonists in interstitial scarring. Kidney Int 1997;63(suppl):111–114. Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK: Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization. J Biol Chem 2001; 276:341–347.

Moriyama/Imai

138

82 Tsujie M, Isaka Y, Ando Y, et al: Gene transfer targeting interstitial fibroblasts by the artificial viral envelope-type hemagglutinating virus of Japan liposome method. Kidney Int 2000;57: 1973–1980. 83 Cattran DC: Idiopathic membranous glomerulonephritis. Kidney Int 2001;59:1983–1994. 84 D’Amico G: Natural history of idiopathic IgA nephropathy: Role of clinical and histological prognostic factors. Am J Kidney Dis 2000;36:227–237. 85 Klahr S, Levey AS, Beck GJ, et al: The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group. N Engl J Med 1994;330:877–884. 86 Jafar TH, Stark PC, Schmid CH, et al: Proteinuria as a modifiable risk factor for the progression of non-diabetic renal disease. Kidney Int 2001;60:1131–1140. 87 Remuzzi G: Nephropathic nature of proteinuria. Curr Opin Nephrol Hypertens 1999;8:655–663. 88 Zoja C, Donadelli R, Colleoni S, et al: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-␬B activation. Kidney Int 1998;53:1608–1615. 89 Wang Y, Chen J, Chen L, Tay YC, Rangan GK, Harris DC: Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 1997;8:1537–1545. 90 Zoja C, Morigi M, Figliuzzi M, et al: Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 1995;26:934–941. 91 Donadelli R, Abbate M, Zanchi C, et al: Protein traffic activates NF-␬B gene signaling and promotes MCP-1-dependent interstitial inflammation. Am J Kidney Dis 2000;36:1226–1241. 92 Mudge SJ, Paizis K, Auwardt RB, Thomas RJ, Power DA: Activation of nuclear factor-␬B by podocytes in the autologous phase of passive Heymann nephritis. Kidney Int 2001;59:923–931. 93 Gomez-Garre D, Largo R, Tejera N, Fortes J, Manzarbeitia F, Egido J: Activation of NF-␬B in tubular epithelial cells of rats with intense proteinuria: Role of angiotensin II and endothelin-1. Hypertension 2001;37:1171–1178. 94 Rangan GK, Wang Y, Tay YC, Harris DC: Inhibition of nuclear factor-␬B activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int 1999;56:118–134. 95 Chen L, Boadle RA, Harris DC: Toxicity of holotransferrin but not albumin in proximal tubule cells in primary culture. J Am Soc Nephrol 1998;9:77–84. 96 Matsuo S, Morita Y, Mizuno M, Nishikawa K, Yuzawa Y: Proteinuria and damage to tubular cells – Is complement a culprit? Nephrol Dial Transplant 1998;13:2723–2726. 97 Nangaku M, Pippin J, Couser WG: Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome. J Am Soc Nephrol 1999;10:2323–2331. 98 Morita Y, Ikeguchi H, Nakamura J, Hotta N, Yuzawa Y, Matsuo S: Complement activation products in the urine from proteinuric patients. J Am Soc Nephrol 2000;11:700–707. 99 Morita Y, Nomura A, Yuzawa Y, et al: The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis. J Am Soc Nephrol 1997;8: 1363–1372. 100 Nomura A, Nishikawa K, Yuzawa Y, et al: Tubulointerstitial injury induced in rats by a monoclonal antibody that inhibits function of a membrane inhibitor of complement. J Clin Invest 1995;96:2348–2356. 101 Nomura A, Morita Y, Maruyama S, et al: Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis. Am J Pathol 1997;151:539–547. 102 Hori Y, Yamada K, Hanafusa N, et al: Crry, a complement regulatory protein, modulates renal interstitial disease induced by proteinuria. Kidney Int 1999;56:2096–2106. 103 Nath KA, Fischereder M, Hostetter TH: The role of oxidants in progressive renal injury. Kidney Int 1994;45(suppl):111–115. 104 Nath KA, Salahudeen AK: Induction of renal growth and injury in the intact rat kidney by dietary deficiency of antioxidants. J Clin Invest 1990;86:1179–1192. 105 Nath KA, Paller MS: Dietary deficiency of antioxidants exacerbates ischemic injury in the rat kidney. Kidney Int 1990;38:1109–1117. 106 Nath K, Grande J, Croatt A, Haugen J, Kim Y, Rosenberg ME: Redox regulation of renal DNA synthesis, transforming growth factor-␤1 and collagen gene expression. Kidney Int 1998;53:367–381. 107 Trachtman H, Del Pizzo R, Futterweit S, et al: Taurine attenuates renal disease in chronic puromycin aminonucleoside nephropathy. Am J Physiol 1992;262:F117–F123.

Myofibroblasts in Renal Diseases

139

108 Trachtman H, Futterweit S, Maesaka J, et al: Taurine ameliorates chronic streptozocin-induced diabetic nephropathy in rats. Am J Physiol 1995;269:F429–F438. 109 Wu Q, Jinde K, Nishina M, et al: Analysis of prognostic predictors in idiopathic membranous nephropathy. Am J Kidney Dis 2001;37:380–387. 110 Alexopoulos E, Seron D, Hartley RB, Cameron JS: Lupus nephritis: Correlation of interstitial cells with glomerular function. Kidney Int 1990;37:100–109. 111 Saito T, Atkins RC: Contribution of mononuclear leucocytes to the progression of experimental focal glomerular sclerosis. Kidney Int 1990;37:1076–1083. 112 Saito T, Yusa A, Soma J, Ootaka T, Sato H, Ito S: Significance of leukocyte infiltration in membranous nephropathy with segmental glomerulosclerosis. Nephron 1998;80:414–420. 113 MacMicking J, Xie QW, Nathan C: Nitric oxide and macrophage function. Annu Rev Immunol 1997;15:323–350. 114 Alfrey AC: Role of iron and oxygen radicals in the progression of chronic renal failure. Am J Kidney Dis 1994;23:183–187. 115 Alfrey AC, Froment DH, Hammond WS: Role of iron in the tubulointerstitial injury in nephrotoxic serum nephritis. Kidney Int 1989;36:753–759. 116 Nankivell BJ, Boadle RA, Harris DC: Iron accumulation in human chronic renal disease. Am J Kidney Dis 1992;20:580–584. 117 Nankivell BJ, Chen J, Boadle RA, Harris DC: The role of tubular iron accumulation in the remnant kidney. J Am Soc Nephrol 1994;4:1598–1607. 118 Kawada N, Moriyama T, Ando A, et al: Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int 1999;56:1004–1013. 119 Becker GJ, Hewitson TD: The role of tubulointerstitial injury in chronic renal failure. Curr Opin Nephrol Hypertens 2000;9:133–138. 120 Hay ED, Zuk A: Transformations between epithelium and mesenchyme: Normal, pathological and experimentally induced. Am J Kidney Dis 1995;26:678–690. 121 Nadasdy T, Laszik Z, Blick KE, Johnson DL, Silva FG: Tubular atrophy in the end-stage kidney: A lectin and immunohistochemical study. Hum Pathol 1994;25:22–28. 122 Jinde K, Nikolic-Paterson DJ, Huang XR, et al: Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis. Am J Kidney Dis 2001;38:761–769. 123 Ng YY, Fan JM, Mu W, et al: Glomerular epithelial-myofibroblast transdifferentiation in the evolution of glomerular crescent formation. Nephrol Dial Transplant 1999;14:2860–2872. 124 Ng YY, Huang TP, Yang WC, et al: Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats. Kidney Int 1998;54:864–876. 125 Yang J, Liu Y: Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 2001;159:1465–1475.

Toshiki Moriyama, MD, PhD, Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine (A8), 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan) Tel. ⫹81 6 68793632, Fax ⫹81 6 68793639, E-Mail [email protected]

Moriyama/Imai

140

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 141–155

Regulation of Renal Extracellular Matrix Metabolism Masanobu Miyazaki, Tomoya Nishino, Katsushige Abe, Akira Furusu, Takehiko Koji, Shigeru Kohno Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan

Introduction

Progression to end-stage kidney disease is accompanied by accumulation of extracellular matrix (ECM) proteins in glomeruli and the interstitium. During the process of glomerular sclerosis, pathological mesangial expansion is characterized by an increase in all intrinsic components of the mesangial matrix, including collagen types IV, V and VI, laminin and fibronectin, as well as additional components of the ECM that are not present in glomeruli under physiological conditions, such as collagen types I and III. Thus, alteration of the quality and quantity of ECM is an important finding during matrix expansion leading to progression of renal injury. Imbalance between ECM production and degradation is an important factor in the process of ECM expansion. Matrix metalloproteinases (MMPs), a group of zinc endopeptidases secreted by a variety of cell types, play a central role in ECM breakdown in various renal diseases. MMP activity is tightly controlled by a specific class of natural inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). Furthermore, the plasminogen-plasmin pathway is also involved in ECM metabolism. Recent molecular biology and molecular pathology findings have revealed that, in addition to ECM components, renal cells could synthesize MMPs, TIMPs and plasminogen activator inhibitor-1 (PAI-1), and that many cytokines and growth factors were involved in the progression of renal fibrosis (glomerular sclerosis and interstitial fibrosis). In this review, we focus on ECM metabolism and relevant factors, such as MMPs, TIMPs and PAI-1, during the progression of

renal fibrosis. We also refer to the possible involvement of bone marrow-derived cells in renal injury.

Production of Extracellular Matrix

In renal tissues with advanced stage injury, abundant ECM proteins are deposited in glomeruli and the interstitium, which can be observed in renal tissue sections by immunohistochemistry using antibodies against various ECM proteins. ECM proteins deposited in the tissue are produced and secreted from cells. Immunochemistry only detects deposited ECM proteins, however advancements in molecular biology techniques have facilitated identification of the cells of origin of ECM components. Eikmans et al. [1] reported dissociation between the levels of mRNAs for collagen types I and IV and accumulation of these proteins in the aging of normal human kidney. Northern blotting and polymerase chain reaction (PCR) studies in vitro revealed that renal cells were capable of producing various ECM mRNAs [2, 3]. Furthermore, in situ hybridization analysis confirmed that renal cells expressed many different mRNAs in vivo [4]. To date, by using PCR and in situ hybridization, renal cells have been found to produce several ECM mRNAs. For example, mesangial cells produce collagen types I, III and IV, fibronectin, and laminin mRNAs, glomerular epithelial cells produce collagen types I, III and IV, fibronectin, and laminin B2 mRNAs, glomerular endothelial cells produce collagen type IV mRNA, and tubular epithelial cells synthesize collagen type III and IV mRNAs. Of the growth factors that promote ECM production, transforming growth factor-␤ (TGF-␤) is the most important, and stimulates many cells to produce ECM components, including collagen types I, III and IV, and fibronectin. TGF-␤ is involved in the progression of renal diseases, including IgA nephropathy [5], diabetic nephropathy [6] and unilateral ureteral obstruction [7]. In addition to TGF-␤, several cytokines and growth factors, including platelet-derived growth factor [8], basic fibroblast growth factor [8], angiotensin II (Ang II) [9], tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-6 [10], connective tissue growth factor (CTGF) [11], IL-1␤ [12], and IL-4 [13] are associated with promoting ECM production. In pathological conditions, these factors are intricately involved in ECM overproduction. A limited number of studies of ECM production have been performed in vivo, particularly in human renal diseases. We describe here collagen production in diabetic nephropathy (DN), because DN is one of the most important renal diseases, suggested by the fact that DN is the leading cause of end-stage renal failure in Japan and the USA. More than 10,000 new patients with DN commence dialysis therapy in Japan every year. Progressive expansion

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

142

of the mesangial matrix is one of the most characteristic pathological features of DN and various other renal diseases. Overproduction of ECM is considered primarily a response to both glomerular and tubulointerstitial changes in DN [14]. Immunohistochemical studies for ECM proteins in DN have shown alteration of ECM metabolism. Heparan sulfate proteoglycan and laminin expression decreased in the capillary walls and collagen type IV and fibronectin tended to increase in the mesangial area [15]. Alteration of ECM expression was observed by immunohistochemistry, however, whether the production of these ECMs was altered, was not determined. PCR and in situ hybridization studies have demonstrated that productions of ␣1 (IV) collagen, collagen type I, collagen type III, laminin B1 and fibronectin are increased in experimental DN and in in vitro studies [16–19]. Collagen type IV is one of the main components of both normal and diseased glomerular and tubulointerstitial ECM. The level of collagen type IV protein is reported to increase in parallel with progression of DN [20]. Several in vitro studies have demonstrated overproduction of collagen type IV by mesangial cells and other cells in the presence of high concentrations of glucose [21–23]. Furthermore, collagen type IV mRNA expression is significantly increased in isolated glomeruli of diabetic rats [24] and in cortical kidney of diabetic mice [25]. Studies employing in situ hybridization have shown that collagen type IV mRNA is confined to the glomeruli and interstitium in human DN [26]. As stated above, many factors impact on ECM production. Recent studies have revealed more precisely the mechanism of collagen overproduction in DN. Protein kinase C (PKC) is now recognized to be important for collagen synthesis under high glucose conditions [27]. Indeed, Koya et al. [28] reported that a PKC inhibitor prevented the progression of experimental DN. Recently, interstitial injury has been recognized as an important factor in deterioration of renal function. With respect to interstitial expression of collagen types III and IV, no collagen type III immunoreactivity was detected in normal renal tubules, while the tubular basement membrane was weakly positive for collagen type IV [29]. In situ hybridization confirmed that normal tubular epithelial cells expressed pro-␣1 (IV) mRNA and pro-␣1 (III) mRNA. In contrast, in damaged tubulointerstitium in DN, both collagen type III and type IV are overexpressed [29]. Razzaque et al. [30] also demonstrated that accumulation of collagen types III and IV might play a significant role in tubulointerstitial damage in benign nephrosclerosis. With respect to other ECM components, reverse transcription (RT)-PCR and in situ hybridization have shown a small amount of proteoglycan in the interstitium and mesangial matrix in normal adult kidney [31]. In human crescentic glomerulonephritis, up-regulation of decorin, biglycan and Col I mRNAs was observed by immunohistochemistry and in situ hybridization [32].

Extracellular Matrix Production and Degeneration

143

Table 1. Matrix metalloproteinase Enzymes

MMP No.

Matrix substrates

Collagenases

MMP-1 MMP-8 MMP-13

Collagens I, II, III, X, gelatins, proteogylcan Collagens I, II, III Collagen I

Gelatinases 72-kD gelatinase A

MMP-2

92-kD gelatinase B

MMP-9

Collagens IV, V, VII, XI, gelatins, fibronectin, elastin, laminin Collagens III, IV, V, gelatins, elastin

Stromelysins Stromelysin 1

MMP-3

Stromelysin 2

MMP-10

Others

MMP-7

Collagens III, IV, VII, IX, fibronecin, laminin, gelatin, proteoglycan Collagens III, IV, V, gelatin, proteoglycan Collagens IV, fibronectin, laminin, gelatin, proteoglycan Fibronectin, laminin, gelatin, proteoglycan Elastin Unknown Enamel, gelatin Amelogenin

MMP-11 MMP-12 MMP-18 MMP-19 MMP-20

Table 2. Membranous type MMP group Enzymes

MMP No.

Matrix substrates

MT1-MMP

MMP-14

MT2-MMP MT3-MMP MT4-MMP MT5-MMP MT6-MMP

MMP-15 MMP-16 MMP-17 MMP-24 MMP-25

Collagens I, II, III, fibronectin, laminin, gelatin, proteoglycan Fibronectin, tenascin, nidogen, aggrican, percan Collagen III, fibronectin, gelatin Unknown Fibronectin Gelatin

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases

Several types of MMP have been identified to date. Based on structural similarity and substrate specificity, MMPs can be divided into five subgroups (tables 1, 2): collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10), membranous type

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

144

PAI-1 Plasminogen

Latent MT1-MMP (⫺)

t-PA, u-PA

Plasmin

(⫺)

Active MT1-MMP Latent MMPs

Active MMPs (⫺)

TIMP

ECM

Degraded ECM

Fig. 1. Control of ECM degradation by MMPs and plasmin. PAI-1: plasminogen activator inhibitor-1; MMPs: matrix metalloproteinases; MT-MMP: membrane type matrix metalloproteinases; t-PA: tissue type plasminogen activator; u-PA: urokinase type plasminogen activator; TIMP: tissue inhibitor metalloproteinase; ECM: extracellular matrix.

MMPs (MT-1-MMP; MMP-14, MT-2-MMP; MMP-15, MT-3-MMP; MMP-16, MT-4-MMP; MMP-17) and others. All MMPs are synthesized as pre-proenzymes and secreted from cells as inactive proenzymes. Inactive MMPs are then activated by other MMPs or plasmin (fig. 1). Many MMPs are involved in the invasion and metastasis of malignant cells, which use MMPs to detach from primary lesions and to invade into remote sites by degrading the ECM. In renal diseases, MMPs are considered to resolve or prevent glomerular sclerosis and interstitial fibrosis. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are also known as 72-kD gelatinase/type IV collagenase and 92-kD gelatinase/type IV collagenase, respectively. They digest gelatins and, to certain extent, native collagens IV and V. MMP-2 is found in many cell types and tumor cells and is activated by membranous type MMPs (MT-MMPs) [33]. Many in vitro studies revealed that renal cells can synthesize MMPs. As shown in table 3, of the renal cell types, mesangial cells, glomerular epithelial and endothelial cells, tubular epithelial cells and infiltrating macrophages can synthesize MMP-2 and MMP-9. MMP-3, which is produced by mesangial cells, tubular epithelial cells and fibroblasts, has broad substrate specificity, including collagen types III, IV and IX, laminin, fibronectin and proteoglycans (tables 1, 3). The renal cells that produce membranous type

Extracellular Matrix Production and Degeneration

145

Table 3. Matrix metalloproteinase Enzymes

MMP No.

Producing renal cells

Collagenases

MMP-1

Mesangial cell, endothelial cell, tubuloepithelial cell, fibroblast, macrophage Neutrophil Unknown

MMP-8 MMP-13 Gelatinases 72-kD gelatinase A

MMP-2

92-kD gelatinase B

MMP-9

Mesangial cell, endothelial cell, epithelial cell, tubuloepithelial cell, fibroblast, macrophage Mesangial cell, endothelial cell, epithelial cell, tubuloepithelial cell, fibroblast, macrophage, neutrophil, T cell

Stromelysins Stromelysin 1 Stromelysin 2

MMP-3 MMP-10

Mesangial cell, tubuloepithelial cell, fibroblast T cell

MMP-7 MMP-11 MMP-12 MMP-18 MMP-19 MMP-20

Mesangial cell Unknown T cell Unknown Unknown Unknown

Others

MMPs (MT-MMPs) have yet to be determined. Only MT-MMP-1 is known to be produced by mesangial cells and fibroblasts. The activity of MMPs is tightly controlled by a specific class of natural inhibitors known as the TIMPs. Four members of this class of inhibitors have been identified to date, designated TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMPs inhibit active forms of MMPs through formation of an enzyme-inhibitor complex with 1:1 stoichiometry. TIMP-1 and TIMP-2 inhibit MMP-9 and MMP-2, respectively, and TIMP-1 is also known to inhibit the activity of MMP-3 [34]. TIMP-1 and TIMP-2 are produced by renal cells, including mesangial and glomerular epithelial cells [35–37].

Factors Associated with Production of MMPs and TIMPs

Many biological factors, such as cytokines and growth factors, affect the production and expression of MMPs and TIMPs. In addition to TGF-␤1, TNF-␣ and IL-1␤ also induce an increase in TIMP-1 mRNA in cultured mesangial cells [38]. Hepatic growth factor (HGF) also influences ECM expansion through alteration of MMP and TIMP expression. HGF markedly increases the

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

146

expression of collagenases, such as MMP-9, as demonstrated by Western blotting and zymography. Other studies also showed that HGF reduces the expression of TIMP-1 and TIMP-2 [39], but did not alter the rate of synthesis of ECM in human proximal tubule cells. HGF was detected in the supernatant of co-cultured mesangial and endothelial cells, suggesting that HGF is locally produced in glomeruli [40]. Moreover, when HGF action was blocked by administration of an anti-HGF antibody, renal fibrosis increased in rats [39]. Nitric oxide decreased IL-1␤-induced expression of MMP-9 and TIMP-1 in rat cultured mesangial cells [41]. Furthermore, 17␤-estradiol increased both MMP-9 mRNA and MMP-9 activity in mesangial cells, indicating that estrogens protect against progression of diabetic glomerulosclerosis [42]. In addition to cytokines and growth factors, hyperglycemia per se also decreases the expression of MMPs and increases expression of TIMPs, suggesting that high glucose itself may induce glomerulosclerosis via enhanced expression of TGF-␤ [43]. Recently, up-regulation of CTGF was reported, and was found to correlate with the degree of interstitial damage [44]. The relationship between expression of CTGF and MMPs and TIMPs remains to be elucidated.

Expression of MMPs and TIMPs in Diseased Kidney

MMP-2 and MMP-9 are expressed in the mesenchyme of embryonic kidneys [45]. MMP-9 is required for branching morphogenesis of the ureteric bud [45]. On the other hand, MMPs and TIMPs are involved in progression of fibrosis, in both glomeruli and the interstitium. In anti-Thy-1.1 nephritis, a model of mesangial proliferative glomerulonephritis, the expression of MT1-MMP, but not MT2- or MT3-MMPs, is increased and correlates with the expression of MMP-2 [46]. In rat crescentic glomerulonephritis, MMP-3 and its transcriptional factor Ets-1 are up-regulated in glomeruli and interstitium during the progression of tissue injury [47]. Involvement of infiltrating cells with glomerular injury has also been reported. Infiltrating macrophages in glomeruli, especially in the mesangial area, expressed MT1-MMP mRNA, and increased MMP-2 activity was observed in glomeruli in crescentic glomerulonephritis [48]. Abnormal expression of MMPs and TIMPs was also reported in a model of diabetes. Reduction in MMP-9 and up-regulation of its inhibitor TIMP-1 was demonstrated in glomeruli of obese Zucker rats, in association with enhanced synthesis of type IV collagen and reduced degradation of ECM [49]. In glomerulonephritis-hereditary nephritic mice (ICGN) mice, lower activity levels of interstitial collagenase (MMP-1) and gelatinases (MMP-2 and MMP-9), but not of stromelysin (MMP-3), were demonstrated compared with control ICGN mice [50]. Unilateral ureteral obstruction (UUO) is often used as a model

Extracellular Matrix Production and Degeneration

147

of interstitial fibrosis. Decreased MMP-9 and MMP-1 enzymatic activity was suggested to participate in the evolution of interstitial fibrosis in UUO [51]. In the same model, TIMP-1 was active in the early phase of the fibrotic process and TIMP-1 mRNA was expressed in interstitial cells [52]. Increased TIMP-1 expression, rather than decreased MMP expression, also contributed to the development of cyclosporine-induced interstitial fibrosis [53]. Tubular MMP-2 activity was also reduced in polycystic kidney disease in rats, due to downregulation of MMP-2 and up-regulation of TIMP-1 and TIMP-2 [54].

Expression of MMPs and TIMPs in Human Renal Diseases

Only a limited number of studies of the expression of MMPs and TIMPs have been reported in human renal diseases. PCR was performed to detect small amounts of mRNA in renal biopsy specimens. Using this technique, increased expression of TIMP-1 and TIMP-2 mRNAs was observed in sclerotic glomeruli [55, 56] and down-regulation of glomerular MMP-2 was reported in type 2 diabetes mellitus. In situ hybridization on renal biopsy specimens from patients with IgA nephropathy (IgAN) revealed that MMP-3 and TIMP-1 mRNAs were expressed in glomerular resident cells, tubular epithelial cells and infiltrated mononuclear cells, and that their expression was enhanced in advanced tissue damage [37]. In particular, the degree of mesangial expansion correlates well with glomerulosclerosis and declining renal function [37]. In renal tissue from type 2 diabetic patients, type IV collagen mRNA, MMP-3 mRNA and TIMP-1 mRNA were expressed in the glomeruli and interstitium, associated with the severity of histopathological changes in DN [26]. Mesangial expression of MMP-3 and TIMP-1 differed between DN and IgAN; MMP-3 and TIMP-1 expression increased during the progression of glomerular injury in IgAN, but decreased in DN, suggesting that these enzymes may play different roles in mesangial expansion in these two conditions. In contrast, the expression levels of MMP-3 and TIMP-1 mRNAs increased in the interstitium with progression of interstitial injury in both IgAN and DN, indicating that MMP-3 and TIMP-1 play similar roles in progression of interstitial damage in these renal diseases, and that the interstitial injury may proceed through a common pathway.

Potential Therapies Based on Regulation of MMPs and TIMPs

The use of MMP inhibitors has now been reported by several investigators. Steinmann-Niggli et al. [57] reported that Ro 31-9790, an inhibitor of MMPs, inhibited the proliferation rate of cultured rat mesangial cells. In an in vivo

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

148

study, BB-1101, another MMP inhibitor, ameliorated tissue injury in antiThy-1.1 nephritis [58]. Although the mechanism through which MMP inhibitors inhibit mesangial proliferation and reduced ECM deposition remains unclear, Daniel et al. [59] recently demonstrated that BB-1101 caused apoptosis of mesangial cells by cell cycle arrest in anti-Thy-1.1 nephritis, resulting in inhibition of mesangial proliferation and an anti-inflammatory effect. Previous studies in TIMP-1-deficient mice have attempted to demonstrate the importance of TIMP-1 in interstitial fibrosis, however, no histological improvement was observed [60, 61]. Eddy et al. [60] showed that interstitial fibrosis was not attenuated in TIMP-1-deficient mice in the model of overload proteinuria. In the UUO model, a genetic deficiency of TIMP-1 could not improve interstitial fibrosis [61]. Taken together, these results indicate the complexity involved in the balance of in vivo ECM metabolism.

Plasminogen-Plasmin Pathway and ECM Metabolism

The plasminogen-plasmin pathway is another important cascade involved in ECM metabolism (fig. 1). Plasmin, an important factor in fibrinolysis, was also reported to directly degrade ECM and to activate the latent forms of some MMPs, which in turn degrade a variety of ECM components [62]. Plasmin is activated from plasminogen by plasminogen activators (PAs), which are serine proteases, and the activation of plasminogen to plasmin is inhibited by PAI-1 [63]. There are two types of PA, urokinase type PA (u-PA) and tissue type PA (t-PA). t-PA synthesis is low in glomeruli compared with that of u-PA [64, 65]. Recent in vitro studies have demonstrated that renal cells, including mesangial, glomerular epithelial and tubular epithelial cells, can also produce u-PA and PAI-1 [66, 67]. PAs and PAI-1 are important for ECM degradation in the plasminogen-plasmin system. PAI-1 synthesis is regulated by several factors; PAI-1 synthesis is stimulated by hyperglycemia [68] and TGF-␤ [69]. In addition, hyperglycemia increases intrarenal levels of Ang II [69]. Ang II directly stimulates PAI-1 mRNA synthesis in rat mesangial cells [70] and Ang II stimulates the synthesis of TGF-␤ [9], leading to PAI-1 production [71]. PAI-1 synthesis is therefore up-regulated, directly or indirectly, through enhanced Ang II production under diabetic conditions. Ang II also stimulates TGF-␤ synthesis [9], which strongly induces synthesis of PAI-1. Moreover, angiotensin IV also stimulates PAI-1 expression in proximal tubular epithelial cells [72]. PAI-1 accumulation was reported in experimental nephropathy for lupus nephritis [73], mixed cryoglobulinemia [73], radiation nephropathy [74] and human renal diseases, such as crescentic glomerulonephritis [67], DN [75], and focal segmental glomerulosclerosis [76]. In most of these diseases, TGF-␤ was associated

Extracellular Matrix Production and Degeneration

149

with increased expression of PAI-1. In an animal model of interstitial fibrosis, Duymelinck et al. [53] reported that expression of PAI-1 and TIMP-1 increased during the late stage of UUO, implicating TIMP-1 and PAI-1 in the progression of tubulointerstitial scarring. Moreover, Oda et al. [77] recently demonstrated an important fibrosis-promoting function of PAI-1 using PAI-1-deficient mice. They developed a UUO model in PAI-1-deficient mice and demonstrated amelioration of interstitial fibrosis [77]. In a model of mesangial proliferative glomerulonephritis, Haraguchi et al. [78] demonstrated a new therapeutic possibility by modulating the plasminogen-plasmin cascade. Injection of human recombinant t-PA into anti-Thy-1.1 nephritis rats increased plasmin generation, and the pathological matrix degradation was promoted without activating latent TGF-␤. Furthermore, an Ang II receptor antagonist, which is used in patients with hypertension and renal diseases, also decreased PAI-1 expression and inhibited glomerular and vascular sclerosis in aging rats [79]. In a preliminary study, we demonstrated that u-PA mRNA, u-PAR mRNA and PAI-1 mRNA were increased in glomeruli of diabetic rats and showed by in situ hybridization that treatment with an angiotensin-converting enzyme inhibitor reduced synthesis of those mRNAs. These results support the existence of an intrarenal plasminogen-plasmin cascade and its importance in the progression of DN.

Bone Marrow-Derived Cells and Fibrosis

Recent studies have shown that bone marrow cells are multipotent and can differentiate into different cell types in various organs. Recently, bone marrowderived cells were shown to migrate into renal tissue and possibly be involved in renal tissue injury [80, 81]. Our preliminary data from the UUO model demonstrated the migration of bone marrow-derived cells, as distinct from infiltrating mononuclear cells, into renal tissue [82]. Using a murine bone marrow transplantation model, we identified bone marrow-derived cells in renal tissue that expressed ␣-smooth muscle actin and collagen type III, suggesting that bone marrow-derived hematopoietic cells were associated with collagen synthesis in the progression of renal injury.

Conclusion

Many interacting factors are involved in the regulation of production and degradation of ECM. A better understanding of the precise mechanism of the cascade of ECM metabolism may allow the design of new therapeutic modalities for glomerular sclerosis and interstitial fibrosis. Gene therapy and

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

150

regeneration medicine might provide novel approaches to solving these clinical problems.

Acknowledgments The authors thank Dr. Kenichi Miyazaki and Dr. Satoshi Funakoshi for their experimental data. This study was supported in part by a Research Grant for ‘Progressive Renal Disease’ from ‘Specially Selected Disease’ by the Ministry of Health and Welfare Research Project, Grant-in-Aid for Scientific Research from Japanese Ministry of Education, Science, Sports and Culture and a Baxter PD Research Fund.

References 1 2 3

4

5

6 7 8

9

10 11

12

13 14

Eikmans M, Baelde HJ, de Heer E, Bruijn JA: Effect of age and biopsy site on extracellular matrix mRNA and protein levels in human kidney biopsies. Kidney Int 2001;60:974–981. Nakamura T, Ebihara I, Shirato I, Tomino Y, Koide H: Modulation of basement membrane component gene expression in glomeruli of aminonucleoside nephrosis. Lab Invest 1991;64:640–647. Nakamura T, Ebihara I, Shirato I, Tomino Y, Koide H: Increased steady-state levels of mRNA coding for extracellular matrix components in kidneys of NZB/W F1 mice. Am J Pathol 1991; 139:437–450. Miyazaki M, Nikolic-Paterson DJ, Endoh M, Nomoto Y, Sakai H, Atkins RC, Koji T: A sensitive method of non-radioactive in situ hybridization for mRNA localization within human renal biopsy specimens: Use of digoxigenin-labeled oligonucleotides. Intern Med 1994;33:87–91. Sakai H, Naka R, Suzuki D, Nomoto Y, Miyazaki M, Nikolic-Paterson DJ, Atkins RC: In situ hybridization analysis of TGF-␤ in glomeruli from patients with IgA nephropathy. Contrib Nephrol. Basel, Karger, 1995, vol 111, pp 107–114. Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, Tomino Y, Koide H: mRNA expression of growth factors in glomeruli from diabetic rats. Diabetes 1993;42:450–456. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-␤1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 1993;44:313–321. Floege J, Eng E, Young BA, Alpers CE, Barrett TB, Bowen-Pope DF, Johnson RJ: Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 1993;92:2952–2962. Kagami S, Border WA, Miller DE, Noble NA: Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-␤ expression in rat glomerular mesangial cells. J Clin Invest 1994;93:2431–2437. Ruef C, Kashgarian M, Coleman DL: Mesangial cell-matrix interactions. Effects on mesangial cell growth and cytokine secretion. Am J Pathol 1992;141:429–439. Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins RG: Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000;11:25–38. Tesch GH, Lan HY, Atkins RC, Nikolic-Paterson DJ: Role of interleukin-1 in mesangial cell proliferation and matrix deposition in experimental mesangioproliferative nephritis. Am J Pathol 1997;151:141–150. Ruger BM, Erb KJ, He Y, Lane JM, Davis PF, Hasan Q: Interleukin-4 transgenic mice develop glomerulosclerosis independent of immunoglobulin deposition. Eur J Immunol 2000;30:2698–2703. Razzaque MS, Koji T, Taguchi T, Harada T, Nakane PK: In situ localization of type III and type IV collagen-expressing cells in human diabetic nephropathy. J Pathol 1994;174:131–138.

Extracellular Matrix Production and Degeneration

151

15 16

17 18

19 20 21

22

23 24 25

26

27

28

29

30

31

32

33 34

Ikeda S, Makino H, Haramoto T, Shikata K, Kumagai I, Ota Z: Changes in glomerular extracellular matrices components in diabetic nephropathy. J Diabet Complications 1991;5:186–188. Yang CW, Vlassara H, Peten EP, He CJ, Striker GE, Striker LJ: Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proc Natl Acad Sci USA 1994;91:9436–9440. Yang CW, Vlassara H, Striker GE, Striker LJ: Administration of AGEs in vivo induces genes implicated in diabetic glomerulosclerosis. Kidney Int 1995;49(suppl):55–58. Van Det NF, Verhagen NA, Tamsma JT, Berden JH, Bruijn JA, Daha MR, van der Woude FJ: Regulation of glomerular epithelial cell production of fibronectin and transforming growth factor-␤ by high glucose, not by angiotensin II. Diabetes 1997;46:834–840. Wang SN, LaPage J, Hirschberg R: Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy. Kidney Int 2000;57:1002–1014. Nerlich A, Schleicher E: Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions. Am J Pathol 1991;139:889–899. Ayo SH, Radnik RA, Glass WF 2nd, Garoni JA, Rampt ER, Appling DR, Kreisberg JI: Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Am J Physiol 1991;260:F185–F191. Haneda M, Kikkawa R, Horide N, Togawa M, Koya D, Kajiwara N, Ooshima A, Shigeta Y: Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 1991;34:198–200. Danne T, Spiro MJ, Spiro RG: Effect of high glucose on type IV collagen production by cultured glomerular epithelial, endothelial and mesangial cells. Diabetes 1993;42:170–177. Fukui M, Nakamura T, Ebihara I, Shirato I, Tomino Y, Koide H: ECM gene expression and its modulation by insulin in diabetic rats. Diabetes 1992;41:1520–1527. Ledbetter S, Copeland EJ, Noonan D, Vogeli G, Hassell JR: Altered steady-state mRNA levels of basement membrane proteins in diabetic mouse kidneys and thromboxane synthase inhibition. Diabetes 1990;39:196–203. Suzuki D, Miyazaki M, Jinde K, Koji T, Yagame M, Endoh M, Nomoto Y, Sakai H: In situ hybridization studies of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1 and type IV collagen in diabetic nephropathy. Kidney Int 1997;52:111–119. Derubertis FR, Craven PA: Activation of protein kinase C in glomerular cells in diabetes. Mechanisms and potential links to the pathogenesis of diabetic glomerulopathy. Diabetes 1994;43:1–8. Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC ␤ inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J 2000;14:439–447. Razzaque MS, Koji T, Horita Y, Nishihara M, Harada T, Nakane PK, Taguchi T: Synthesis of type III collagen and type IV collagen by tubular epithelial cells in diabetic nephropathy. Pathol Res Pract 1995;191:1099–1104. Razzaque MS, Cheng M, Horita Y, Nishihara M, Harada T, Taguchi T: Immunohistochemical analysis of type III and IV collagens in tubulointerstitial damage in human benign nephrosclerosis. J Int Med Res 1995;23:480–486. Schaefer L, Grone HJ, Raslik I, Robenek H, Ugorcakova J, Budny S, Schaefer RM, Kresse H: Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin and lumican. Kidney Int 2000;58:1557–1568. Stokes MB, Hudkins KL, Zaharia V, Taneda S, Alpers CE: Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis. Kidney Int 2001; 59:532–542. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seki M: A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 1994;370:61–65. Ward RV, Hembry RM, Reynolds JJ, Murphy G: The purification of tissue inhibitor of metalloproteinases-2 from its 72-kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1. Biochem J 1991;278:179–187.

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

152

35 36 37

38 39 40

41 42

43

44 45

46

47

48

49

50

51

52

53 54

55

Martin J, Knowlden J, Davies M, Williams JD: Identification and independent regulation of human mesangial cell metalloproteinases. Kidney Int 1994;46:877–885. Knowlden J, Martin J, Davies M, Williams JD: Metalloproteinase generation by human glomerular epithelial cells. Kidney Int 1995;47:1682–1689. Miyazaki M, Koji T, Furusu A, Abe K, Ozono Y, Harada T, Suzuki D, Yagame M, Endoh M, Nomoto Y, Hara K, Nakane PK, Sakai H: In situ hybridization studies of stromelysin and tissue inhibitor of metalloproteinase-1 in IgA nephropathy. Nephrology 1995;1:119–127. Pawluczyk IZ, Harris KP: Cytokine interactions promote synergistic fibronectin accumulation by mesangial cells. Kidney Int 1998;54:62–70. Liu Y, Rajur K, Tolbert E, Dworkin LD: Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. Kidney Int 2000;58:2028–2043. Yo Y, Morishita R, Yamamoto K, Tomita N, Kida I, Hayashi S, Moriguchi A, Kato S, Matsumoto K, Nakamura T, Higaki J, Ogihara T: Actions of hepatocyte growth factor as a local modulator in the kidney: Potential role in pathogenesis of renal disease. Kidney Int 1998;53:50–58. Eberhardt W, Beeg T, Beck KF, Walpen S, Gauer S, Bohles H, Pfeilschifter J: Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. Kidney Int 2000;57:59–69. Potier M, Elliot SJ, Tack I, Lenz O, Striker GE, Striker LJ, Karl M: Expression and regulation of estrogen receptors in mesangial cells: Influence on matrix metalloproteinase-9. J Am Soc Nephrol 2001;12:241–251. McLennan SV, Fisher E, Marteel SY, Death AK, Williams PF, Lyons JG, Yue DK: Effects of glucose on matrix metalloproteinase and plasmin activities in mesangial cells: Possible role in diabetic nephropathy. Kidney Int 2000;77(suppl):81–87. Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, Goldschmeding R: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998;53:853–861. Lelongt B, Trugnan G, Murphy G, Ronco PM: Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro. J Cell Biol 1997;136:1363–1373. Hayashi K, Osada S, Shofuda K, Horikoshi S, Shirato I, Tomino Y: Enhanced expression of membrane type-1 matrix metalloproteinase in mesangial proliferative glomerulonephritis. J Am Soc Nephrol 1998;9:2262–2271. Naito T, Razzaque MS, Nazneen A, Liu D, Nihei H, Koji T, Taguchi T: Renal expression of the Ets-1 proto-oncogene during progression of rat crescentic glomerulonephritis. J Am Soc Nephrol 2000;11:2243–2255. Hayashi K, Horikoshi S, Osada S, Shofuda K, Shirato I, Tomino Y: Macrophage-derived MT1MMP and increased MMP-2 activity are associated with glomerular damage in crescentic glomerulonephritis. J Pathol 2000;191:299–305. Schaefer L, Han X, August C, Matzkies F, Lorenz T, Schaefer RM: Differential regulation of glomerular gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase-1 in obese Zucker rats. Diabetologia 1997;40:1035–1043. Uchio K, Manabe N, Tamura K, Miyamoto M, Yamaguchi M, Ogura A, Yamamoto Y, Miyamoto H: Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain). Nephron 2000;86:145–151. Gonzalez-Avila G, Iturria C, Vadillo-Ortega F, Ovalle C, Montano M: Changes in matrix metalloproteinases during the evolution of interstitial renal fibrosis in a rat experimental model. Pathobiology 1998;66:196–204. Duymelinck C, Dauwe SE, De Greef KE, Ysebaert DK, Verpooten GA, De Broe ME: TIMP-1 gene expression and PAI-1 antigen after unilateral ureteral obstruction in the adult male rat. Kidney Int 2000;58:1186–201. Duymelinck C, Deng JT, Dauwe SE, De Broe ME, Verpooten GA: Inhibition of the matrix metalloproteinase system in a rat model of chronic cyclosporine nephropathy. Kidney Int 1998;54:804–818. Schaefer L, Han X, Gretz N, Hafner C, Meier K, Matzkies F, Schaefer RM: Tubular gelatinase A (MMP-2) and its tissue inhibitors in polycystic kidney disease in the Han:SPRD rat. Kidney Int 1996;49:75–81. Esposito C, Patel A, Liu ZH, Striker GE, Striker LJ: Involvement of synthesis and degradation pathways of collagen type IV in human glomerulosclerosis: Molecular analysis by in situ reverse

Extracellular Matrix Production and Degeneration

153

56

57

58 59

60 61

62 63 64 65 66 67 68

69 70 71

72

73

74

75 76

transcription and competitive polymerase chain reaction. Contrib Nephrol. Basel, Karger, 1996, vol 118, pp 12–16. Carome MA, Striker LJ, Peten EP, Moore J, Yang CW, Stetler-Stevenson WG, Striker GE: Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA. Am J Physiol 1993;264: F923–F929. Steinmann-Niggli K, Lukes M, Marti HP: Rat mesangial cells and matrix metalloproteinase inhibitor: Inhibition of 72-kD type IV collagenase (MMP-2) and of cell proliferation. J Am Soc Nephrol 1997;8:395–405. Steinmann-Niggli K, Ziswiler R, Kung M, Marti HP: Inhibition of matrix metalloproteinases attenuates anti-Thy-1.1 nephritis. J Am Soc Nephrol 1998;9:397–407. Daniel C, Duffield J, Brunner T, Steinmann-Niggli K, Lods N, Marti HP: Matrix metalloproteinase inhibitors cause cell cycle arrest and apoptosis in glomerular mesangial cells. J Pharmacol Exp Ther 2001;297:57–68. Eddy AA, Kim H, Lopez-Guisa J, Oda T, Soloway PD: Interstitial fibrosis in mice with overload proteinuria: Deficiency of TIMP-1 is not protective. Kidney Int 2000;58:618–628. Kim H, Oda T, Lopez-Guisa J, Wing D, Edwards DR, Soloway PD, Eddy AA: TIMP-1 deficiency does not attenuate interstitial fibrosis in obstructive nephropathy. J Am Soc Nephrol 2001;12: 736–748. Baricos WH, Cortez SL, el-Dahr SS, Schnaper HW: ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade. Kidney Int 1995;47:1039–1047. Vassalli JD, Sappino AP, Belin D: The plasminogen activator/plasmin system. J Clin Invest 1991;88:1067–1072. Iwamoto T, Nakashima Y, Sueishi K: Secretion of plasminogen activator and its inhibitor by glomerular epithelial cells. Kidney Int 1990;37:1466–1476. Wilson HM, Haites NE, Reid FJ, Booth NA: Interleukin-1␤ up-regulates the plasminogen activator/plasmin system in human mesangial cells. Kidney Int 1996;49:1097–1104. Keeton M, Ahn C, Eguchi Y, Burlingame R, Loskutoff DJ: Expression of type 1 plasminogen activator inhibitor in renal tissue in murine lupus nephritis. Kidney Int 1995;47:148–157. Rondeau E, Mougenot B, Lacave R, Peraldi MN, Kruithof EK, Sraer JD: Plasminogen activator inhibitor-1 in renal fibrin deposits of human nephropathies. Clin Nephrol 1990;33:55–60. Maiello M, Boeri D, Podesta F, Cagliero E, Vichi M, Odetti P, Adezati L, Lorenzi M: Increase expression of tissue plasminogen activator and its inhibitor and reduced fibrinolytic potential of human endothelial cells cultured in elevated glucose. Diabetes 1992;41:1009–1015. Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, Tomino Y, Koide H: mRNA Expression of growth factors in glomeruli from diabetic rats. Diabetes 1993;42:450–456. Kagami S, Kuhara T, Okada K, Kuroda Y, Border WA, Noble NA: Dual effects of angiotensin II on the plasminogen/plasmin system in rat mesangial cells. Kidney Int 1997;51:664–671. Maiello M, Boeri D, Podesta F, Cagliero E, Vichi M, Odetti P, Adezati L, Lorenzi M: Increase expression of tissue plasminogen activator and its inhibitor and reduced fibrinolytic potential of human endothelial cells cultured in elevated glucose. Diabetes 1992;41:1009–1015. Gesualdo L, Ranieri E, Monno R, Rossiello MR, Colucci M, Semeraro N, Grandaliano G, Schena FP, Ursi M, Cerullo G: Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells. Kidney Int 1999;56:461–470. Moll S, Menoud PA, Fulpius T, Pastore Y, Takahashi S, Fossati L, Vassalli JD, Sappino AP, Schifferli JA, Izui S: Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis. Kidney Int 1995;48:1459–1468. Oikawa T, Freeman M, Lo W, Vaughan DE, Fogo A: Modulation of plasminogen activator inhibitor-1 in vivo: A new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition. Kidney Int 1997;51:164–172. Tamaki K, Okuda S: Role of TGF-␤ in the progression of renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 44–65. Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border WA: Expression of transforming growth factor-␤ isoforms in human glomerular diseases. Kidney Int 1996;49: 461–469.

Miyazaki/Nishino/Abe/Furusu/Koji/Kohno

154

77 78 79 80

81

82

Oda T, Jung YO, Kim HS, Cai X, Lopez-Guisa JM, Ikeda Y, Eddy AA: PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction. Kidney Int 2001;60:587–596. Haraguchi M, Border WA, Huang Y, Noble NA: t-PA promotes glomerular plasmin generation and matrix degradation in experimental glomerulonephritis. Kidney Int 2001;59:2146–2155. Ma LJ, Nakamura S, Whitsitt JS, Marcantoni C, Davidson JM, Fogo AB: Regression of sclerosis in aging by an angiotensin inhibition-induced decrease in PAI-1. Kidney Int 2000;58:2425–2436. Imasawa T, Utsunomiya Y, Kawamura T, Zhong Y, Nagasawa R, Okabe M, Maruyama N, Hosoya T, Ohno T: The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells. J Am Soc Nephrol 2001;12:1401–1409. Grimm PC, Nickerson P, Jeffery J, Savani RC, Gough J, McKenna RM, Stern E, Rush DN: Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renalallograft rejection. N Engl J Med 2001;345:93–97. Miyazaki M, Nishino T, Funakoshi S, Nakamura T, Matsuda M, Harada T, Ozono Y, Koji T, Kohno S: Possible transdifferentiation of bone marrow-derived cells into myofibroblasts in renal inflammatory lesion (abstract). J Am Soc Nephrol 2000;11:511.

Masanobu Miyazaki, MD, PhD, Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501 (Japan) Tel./Fax ⫹81 95 8497282, E-Mail [email protected]

Extracellular Matrix Production and Degeneration

155

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 156–172

Implication of Apoptosis in Progression of Renal Diseases Naoki Kashiharaa, Hitoshi Sugiyamab, Hirofumi Makinob a

Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, and bDepartment of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Introduction

Glomerulosclerosis is the process featured by progressive accumulation of the extracellular matrix (ECM) and glomerular cell loss [1]. The mechanism of ECM accumulation has been well explored in recent years. A large body of information has been obtained. The major players implicated in this process have been defined. Among them, growth factors, such as transforming growth factor-␤, have been demonstrated to play an important role in this mechanism [2, 3]. In contrast, the mechanism of cell death in the process of glomerulosclerosis has not been fully elucidated. Apoptosis is a specific form of cell death defined by characteristic morphological and biochemical alterations. Apoptosis is a type of cell death that plays an important role not only in physiological processes such as embryogenesis, normal cell turnover and endocrine-dependent tissue atrophy, but also in some pathologic conditions such as tumor regression and degenerative diseases [4, 5]. Apoptosis is an active form of cell death, that is, it requires some specific gene expressions. Apoptosis in renal diseases has mainly been studied in tubular injuries and renal injuries by nephrotoxic agents [6]. Cell deletion by apoptosis occurred in hydronephrosis and ischemic-reperfusion injuries [7]. Apoptosis has also been demonstrated in the diseased glomerulus [8, 9]. Two groups reported that apoptosis was the major mechanism for the resolution of glomerular hypercellularity in experimental proliferative form of glomerulonephritis, Thy-1.1 glomerulonephritis model [10, 11]. They also demonstrated

(⫹) Acute glomerulonephritis Glomerulosclerosis

Glomerular cell number

Chronic renal diseases

(⫹)

(⫺)

(⫺)

Fig. 1. The change of glomerular cell number in renal diseases. The glomerular cell number is regulated by the balance between the rate of proliferation and cell death by apoptosis in the diseased kidney. In the self-limited glomerulonephritis, the surplus cells are deleted by apoptosis. Apoptosis is the major mechanism in the resolution of this type of renal disease. In the late stage of chronic renal disease, the number of apoptotic cells increased with the progression of glomerulosclerosis.

that mesangial apoptosis may be engaged as a homeostatic mechanism controlling the size of the glomerular cell population and promoting resolution of the mesangial proliferation. These results suggested that cell deletion by apoptosis might be beneficial for the glomerulus in self-limiting proliferative glomerulonephritis [12, 13]. Furthermore, glucocorticoid, one of the major therapeutic agents for glomerulonephritis, proved to accelerate the resolution of glomerulonephritis by sensitizing mesangial cells to apoptosis. This effect was associated with suppression of a transcription factor, nuclear factor-␬B (NF-␬B) [14, 15]. However, apoptosis may also play a pathological role, which is not beneficial (fig. 1). We hypothesized that apoptosis may be involved in the glomerular cell deletion of progressive glomerulosclerosis. This hypothesis was tested in the rat remnant kidney model and in human glomerulonephritis [16].

Apoptosis in Glomerulosclerosis

Apoptosis in Subtotal Nephrectomized Model Firstly, the implication of apoptosis was examined in the rat remnant kidney model. Progressive glomerular hypertrophy and subsequent sclerosis developed after nephrectomy. The rats were killed at week 4 and week 8. Renal tissues were

Apoptosis in Renal Diseases

157

Fig. 2. Apoptosis in the glomerulus of subtotal nephrectomized rats. CL ⫽ Capillary Lumen; Ep ⫽ Epithelial Cell; En ⫽ Endothelial Cell; arrow ⫽ Apoptotic Cell.

examined by light and electron microscopy, and the TdT-mediated nick endlabeling (TUNEL) method to detect apoptosis. Light microscopic studies revealed the presence of apoptotic cells in the glomeruli from experimental rats. The apoptotic cells, with condensed chromatin and surrounding vacuole, were found in the sclerosis and in the expansion of the matrix. Apoptosis was not found in the glomeruli from the control group at any time point studied. Given the lack of definitive molecular markers, morphologic examination by electron microscopy is most definitive in confirming apoptosis. An apoptotic cell containing condensed chromatin was observed in the mesangium with a sclerotic lesion at 8 weeks after nephrectomy by electron microscopy (fig. 2). Apoptosis of tubular epithelial cells was also identified in tubules of nephrectomized rats [17]. In the interstitium of the renal cortex, apoptotic cells, most likely infiltrating cells, were recognized. After obtaining morphologic evidence of apoptosis, the TUNEL method was employed to detect DNA fragmentation which is the biochemical characteristic of apoptosis. No staining was present in the glomeruli of rats in the control group. The glomeruli of the remnant kidneys demonstrated a few positive stainings at week 8 after nephrectomy (fig. 3). The TUNEL-positive cells were recognized in the tubules, too. Some of them were detached in the tubular lumen. The apoptotic cells with a halo were present in dilated and atrophic

Kashihara/Sugiyama/Makino

158

Fig. 3. Apoptotic cell detected by the TUNEL method in the glomerulus of subtotal nephrectomized rats. A TUNEL-positive cell (arrow) is observed in the glomerulus from a remnant kidney rat at 8 weeks after nephrectomy.

tubules. More than 30 glomerular cross sections and more than 200 tubular cross sections were examined for each animal in each time point. The number of positive-staining nuclei per tubular lumen in each section was evaluated as the tubular TUNEL score. The number of apoptotic cells significantly increased in the glomerulus at week 8 and in the tubules at week 4 and week 8. These data indicated that apoptosis of both glomerular and tubular cells was associated with cell deletion and matrix accumulation in the late stages of glomerulosclerosis after the early transient cell proliferation in the rat remnant kidney model. Apoptosis in Human Renal Diseases The apoptotic cells in the human kidney specimens were examined. Then the role of apoptosis was investigated in human renal diseases leading to glomerulosclerosis [18]. Tissue samples were taken from 9 patients with lupus nephritis and 24 patients with IgA nephropathy. Glomerular and tubular TUNEL sores were compared to the histological indices and clinical parameters including serum creatinine concentration, creatinine clearance and daily urinary protein excretion at the time of the renal biopsy. A TUNEL-positive cell was observed in the sclerotic lesions of the renal tissues from the patient with IgA nephropathy and lupus nephritis. This TUNEL-positive cell was surrounded by a clear vacuole in hematoxylin and eosin section. TUNEL-positive cells were recognized in 7 of 9 patients (78%) with lupus nephritis and in 14 of 24 patients (58%) with IgA nephropathy. The glomerular TUNEL score correlated significantly with the glomerular sclerosis index in 24 patients with IgA nephropathy and 9 patients with lupus nephritis. This score also correlated with 24-hour creatinine clearance in patients with IgA nephropathy and with serum creatinine concentration in patients with lupus

Apoptosis in Renal Diseases

159

nephritis. There was no significant correlation between the glomerular TUNEL score, proliferation index or daily protein excretion. The cell type of apoptosis was difficult to be determined, because the morphology of the apoptotic cell is uniform regardless of cell type. Moreover, apoptotic cells are so rapidly phagocytosed and ingested that they do not maintain their original structures and distinctive markers of cell origin. Apoptosis was demonstrated in the glomerular mesangium with matrix accumulation but not in the glomerular capillaries. This observation suggests that at least one of the apoptotic cells is the mesangial cell. These results indicate that apoptosis is, at least in part, involved in the cell deletion of both experimental model and human glomerulonephritis leading to sclerosis.

Mechanisms of Mesangial Cell Apoptosis

Reactive Oxygen Species (ROS) Because apoptosis is responsible for cell loss in glomerulosclerosis, elucidation of the mechanism of apoptosis in glomerular cells is important in developing new approaches to treating glomerular disease. ROS have been implicated in the pathophysiology of immune and non-immune-mediated glomerular injuries [19, 20]. Infiltrative leukocytes generate ROS and induce proteinuria in many types of experimental glomerulonephritis, including anti-glomerular basement membrane antibody-mediated nephritis and immune-complex glomerulonephritis. In these models, inhibitors or scavengers of ROS reduce the amount of tissue damage and proteinuria. ROS are demonstrated to inhibit the production of proteins relevant to permselectivity of basement membrane, such as heparan sulfate proteoglycan. Resident glomerular cells as well as infiltrating leukocytes can produce ROS. Among them, the mesangial cell is able to generate ROS in response to proinflammatory stimuli, such as interleukin-1␣ and tumor necrosis factor-␣. ROS is also implicated in the pathophysiology of diabetic nephropathy. Glucose autooxidation and glycated proteins generate ROS, such as hydroxy radicals and superoxide. A large body of evidence has suggested that ROS are involved in the induction of apoptosis. Hydrogen peroxide induces apoptosis in a variety of cell types. Depletion of cellular antioxidants can induce apoptosis in some cell types. ROS play a role as mediators of apoptosis. TNF can induce apoptosis in various cell types. TNF treatment causes a rapid increase in the intracellular concentration of ROS. TNF-induced apoptosis can be inhibited by free radical scavenger. Thus, ROS induce apoptosis and mediate intracellular apoptotic signals. ROS could play a role in the induction of apoptosis in mesangial cells.

Kashihara/Sugiyama/Makino

160

Fig. 4. Apoptotic cultured mesangial cell. An electron micrograph of human mesangial cells treated with H2O2. A mesangial cell with fragmented nucleus and condensed chromatin is seen.

To test this hypothesis, a morphological examination was performed after treatment of cultured human mesangial cells with ROS [21]. After exposure to 20 mM H2O2 for 4 h, mesangial cells showed a typical apoptotic morphology, with marked chromatin condensation, nuclear fragmentation into spherical structures, and cytoplasmic compaction, but they were still surrounded by the cell membrane (fig. 4). Next, the effects of ROS on DNA fragmentation were examined. Apoptosis induced by ROS was demonstrated by the TUNEL method in cultured human mesangial cells. The number of TUNEL-positive cells was significantly increased by incubation of cells in the presence of H2O2. A pattern of inter-nucleosomal DNA cleavage into a ladder of regular subunits of about 200 bp was apparent after incubation of cells for 4 h with 2 mM H2O2. We also exposed mesangial cells to xanthine and xanthine oxidase, which generated all the members of ROS, and observed a similar pattern of DNA fragmentation. The incubation of mesangial cells with ROS resulted in loss of viability and cytolysis with release of LDH into the medium. The cell death induced by ROS increased in a concentration- and time-dependent manner. The survival of cells requires an appropriate balance between oxidants and antioxidants. Catalase inhibited the release of LDH both in H2O2 and in Xa/XaOtreated mesangial cells, while superoxide dismutase did not inhibit the LDH release. These results suggest that ROS derived from hydrogen peroxide may mediate mesangial cell apoptosis. These results indicate ROS at the concentrations used in these studies induce apoptosis in cultured human mesangial cells.

Apoptosis in Renal Diseases

161

Apoptosis plays physiological and pathological roles. Whether ROSinduced mesangial cell apoptosis in vivo serves a beneficial function by maintaining glomerular cell number or acts in a harmful manner by decreasing the number of glomerular cells is not known. ROS produced during nonenzymatic glycation in diabetic nephropathy in the absence of cellular proliferation might induce mesangial cell apoptosis and promote the development of glomerulosclerosis. ROS are demonstrated to be generated in the process leading to glomerulosclerosis. Thus, ROS may contribute to the loss of mesangial cells in the process of development of glomerulosclerosis. Cell-Matrix Interactions It is well known that cell phenotype, such as proliferation and differentiation, is greatly influenced by the ECM surrounding the cells [22]. Cell-matrix interaction also regulates cell death [23, 24]. Certain cell types including epithelial and endothelial cell undergo apoptosis (‘anoikis’) when detached from ECM. The survival signal could be transmitted to the cells through the integrin molecule. The specific integrin suppresses apoptosis in certain cell types. The endothelial cells are particularly dependent on the ␣5␤1 integrin. The intracellular signal transduction pathway was not fully explored. Focal adhesion kinase (FAK) is demonstrated to be implicated in this mechanism. FAK becomes activated upon integrin-mediated cell attachment to ECM and initiates a signaling cascade which leads to the activation of MAP kinases. Activation of FAK also leads to the activation of phosphoinositide 3-kinase (PI3K) and the kinase AKT. In diseased conditions, the mesangial matrix is altered both quantitatively and qualitatively [25]. The increased ECM includes not only normal components but also de novo induction of type I and III collagens, which are not normally expressed in the glomerulus. Alteration of ECM components are associated with phenotypic change of mesangial cells [26]. Several studies suggested that the alteration of the composition of the ECM affected the behavior of the mesangial cells including proliferation, migration and differentiation [27]. Several studies revealed that cell attachment to ECM is required for suppression of apoptosis [28]. It is, therefore, of interest to determine whether cellmatrix interactions may influence apoptosis of the mesangial cells. The normal ECM may support the survival of mesangial cells and prevent their death. Alteration in ECM constituents may lessen the survival signals to mesangial cells and increase their susceptibility to stimuli that induce apoptosis. To test this hypothesis, the difference in the susceptibility to apoptotic stimuli was examined in the mesangial cells cultured on various ECM components [29]. The mesangial cells were cultured either on plastic culture dish, type I collagen, fibronectin or basement membrane matrix-coated dish. Apoptotic stimuli,

Kashihara/Sugiyama/Makino

162

50

BM PL

* * *

40

Apoptosis (%)

FN Col I

30

20

10

0 0

8

16

24

Time (h)

Fig. 5. Effects of ECM on the susceptibility to apoptotic stimuli in mesangial cell. The mesangial cells were cultured on the different ECM. Apoptosis was induced by serum deprivation from the culture media. Cellular apoptosis was detected by nuclear DNA staining. BM ⫽ Basement membrane matrix; PL ⫽ plastic; FN ⫽ fibronectin; Col I ⫽ type I collagen; * ⫽ p ⬍ 0.05.

either serum deprivation or H2O2 were applied to these cells. After deprivation of serum from the medium, apoptotic cell death was observed in the cell cultured on plastic and type I collagen (fig. 5). The DNA extracted from mesangial cells cultured on plastic dish and type I collagen, showed ladder pattern on gel electrophoresis, biochemical characteristic of apoptosis. The release of LDH into the medium increased and the viability of cells decreased under such conditions. In contrast, the basement membrane matrix did not induce cleavage of DNA or increase the release of LDH from the cells. Similar difference was observed when the mesangial cells were exposed to ROS. Cells cultured on plastic type I collagen, or fibronectin, showed increased susceptibility to apoptotic stimuli. The results suggest that basement membrane matrix prevented mesangial cells from undergoing apoptosis and promoted the survival of mesangial cells after serum deprivation or H2O2 treatment, as compared with type I collagen or fibronectin matrix. This indicates that the matrix influences susceptibility to proapoptotic stimuli of mesangial cells and is involved in the control of cell survival and death. Kitamura’s group [30] reported that heparin suppressed apoptosis induced by ROS in the glomerular cells. Heparin is a sulfated glycosaminoglycan and

Apoptosis in Renal Diseases

163

similar in structure to heparan sulfate proteoglycan (HS-PG). HS-PG is an intrinsic structural protein of glomerulus and plays an important role in the regulation of permselectivity of basement membrane. The amount of HS-PG in the glomerulus changes in the experimental disease models including streptzotocin-induced diabetes, puromycin aminonucleoside nephrosis and anti-Thy-1 glomerulonephritis. Furthermore, an ex vivo study revealed that synthesis of HS-PG was suppressed by ROS. These findings indicate that HS-PG affects the survival of glomerular cells. Implications of Mitochondria in Apoptosis Mitochondria produce most of the cell’s energy by oxidative phosphorylation, a process that requires the coordinated actions of five respiratory enzyme complexes located in the mitochondrial inner membrane. On the other hand, emerging evidence demonstrates that mitochondria play a central role in the regulation of apoptosis. Mitochondria are regarded to be essential for cell life and death [31–33]. Mitochondria can induce apoptosis in at least three ways: (1) disruption of electron transport and energy production through oxidative phosphorylation and resulting adenosine triphosphate production; (2) release and activation of proteins that mediate apoptosis, such as cytochrome c, and (3) alteration of cellular redox potential. Cytochorome c is released from mitochondria into cytosol during apoptosis induced by apoptogenic mediators such as UV irradiation, oxidants and overexpression of Bax. Released cytochrome c combines with Apaf-1 and procaspase-9, thereby triggering caspase-9 activation and initiating the proteolytic cascade of caspase activations [34, 35]. The outer side of the inner mitochondrial membrane is charged positively and thus forms mitochondrial transmembrane potential (⌬⌿m). A dissipation of the ⌬⌿m often precedes other manifestations of apoptosis, including caspase activation. Collapse of ⌬⌿m is induced by the opening of a large conductance channel known as the mitochondrial permeability transition (PT) pore. The PT pore is the multiprotein complex formed at the contact site between the inner and outer mitochondrial membranes. The constituents include proteins from the outer membrane, such as the voltage-dependent anion-channel, and from the inner membrane, such as the adenine translocase (ANT). Opening of this channel in the inner membrane allows for equilibration of ions within the matrix and inter-membrane space. The PT pore opening may result in expansion of the matrix space due to hyperosmolality of the matrix. The matrix expansion will cause outer membrane rupture and eventually release cytochrome c into the cytosol. Thus, PT seems to play an important role in the regulation of apoptosis. Opening of PT pore is increased by intramitochondrial Ca2⫹ and probably cellular ROS. Cyclosporin A binds cyclophilin D, which is associated with the ANT,

Kashihara/Sugiyama/Makino

164

ROS

Ca2⫹

Ceramide

ANT VDAC Bcl-2 CyA

Bax

Cytochrome c Apaf-1 Pro-caspase-9 Caspase-9

Activation of caspases

Apoptosis

Fig. 6. Implication of mitochondria in the process of apoptotic cell death. Multiple stimuli such as ROS, Ca2⫹ and ceramide can trigger the PT pore of mitochondria to open and release cytochrome c. Cytochrome c activates caspases by binding to Apaf-1.

and inhibits PT pore opening. The Bcl-2 family proteins are deeply involved in both the inhibition and induction of cellular apoptosis [36, 37]. Emerging findings indicate that these proteins exert either anti-apoptotic (Bcl-2 or Bcl-XL) or pro-apoptotic (Bax or Bcl-xS) effects through regulating opening of the PT pore. Bcl-2 protein appears to inhibit PT pore opening induced by a number of agents. In contrast, Bax protein can induce A⌿m dissipation and subsequent cytochrome c release to cytosol (fig. 6). Oxidative Modifications of Mitochondrial DNA Mitochondria possess a distinct DNA, which consists of 16,500 base pairs. Mitochondrial DNA (mtDNA) encodes a part of mitochondrial respiratory enzymes. ROS is generated during mitochondrial oxidative phosphorylation. Thus, mitochondria are a major cellular site of ROS production. In addition, mitochondria do not possess protective proteins such as histones or DNAbinding proteins. Accordingly, mtDNA is exposed to a high steady-state level of ROS and is more susceptible to oxidative damage than nuclear DNA. Oxidative modification and mutation of mtDNA occur with great ease. Accumulation of oxidative modification of mtDNA was demonstrated to increase exponentially with age [38–40].

Apoptosis in Renal Diseases

165

ROS can cause a wide variety of oxidative modifications on DNA, including oxidized bases, abasic sites and DNA strand break [41–43]. The production of 8-hydroxydeoxyguanosine (8-OHdG) by the hydroxylation of carbon-8 of guanine is particularly of note. When DNA replicates, adenine exhibits a 50% probability of mis-pairing with 8-OHdG. Consequently, G:C produces a T:A transversion. As a result, a point mutation could be generated with a probability of 50%. In addition, accumulation of 8-OHdG has been reported to lead to increased frequency of deletion in mtDNA. 8-OHdG is commonly used as the marker of oxidative damage of DNA. Accumulation of oxidative damage of DNA was reported in various disease conditions including diabetes [44]. The accumulation of 8-OHdG in renal tissue from 5/6 nephrectomized rat was examined immunohistochemically, using monoclonal antibody. Positive staining was observed in the cytosol of glomerular epithelial and endothelial cells. Mesangial cells were also stained to some extent. To determine subcellular localization of 8-OHdG, we performed immunoelectron microscopical examination using immunogold-labeled antibody. The gold particles were localized in the mitochondria. Urinary excretion of 8-OhdG was measured in nephrectomized rat. After 20 weeks of nephrectomy, a significant increase in urinary 8-OHdG levels was observed in comparison to controls. These observations indicated that oxidative modifications occurred in mtDNA of renal cells during progression of renal injuries. A variety of mtDNA modifications could occur upon exposure to ROS. They include point mutations and deletions. Mitochondial DNA damage was assessed using a quantitative polymerase chain reaction assay that relies on the principle that oxidative DNA lesions inhibit DNA polymerases, resulting in decreased amplification of the target sequence [45]. DNA damages were quantified by comparing the relative efficiency of amplification of whole mitochondrial DNA. Amplification efficiency was suppressed in the nephrectomized rat comparing to control rat. These results indicate a possibility that oxidative modification may accumulate in mtDNA of nephrectomized rat. The mtDNA encodes some of the respiratory chain enzymes. The expressions of some of the mitochondrial genes, NADH dehydrogenase-2 and cytochrome b genes, which encode structural proteins in complexes I and III, were examined respectively. These gene expressions were inhibited in the glomeruli from the 5/6 nephrectomized experimental model. The accumulation of oxidative modifications of mtDNA and altered expressions of mtDNA were recognized in the 5/6 nephrectomized experimental models. This change may result in alterations of enzymatic activities of mitochondria. The specific inhibitors were utilized to elucidate the results of deterioration of mitochondrial respiratory functions. The inhibitors for respiratory enzyme complexes, retone, TTFA, antimycin for complexes I, II and III,

Kashihara/Sugiyama/Makino

166

Glomerular injuries

ROS production Accumulation of 8-OHdG mtDNA damage ROS Vicious cycle

Altered expressions of mitochondrial mRNA

Deterioration of respiratory function

Dissipation of ⌬⌿m

Deterioration of cellular functions

Induction of apoptosis

Fig. 7. Hypothetical scheme of possible implication of oxidative stress and mitochondria.

respectively, were utilized to suppress the specific functions. The ROS production was measured in the cultured human mesangial cells treated with these inhibitors using flow cytometry and dichlorofluorescein. Rotenone and antimycin, which inhibit complexes I and III, respectively, increased ROS production. These results indicate that oxidative modification of mtDNA further augment generation of ROS, which in turn elevate oxidative stress to mitochondria. This vicious cycle may operate in renal tissues of progressive forms of renal diseases (fig. 7). Role of Mitochondria in Apoptosis of Renal Cells Recent studies now emphasize the role of mitochondria in apoptosis signaling as mentioned above. The role of the mitochondrial pathway in apoptosis of renal cells was not fully elucidated. Possible implication of mitochondrial cytochrome c was investigated in the hypertensive renal injuries in the kidney of Dhal/Rapp salt-sensitive rat [46, 47]. Apoptosis of renal cells was recognized and associated with release of cytochrome c and activations of caspase-9 and caspase-3. The cells undergoing apoptosis show an early reduction of the ⌬⌿m, with concomitant release of the mitochondrial protein cytochrome c [48]. In the cytosol, cytochrome c in combination with Apaf-1 activates caspase-9, which finally leads to activation of caspase-3 and DNA fragmentation. We examined the possible implication of mitochondria in the mechanism of apoptosis induced by ROS. Mesangial cells were treated with H2O2. Changes in mitochondrial

Apoptosis in Renal Diseases

167

a

b Fig. 8. The changes in mitochondrial membrane potential of mesangial cells. Mitochondrial membrane potential was examined by membrane potential-sensitive dye and confocal microscopy. Mitochondrial staining was observed in the control mesangial cells (a). The mesangial cells treated with H2O2 lost this staining (b).

transmembrane potential were assessed by potential-sensitive dye JC-1. In control mesangial cells, JC-1 uptake occurred in mitochondria due to the retention of the membrane potential. In contrast, the mesangial cells incubated with hydrogen peroxide were unable to absorb this dye (fig. 8). The number of cells which maintain the mitochondrial membrane potential decreased in a timedependent manner. The occurrence of apoptotic cell death followed the loss of membrane potential (fig. 9). Cyclosporin A (CyA), which is used as an immunosuppressive agent, has been shown to stabilize the mitochondrial transmembrane potential through binding to cyclophilin, thereby inhibiting apoptosis induced by different stimuli [49]. The influence of CyA was investigated in the induction of apoptosis on mesangial cells by ROS. Mesangial cells were incubated with 20 mM H2O2 in the presence of a series of CyA. 0.5 mg/ml of CyA effectively inhibit ROSinduced apoptosis in mesangial cells. However, higher concentration of CyA did not show such anti-apoptotic effect. CyA has nephrotoxic effects, too. Chronic CyA nephropathy is characterized by tubular atrophy and interstitial fibrosis. Apoptosis was demonstrated to be involved in the loss of tubular and interstitial cells in this condition. The precise mechanism of apoptotic cell death in CyA nephropathy was not explored. CyA causes vascular injuries and resultant tissue ischemia. Therefore, the apoptotic cell death observed in CyA nephropathy may not be caused by a direct effect of CyA. We could expect an anti-apoptotic effect by CyA in the specific concentration based on the in vitro data.

Kashihara/Sugiyama/Makino

168

a

b Fig. 9. Apoptosis in cultured mesangial cells. Condensed and fragmented nuclei were observed in the mesangial cells treated with H2O2 for 4 h (b). Fine staining in the nuclei was observed in the control cells (a). Nuclear DNA was stained with Hoechst dye.

DM

Glomerulonephritis

Hypertension

Glomerular injuries

Phenotypic changes of mesangial cells

Increased susceptibility for apoptotic stimuli

Alterations of ECM

Apoptotic stimuli ROS, etc. Accumulation of ECM

Apoptosis

Glomerulosclerosis

Fig. 10. Hypothetical scheme of the mechanism of progression of glomerulosclerosis. In the process leading to glomerulosclerosis, mesangial matrix alters quantitatively and qualitatively. An altered matrix increases the susceptibility for apoptotic stimuli, such as ROS, of glomerular cells.

Apoptosis in Renal Diseases

169

Conclusion

Our hypothetical scheme of the mechanism of progression of glomerulosclerosis is summarized in figure 10. Accumulation of ECM and progressive cell loss are the most prominent features of glomerulosclerosis. ECM components are altered both quantitatively and qualitatively in the process leading to sclerosis. Altered matrix components may influence the susceptibility to apoptotic stimuli of mesangial cells, such as ROS. In such a situation, glomerular cells are easily lost by apoptosis. Oxidative stress relevant to apoptosis could be augmented further by oxidative modification of mitochondria [50]. The mechanism of glomerular cell apoptosis requires further study to gain new insights into the treatment of renal diseases and prevention of subsequent glomerular scarring.

References 1

2 3 4 5 6 7 8 9 10 11

12 13

14

15

16

Floege J, Johnson RJ, Gordon K, Iida H, Pritzl P, Yoshimura A, Campbell C, Alpers CE, Couser WG: Increased synthesis of extracellular matrix in mesangial proliferative nephritis. Kidney Int 1991;40:477–488. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor ␤1. Nature 1990;346:371–374. Tamaki K, Okuda S: Role of TGF-␤ in the progression of renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 44–65. Vaux DL, Korsmeyer SJ: Cell death in development. Cell 1999;96:245–254. Desmouliere A, Redard M, Darby I, Gabbiani G: Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 1995;146:56–66. Lau AH: Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int 1999;56:1295–1298. Gobe G, Willgoss D, Hogg N, Schoch E, Endre Z: Cell survival or death in renal tubular epithelium after ischemia-reperfusion injury. Kidney Int 1999;56:1299–1304. Savill J: Apoptosis and the kidney. J Am Soc Nephrol 1994;5:12–21. Savill J: Apoptosis in disease. Eur J Clin Invest 1994;24:715–723. Shimizu A, Kitamura H, Masuda Y, Ishizaki M, Sugisaki Y, Yamanaka N: Apoptosis in the repair process of experimental proliferative glomerulonephritis. Kidney Int 1995;47:114–121. Baker A, Mooney A, Hughes J, Lombardi D, Johnson R, Savil J: Mesangial cell apoptosis: The major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. J Clin Invest 1994;94:2105–2116. Savill J: Regulation of glomerular cell number by apoptosis. Kidney Int 1999;56:1216–1222. Sugiyama H, Kashihara N, Onbe T, Yamasaki Y, Wada J, Sekikawa T, Okamoto K, Kanao K, Maeshima Y, Makino H: Bcl-2 expression and apoptosis in nephrotoxic nephritis. Exp Nephrol 1997;5:481–489. Maruyama K, Kashihara N, Yamasaki Y, Sato M, Sugiyama H, Okamoto K, Maeshima Y, Odawara M, Sasaki J, Makino H: Methylprednisolone accelerates the resolution of glomerulonephritis by sensitizing mesangial cells to apoptosis. Exp Nephrol 2001;9:317–326. Sugiyama H, Savill JS, Kitamura M, Zhao L, Stylianou E: Selective sensitization to tumor necrosis factor-␣-induced apoptosis by blockade of NF-␬B in primary glomerular mesangial cells. J Biol Chem 1999;274:19532–19537. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z: Apotosis in glomerular sclerosis. Kidney Int 1996;49:103–111.

Kashihara/Sugiyama/Makino

170

17

18

19

20 21 22 23 24 25

26

27

28

29 30 31 32 33 34 35

36 37 38

39 40 41 42

Makino H, Sugiyama H, Kashihara N, Sekikawa T, Onbe T, Nakajima T, Yamasaki Y, Ota Z: Apoptosis of tubular epithelial cells in rats with chronic renal failure. Med Electron Microsc 1995; 28:210–212. Makino H, Kashihara N, Sugiyama H: Role of apoptosis in the progression of glomerulosclerosis; in Koide H, Ichikawa I (eds): Progression of Chronic Renal Diseases. Contrib Nephrol. Basel, Karger, 1996, vol 118, pp 41–47. Kashihara N, Watanabe Y, Makino H, Wallner EI, Kanwar YS: Selective decreased de novo synthesis of glomerular proteoglycans under the influence of reactive oxygen species. Proc Natl Acad Sci USA 1992;89:6309–6313. Nath K, Croatt A, Hostetter T: Oxygen consumption and oxidant stress in surviving nephrons. Am J Physiol 1990;258:F1354–F1362. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z: Reactive oxygen species induce apoptosis in cultured human mesangial cells. J Am Soc Nephrol 1996;7:2357–2363. Giancotti FG, Ruoslahti E: Integrin signaling. Science 1999;285:1028–1032. Ruoslahti E, Reed JC: Anchorage dependence, integrin and apoptosis. Cell 1994;77:477–478. Frisch SM, Ruoslahti E: Integrins and anoikis. Curr Opin Cell Biol 1997;9:701–706. Okamoto K, Kashihara N, Yamasaki Y, Kanao K, Maeshima Y, Sekikawa T, Sugiyama H, Murakami T, Makino H: Caldesmon isoform associated with phenotypic modulation of mesangial cells. Exp Nephrol 2000;8:20–27. Johnson RJ, Iida H, Alpers CE, Majesky MW, Schwartz SM, Pritzl P, Gordon K, Gown AM: Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. J Clin Invest 1991;87:847–858. Makino H, Kashihara N, Sugiyama H, Kanao K, Sekikawa T, Okamoto K, Maeshima Y, Ota Z, Nagai R: Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes. Diabetes 1996;45:488–495. Makino H, Kashihara N, Sugiyama H, Kanao K, Sekikawa T, Okamoto K, Maeshima Y, Ota Z, Nagai R: Phenotypic changes of the mesangium in diabetic nephropathy. J Diabetes Complications 1995;9:282–284. Sugiyama H, Kashihara N, Sekikawa T, Makino H, Ota Z: Cell-matrix interactions modulate mesangial apoptosis. J Am Soc Nephrol 1995;6:912. Ishikawa Y, Kitamura M: Inhibition of glomerular cell apoptosis by heparin. Kidney Int 1999;56:954–963. Green DR, Reed JC: Mitochondria and apoptosis. Science 1998;281:1309–1312. Gross A, McDonnell JM, Korsmeyer SJ: Bcl-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899–1911. Brenner C, Kroemer G: Apoptosis. Mitochondria – the death signal integrators. Science 2000;289: 1150–1151. Kroemer G, Reed JC: Mitochondrial control of cell death. Nat Med 2000;6:513–519. Li H, Kolluri SK, Gu J, Dawson MI, Cao X, Hobbs PD, Lin B, Chen G, Lu J, Lin F, Xie Z, Fontana JA, Reed JC, Zhang X: Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 2000;289:1159–1164. Chao DT, Korsmeyer SJ: Bcl-2 family: Regulators of cell death. Annu Rev Immunol 1998;16:395–419. Tsujimoto Y, Shimizu S: Bcl-2 family: Life-or-death switch. FEBS Lett 2000;466:6–10. Wei YH, Lu CY, Lee HC, Pang CY, Ma YS: Oxidative damage and mutation to mitochondrial DNA and age-dependent decline of mitochondrial respiratory function. Ann NY Acad Sci 1998;854:155–170. Sastre J, Pallardo FV, Garcia de la Asuncion J, Vina J: Mitochondria, oxidative stress and aging. Free Radic Res 2000;32:189–198. Richter C: Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol 1995;27:647–653. Cadenas E, Davies KJ: Mitochondrial free radical generation, oxidative stress and aging. Free Radic Biol Med 2000;29:222–230. Lenaz G: Role of mitochondria in oxidative stress and ageing. Biochim Biophys Acta 1998;1366: 53–67.

Apoptosis in Renal Diseases

171

43 44 45

46 47 48 49

50

Ashok BT, Ali R: The aging paradox: Free radical theory of aging. Exp Gerontol 1999;34: 293–303. Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, Nicotera T: Oxidative damage to DNA in diabetes mellitus. Lancet 1996;347:444–445. Ballinger SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van HB, Ballinger CA, Freeman BA, Runge MS: Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 2000;86:960–966. Ying WZ, Wang PX, Sanders PW: Induction of apoptosis during development of hypertensive nephrosclerosis. Kidney Int 2000;58:2007–2017. Ying WZ, Sanders PW: Cytochrome c mediates apoptosis in hypertensive nephrosclerosis in Dahl/Rapp rats. Kidney Int 2001;59:662–672. Li AE, Ito H, Rovira II, Kim KS, Takeda K, Yu ZY, Ferrans VJ, Finkel T: A role for reactive oxygen species in endothelial cell anoikis. Circ Res 1999;85:304–310. Walter DH, Haendeler J, Galle J, Zeiher AM, Dimmeler S: Cyclosporin A inhibits apoptosis of human endothelial cells by preventing release of cytochrome c from mitochondria. Circulation 1998;98:1153–1157. Kashihara N, Sugiyama H, Makino H: Mechanisms for induction of apoptosis and glomerular disease. Nephrol Dial Transplant 1999;1:51–54.

Naoki Kashihara, MD, Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Matsushima 577, Kurashiki City, Okayama 701-0192 (Japan) Tel. ⫹81 86 4621111, Fax ⫹81 86 4621199, E-Mail [email protected]

Kashihara/Sugiyama/Makino

172

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 173–186

Factors Regulating the Progression of Hypertensive Nephrosclerosis Mohammed S. Razzaquea,c, Abdallah Azouzb, Tatsuo Shinagawad, Takashi Taguchic a Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Mass.; bDepartment of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass., USA; cDepartment of Pathology, Nagasaki University Graduate School of Medical Sciences, Nagasaki, and d Department of Medicine, Kawatana National Hospital, Kawatana, Japan

Introduction

Hypertensive nephrosclerosis is one of the commonest causes of end-stage renal disease (ESRD). The exact incidence of nephrosclerosis within the hypertensive population with chronic renal diseases is not known. Since a significant number of patients with hypertension also have other renal diseases, such as diabetic nephropathy and chronic glomerulonephritis. The extent of loss of renal function due to prolonged hypertension in these patients with overlapping glomerular diseases is difficult to estimate. However, a higher incidence of nephrosclerosis has been documentated in African-American populations, compared to Caucasians. African-American hypertensive patients develop ESRD at a severalfold higher rate than white patients [1]. According to the US Renal Data System (USRDS), approximately 50,000 new patients developed ESRD due to hypertension during the period 1988–1991 [2]. Hypertension accounts for 38% of ESRD in African-Americans, and 25% of ESRD in whites [3]. Similarly, around 20% of patients accepted for regular dialysis treatment in Europe are thought to have ESRD due to hypertensive nephrosclerosis [4]. In Japan, a total 8.2% of nephrosclerotic patients undergoing hemodialysis are reported to be associated with some form of hypertension [5]. According to a more recent report in 1999 from the USRDS, hypertensive nephrosclerosis accounts for as

many as 34% of patients reaching ESRD each year in the USA; hypertensive nephrosclerosis is the second most common cause of ESRD in white people (21%) and is the leading cause of ESRD in black people (34%). The exact cause of the higher incidence of hypertension-related ESRD in a particular race, such as African-Americans, is not known. However, a different renal architecture, renal circulation and hyperresponsiveness of vascular smooth muscle cells to growth factors have been speculated as possible contributing factors [6, 7]. Genetic factors might also partly explain why African-Americans are more susceptible to developing nephrosclerosis. Specific polymorphisms in genes encoding angiotensin-converting enzyme (ACE), angiotensinogen and the angiotensin type 1 receptor have been linked with hypertension, myocardial infarction and cardiac hypertrophy [8]. In African-Americans with essential hypertension, mutations in genes encoding angiotensinogen were more frequent, compared to Caucasian hypertensive patients [9]. Similarly, polymorphism of the ACE gene was shown to be more frequent in African-American populations than in Caucasians [10, 11]. These polymorphisms may significantly alter the activity of the renin-angiotensin system, and may thereby be able to modulate the effectiveness of ACE inhibitors [12–14]. Excellent and extensive reviews detailing etiopathology and treatment options for hypertension have been published. This article, therefore, will be limited to hypertensive renal injury, emphasizing the molecular mechanisms of nephrosclerosis.

Nephrosclerosis

Hypertensive nephrosclerosis is a multifactorial process. The early changes may be associated with an increased intravascular volume, due to a gradual decrease in the glomerular filtration rate, resulting in decreased renal excretion of sodium and water. Intravascular volume expansion may activate both sympathetic tone and the renin-angiotensin system to initiate a cascade of events that eventually lead to nephrovascular sclerosis. In addition, decreased activities of vasorelaxants (nitric oxide) and increased activity of vasoconstrictors (endothelin) may have a determinant effect on subsequent nephrosclerosis. The narrowing of preglomerular arteries and arterioles due to prolonged hypertension may result in reduced glomerular blood flow; glomerular ischemia is one of the main contributory factors in hypertensive nephrosclerosis. Focal loss of glomeruli due to hypertensive sclerosis may have compensatory effects on other non-sclerotic glomeruli to restore the loss of renal function; the remaining nephrons become hyperfunctioning, and the preglomerular arterioles undergo vasodilatation, with an increase in renal blood flow and glomerular filtration. The resultant glomerular hypertension and glomerular hyperfiltration

Razzaque/Azouz/Shinagawa/Taguchi

174

lead to further deterioration of renal function, with an increase in the number of sclerotic glomeruli. In a recent retrospective study of 7,108 adult renal biopsy cases, benign nephrosclerosis was identified in 590 (8.3%) cases. The presence of globally sclerotic glomeruli in the biopsy was reported in about 41% of cases, and correlated with a clinically poor prognostic outcome [15]. The molecular and cellular events occurring during the development of hypertensive nephrosclerosis are not yet clear, but experimental models of hypertensive nephrosclerosis have provided vital information relevant to human nephrosclerosis.

Factors Regulating Nephrosclerosis

The multistep and multifactorial process of nephrosclerosis is regulated by complex cellular and molecular interactions. Mediators, such as cytokines, chemokines and growth factors, released by resident renal cells or infiltrating inflammatory cells, play an important role in remodeling of ECM in the kidney. In addition to the above-mentioned factors, oxidative stress has a definitive role in most tissue damage, including hypertensive renal injuries. Oxidative stress, associated with increased plasma isoprostane and reductions in plasma glutathione, has been shown to cause severe hypertension in normal rats [16]. Modulation of oxidative stress with phytoestrogens was shown to attenuate oxidative DNA damage in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats [17]. Similarly, in chronic experimental congestive heart failure, the use of probucol, an antioxidant, resulted in beneficial morphological effects on left ventricular remodeling and function; it also reduced neurohumoral activation, and helped in maintaining renal function [18]. Changes in microenvironments due to prolonged hypertension could also facilitate and/or intensify the local immunoinflammatory responses and subsequent nephrosclerosis. Microenvironments in hypertensive kidney may influence the phenotypic changes of glomerular cells (mesangial and podocytes), interstitial cells and tubular epithelial cells [19]. These phenotypically altered renal cells are capable of producing increased levels of inflammatory and fibrogenic factors that contribute to both early (inflammatory) and late (fibrotic) events of nephrosclerosis induced by hypertension. The early stage of hypertensive renal injury is associated with inflammatory changes with increased infiltration of inflammatory cells, including macrophages. The degree of renal accumulation of macrophages was shown to be closely associated with the severity of tissue injuries. Increased expression of certain inflammatory cell-recruiting molecules has been detected in the kidneys of both human and experimental models of hypertension. For example,

Hypertensive Nephrosclerosis

175

using double transgenic hypertensive rats harboring both human renin and human angiotensinogen genes, increased expression of surface adhesion molecules (intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)) was observed in the interstitium, intima and adventitia of the small renal vessels, while their corresponding ligands were detected on circulating leukocytes. Leukocyte infiltration in the vascular wall was associated with expression of plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor. The expression of transforming growth factor (TGF)-␤ and accumulation of ECM proteins was accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys of these transgenic hypertensive rats [20, 21]. ACE inhibition, angiotensin II type 1 (AT1) receptor blockade, and human renin inhibition resulted in prevention of monocyte/macrophage infiltration and decreased expression of adhesion molecules in the kidneys of double transgenic hypertensive rats [20]. Increased expression of MCP-1 was detected in the glomeruli in 5 of 6 nephrectomized rats, in a model of progressive glomerular sclerosis associated with glomerular hypertension [22]. Subsequent studies have shown that exogenous pressure on cultured mesangial cells can induce MCP-1 via activation of the MAP kinase pathway [22, 23]. AT1 receptor blockade has been shown to prevent the expression of MCP-1 and accumulation of macrophages in the kidneys of experimental models of hypertensive rats [24]. Further studies should determine the roles of other inflammatory cell-recruiting molecules, such as macrophage colony-stimulating factor (m-CSF) and macrophage migration inhibitory factor (MIF) in hypertensive nephrosclerosis. Increased expression of m-CSF and MIF was shown to be closely associated with increased accumulation and proliferation of macrophages in various human and experimental fibrotic diseases, including experimental models of nephritis [25–27]. Accumulated inflammatory infiltrates, mainly macrophages, might play an important role in subsequent nephrosclerosis, by triggering increased production of certain fibrogenic cytokines responsible for inducing and/or propagating the fibrotic cascade. TGF-␤1 is an important factor actively involved in the synthesis of ECM proteins such as fibronectin and collagens [28, 29], and increased renal expression of TGF-␤1 has been detected in various human and experimental fibrotic renal diseases [30]. Furthermore, in vivo suppression of the bioactivities of TGF-␤1 by using its neutralizing antibody was effective in delaying the glomerulosclerosis in experimental glomerulonephritic rats [31]. Increased glomerular expression of TGF-␤1 and phenotypic modulation of renal cells have been documented in an experimental model of hypertension, during the progression of glomerulosclerosis and tubulointerstitial fibrosis [19]. Increased renal expression of TGF-␤1 with increased deposition of interstitial collagens

Razzaque/Azouz/Shinagawa/Taguchi

176

has also been detected in renal biopsy sections of human hypertensive nephrosclerosis [32, 33]. By using in situ hybridization, intraglomerular cells and interstitial cells were detected as collagen-producing cells in renal biopsy sections obtained from patients with hypertensive nephrosclerosis [34]. Recently, the plasma levels of active and latent TGF-␤1 were determined in 85 patients with essential hypertension, and compared with levels in 30 normotensive individuals; plasma levels of active and latent TGF-␤1 were markedly increased in patients with hypertension [35]. Dahl salt-sensitive hypertensive rats treated with an intraperitoneal injection of anti-TGF-␤ antibody every other day, exhibited significant reductions in blood pressure, proteinuria, and the degree of glomerulosclerosis and renal medullary fibrosis [36]. In patients with essential hypertension, an association has been documented between increased levels of TGF-␤1, with increased synthesis and decreased degradation of type I collagen, and subsequent cardiorenal damage [37]. It is likely that overexpression of TGF-␤1 may play a central role in hypertensive nephrosclerosis by inducing increased synthesis of matrix proteins, and blocking the bioactivities of TGF-␤1 might partly modulate hypertensive nephrovascular sclerosis. However, TGF-␤ not only has the ability to induce matrix proteins, but it also has diverse physiological effects on various essential cellular functions. It suppresses the growth of epithelial cells, inhibits keratinocyte proliferation, enhances neovascularization, acts as a chemoattractant for monocytes and fibroblasts, and has a role in immunosuppression [38, 39]. Although blocking the bioactivity of TGF-␤ has some beneficial effects on delaying the fibrotic process, it could disrupt other non-fibrotic physiologic functions of TGF-␤. Thus, identifying fibrosis-specific factors and their blockade will be ideal for the design of future therapeutic approaches. Connective tissue growth factor (CTGF) is a TGF-␤1-inducible gene that promotes proliferation, and chemotaxis by mesenchymal cells. CTGF is a potent inducer of collagen synthesis. CTGF is thought to influence the terminal events of TGF-␤-induced fibrogenesis. Although increased levels of CTGF with excessive accumulation of collagens have been detected in numerous fibrotic disorders, including renal fibrotic diseases [40, 41], its role in hypertensive nephrosclerosis is not yet clear. Recent studies in experimental cyclosporin-induced hypertension showed that myocardial changes were associated with increased myocardial expression levels of CTGF, type I collagen and type III collagen by 91, 198 and 151%, respectively [42]. Moreover, blockade of the renin-angiotensin system prevented vascular damage and cyclosporin-induced CTGF, type I collagen and type III collagen mRNA overexpression in the heart [42]. Further experiments that focus on the relationship and interaction between TGF-␤1 and CTGF in the regulation and synthesis of collagens and ECM proteins in hypertensive nephrosclerosis should provide

Hypertensive Nephrosclerosis

177

50␮m

50␮m

a

b Fig. 1. Periodic acid methenamine silver staining on paraffin sections prepared from kidneys obtained from Dahl salt-sensitive rat fed with regular diet (a), and an age-matched rat fed with high salt diet (8% NaCl) for 5 weeks (b). Note severe glomerulosclerosis (arrows), tubular basement membrane thickening (open arrows) and tubulointerstitial fibrosis in the kidneys obtained from hypertensive rats.

information on the role of CTGF, and its therapeutic potential in the treatment of hypertensive renal damage. By blocking the effects of CTGF, it may be possible to reduce TGF-␤-induced hypertensive nephrosclerosis, without interfering with the other essential physiologic functions of TGF-␤. Heat-shock protein 47 (HSP47) plays a crucial role in the intracellular processing, assembly and synthesis of procollagens. Increased expression of HSP47 with increased deposition of collagens has been detected in a number of human and experimental fibrotic diseases, including hypertensive nephrosclerosis [43–49]. Using hypertensive Dahl rats, where nephrovascular sclerosis is a severe morphological feature (fig. 1), phenotypically altered ␣-smooth muscle actin-positive glomerular and interstitial cells, desmin-positive glomerular epithelial cells, and vimentin-positive tubular epithelial cells were detected in kidneys obtained from hypertensive rats, compared to control normotensive rats. In these hypertensive rat kidneys, similar to the increased expression and deposition of collagens (fig. 2), an increased level of HSP47 was detected in the sclerotic glomeruli, fibrotic interstitium and thickened blood vessel walls (fig. 3). By dual immunostaining, all the phenotypically altered renal cells in the kidneys of hypertensive rats were found to express increased levels of HSP47 (fig. 4). Furthermore, increased expression of HSP47 was associated with increased accumulation of collagens in the sclerotic glomeruli, and in the fibrotic interstitium of kidneys of hypertensive Dahl rats (fig. 4). Preliminary studies have shown that modulating the expression of HSP47 could suppress and/or slow down the accumulation of collagens, and thereby could alter the course of fibrosis [50]. Further studies will determine its therapeutic potential in the modulation of hypertensive nephrosclerosis. It will be interesting to

Razzaque/Azouz/Shinagawa/Taguchi

178

50␮m

50␮m

a

b

50␮m

50␮m

c

d

50␮m

50␮m

e

f Fig. 2. Immunohistochemistry on paraffin sections prepared from kidneys obtained from normotensive (a, c, e) and hypertensive (b, d, f) Dahl rats, for type IV collagen (a, b), type I collagen (c, d) and type III collagen (e, f). Compared to normotensive rat kidneys (a), an increased expression of type IV collagen is detected in thickened TBM (open arrows) and sclerotic glomeruli (solid arrows) in kidneys obtained from hypertensive Dahl rats (b). A similar increase in the expression of type I collagen (d) and type III collagen (f) is detected in the fibrotic interstitium of hypertensive rat kidneys (arrows), compared to control normotensive rat kidneys (c, e).

clarify whether certain antihypertensive drugs could modulate the bioactivity of HSP47 in achieving the beneficial effects. In addition to the aforementioned factors, expression of endothelin (ET-1), interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF-␣), granulocyte-macrophage

Hypertensive Nephrosclerosis

179

50␮m

50␮m

a

b

50␮m

50␮m

c

d Fig. 3. Immunohistochemistry on paraffin sections prepared from kidneys obtained from normotensive (a) and hypertensive (b, c) Dahl rats for HSP47. Note the increased expression of HSP47 in the glomerular cells (arrowheads), interstitial cells (open arrows) and cells in the thickened blood vessel walls (arrows) in kidneys obtained from hypertensive Dahl rats (b, c). No specific staining was detected when monoclonal antibody to HSP47 was replaced with mouse IgG (d).

colony-stimulating factor, basic-fibroblast growth factor and ICAM-1 have been reported to play roles in hypertensive nephrosclerosis [51]. Of these, the role of ET-1, a potent vasoconstrictor, in the pathogenesis of various cardiovascular and renal diseases has been extensively studied. The expression and activity of ET-1 are increased in the kidneys of human and experimental animals. In transgenic mice overexpressing the ET-1 gene, renal injuries, including glomerulosclerosis, interstitial fibrosis, renal cysts and narrowing of arterioles, were detected in 12-month-old mice [52]. Moreover, the fibrogenic role of ET-1 and endothelin-converting enzyme-1 has been reported in patients with idiopathic pulmonary fibrosis; it promotes growth and differentiation of epithelial cells and fibroblasts and induces collagen synthesis [53]. Furthermore, modulation of the biological activities of ET-1 by blocking its receptors resulted in inhibition of fibrosis in a rat model of bleomycin-induced pulmonary fibrosis [54]. Similar studies using hypertensive animal models provided evidence of ET-1 as

Razzaque/Azouz/Shinagawa/Taguchi

180

a

b

c

d Fig. 4. Double immunohistochemistry on paraffin sections prepared from kidneys obtained from hypertensive Dahl rats. HSP47 is stained as black, and ␣-smooth muscle actin (a), vimentin (b), desmin (c), and type III collagen (d) are stained as red. Note the coexpression of HSP47 with ␣-smooth muscle actin-positive interstitial cells (arrows) (a), vimentin-positive tubular epithelial cells (arrows) and interstitial cells (open arrows) (b), and desmin-positive glomerular epithelial cells (arrows) (c). Colocalization of increased interstitial accumulation of type III collagen (red) is associated with increased tubular (arrows) and interstitial (open arrows) expression of HSP47 (black) in kidneys obtained from hypertensive Dahl rats (d).

one of the important inducers of collagen synthesis in scarred kidneys [55], and blocking the bioactivities of ET-1 could modulate the process of fibrosis in the kidney. PDGF has been shown to play a role in fibrogenesis, either by inducing mitogenic effects on matrix-producing cells, and/or possibly by regulating a number of pro-fibrotic mediators, such as TNF-␣, TGF-␤1, IL-1 and fibroblast growth factor. Compared to the level of expression of both PDGF and its receptors in normal adult lungs, increased expression was noted in idiopathic pulmonary fibrosis [56]. Furthermore, overexpression of the PDGF-B gene produces histopathological features of fibrosis in experimental animals [57]. In experimental models of hypertension, increased PDGF-A expression correlated with blood pressure and remodeling of large and small arteries [58]. In stroke-prone spontaneously

Hypertensive Nephrosclerosis

181

hypertensive rats, infusion of antisense oligodeoxynucleotides to the PDGF A-chain improved the arterial and renal tissue damage [59]. The imbalance between production of matrix proteins and their inadequate degradation or clearance results in increased deposition of matrix proteins in the involved tissues and organs, resulting in inability to perform their physiological functions. Recent research has focused on the role of various proteolytic enzymes in matrix remodeling during fibrogenesis. For example, increased levels of matrix metalloproteinases (MMPs-1, -2, -8 and -9) and their inhibitory enzymes, tissue inhibitor of metalloproteinases (TIMPs-1 to -4), have been detected in pulmonary fibrosis [60–62]. It is likely that expression of TIMPs could neutralize the collagenolytic activities of MMPs, and the end result is matrix accumulation in diseases associated with pulmonary fibrosis. Likewise, to understand the matrix remodeling in hypertensive nephrosclerosis, it is important to determine the specific numbers of proteolytic enzymes and inhibitors. Recently, elevated levels of TIMP-1 were found to correlate with left ventricular dysfunction and cardiac fibrosis in patients with hypertension [63]. In addition, the plasminogen system was shown to actively contribute to hypertensive nephrosclerosis. Plasminogen activators cleave plasminogen to plasmin, and also activate MMPs. PAI-1 inhibits fibrinolysis and proteolysis by inhibiting activation of t-PA (tissue-type) and u-PA (urokinase-type PA). Recently, a role for PAI-1 has been suggested in human and experimental models of hypertensive nephrosclerosis [64], similar to various other fibrotic diseases, where a role PAI-1 has been demonstrated during matrix remodeling [65].

Conclusion

Despite the etiological differences, once hypertension develops, a series of mechanical, cellular and molecular events in the kidneys eventually contribute to the development of nephrosclerosis, which further exacerbates the hypertension; the resultant effect is irreversible renal failure. It is, to some extent, difficult to estimate if nephrosclerosis in patients with hypertension is a cause or consequence. Understanding the molecular mechanisms of nephrosclerosis, identifying the important and relevant molecules, their interactions, and signaling cascades may provide information that could form the basis for designing future therapeutic strategies to slow down and/or halt the progression of nephrosclerosis in patients with hypertension. In this brief review, we discussed the pathomechanisms of nephrosclerosis. We realize that hypertensive nephrosclerosis is a multistep and multifactorial phenomenon. To that end, we tried to provide basic information of a limited number of essential molecules that could be important in the early and late events of nephrosclerosis.

Razzaque/Azouz/Shinagawa/Taguchi

182

Comprehensive identification of such molecules and factor(s) that regulate the fibrotic process may provide investigators with the tools to identify sitespecific therapies and intervention.

References 1 2 3 4 5

6 7 8 9 10 11

12

13

14

15 16 17

18

19

Klag MJ, Whelton PK, Randall BL, et al: Blood pressure and end-stage renal disease in men. N Engl J Med 1996;334:13–18. US Renal Data System; in USRDS 1991 Annual Data Report. Bethesda, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Disease, 1991. US Renal Data System: Annual Data Report 1994. Incidence and causes of treated ESRD. Am J Kidney Dis 1994;24(suppl 2):48–56. Brunner FP, Selwood NH, on behalf of the EDTA Registration Committee: Profile of patients on RRT in Europe and death rates groups. Kidney Int 1992;42(suppl 38):4–15. Nakai S, et al: The current state of chronic dialysis treatment in Japan. Patient Registration Committee, Japanese Society for Dialysis Therapy, Tokyo, Japan. J Jpn Soc Dial Ther 2002;35: 1155–1184. Luke RG: Essential hypertension: A renal disease. A review and update of the evidence. Hypertension 1993;21:380–390. Dustan HP: Growth factors and racial differences in severity of hypertension and renal diseases. Lancet 1992;339:1339–1340. Teio KK: Angiotensin-converting enzyme genotypes and disease. BMJ 1995;311:76–77. Bloem LJ, Manatunga AK, Tewksbury DA, Pratt JH: The serum angiotensinogen concentration and variants of the angiotensinogen gene in white and black children. J Clin Invest 1995;95:948–953. Rutledge DR, Kubilis P, Browe CS, Ross EA: Polymorphism of the angiotensin I converting enzyme gene in essential hypertensive patients. Biochem Mol Biol Int 1995;35:661–668. Duru K, Farrow S, Wang JM, Lockette W, Kurtz T: Frequency of a deletion polymorphism in the gene for angiotensin-converting enzyme is increased in African-Americans with hypertension. Am J Hypertens 1994;7:759–762. Yoshida H, Mitarai T, Kawamura T, Kitajima T, Kanai T, Miyazaki Y, Kawaguchi Y, Kubo H, Nagasawa R, Ichikawa I, Sakai O: Role of the deletion polymorphism of the angiotensinconverting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. J Clin Invest 1995;96:2162–2169. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F: An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86:1343–1346. Ueda S, Elliott HL, Morton JJ, Connell JMC: Enhanced pressor response to angiotensin I in normotensive men with the deletion genotype (DD) for angiotensin-converting enzyme. Hypertension 1995;25:1266–1269. Takebayashi S, Kiyoshi Y, Hisano S, Uesugi N, Sasatomi Y, Meng J, Sakata N: Benign nephrosclerosis: Incidence, morphology and prognosis. Clin Nephrol 2001;55:349–356. Vaziri ND, Wang XQ, Oveisi F, Rad B: Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 2000;36:142–146. Mizutani K, Ikeda K, Nishikata T, Yamori Y: Phytoestrogens attenuate oxidative DNA damage in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. J Hypertens 2000;18:1833–1840. Sia YT, Lapointe N, Parker TG, Tsoporis JN, Deschepper CF, Calderone A, Pourdjabbar A, Jasmin JF, Sarrazin JF, Liu P, Adam A, Butany J, Rouleau JL: Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat. Circulation 2002;105:2549–2555. Hamaguchi A, Kim S, Ohta K, Yagi K, Yukimura T, Miura K, Fukuda T, Iwao H: Transforming growth factor-␤1 expression and phenotypic modulation in the kidney of hypertensive rats. Hypertension 1995;26:199–207.

Hypertensive Nephrosclerosis

183

20

21

22

23

24

25

26

27 28 29 30 31

32

33

34

35

36 37 38 39 40

Mervaala EM, Muller DN, Park JK, Schmidt F, Lohn M, Breu V, Dragun D, Ganten D, Haller H, Luft FC: Monocyte infiltration and adhesion molecules in a rat model of high human rennin hypertension. Hypertension 1999;33:389–395. Luft FC, Mervaala E, Muller DN, Gross V, Schmidt F, Park JK, Schmitz C, Lippoldt A, Breu V, Dechend R, Dragun D, Schneider W, Ganten D, Haller H: Hypertension-induced end-organ damage: A new transgenic approach to an old problem. Hypertension 1999;33:212–218. Suda T, Osajima A, Tamura M, Kato H, Iwamoto M, Ota T, Kanegae K, Tanaka H, Anai H, Kabashima N, Okazaki M, Nakashima Y: Pressure-induced expression of monocyte chemoattractant protein-1 through activation of MAP kinase. Kidney Int 2000;60:1705–1715. Rovin BH, Wilmer WA, Danne M, Dickerson JA, Dixon CL, Lu L: The mitogen-activated protein kinase p38 is necessary for interleukin-1␤-induced monocyte chemoattractant protein-1 expression by human mesangial cells. Cytokine 1999;11:118–126. Hilgers KF, Hartner A, Porst M, Veelken R, Mann JF: Angiotensin II type 1 receptor blockade prevents lethal malignant hypertension: Relation to kidney inflammation. Circulation 2001;104: 1436–1440. Lan HY, Nikolic-Paterson DJ, Mu W, Atkins RC: Local macrophage proliferation in the progression of glomerular and tubulointerstitial injury in rat anti-GBM glomerulonephritis. Kidney Int 1995;48:753–760. Lan HY, Yang N, Nikolic-Paterson DJ, Yu XQ, Mu W, Isbel NM, Metz CN, Bucala R, Atkins RC: Expression of macrophage migration inhibitory factor in human glomerulonephritis. Kidney Int 2000;57:499–509. Razzaque MS, Foster CS, Ahmed AR: Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2002;43:2977–2983. Border WA, Noble NA: Cytokines in kidney disease: The role of transforming growth factor-␤. Am J Kidney Dis 1993;22:105–113. Barnard JA, Lyons RM, Moses HL: The cell biology of transforming growth factor-␤. Biochim Biophys Acta 1990;1032:79–87. Tamaki K, Okuda S: Role of TGF-␤ in the progression of renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, Vol 139, pp 44–65. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor-␤1. Nature 1990;346: 371–374. Razzaque MS, Harada T, Taguchi T: Significance of increased accumulation of type VI collagen and transforming growth factor-␤1 in tubulointerstitial damage in hypertensive nephrosclerosis: An immunohistochemical study. J Int Med Res 1996;24:199–208. Razzaque MS, Cheng M, Horita Y, Nishihara M, Harada T, Taguchi T: Immunohistochemical analysis of type III and IV collagens in tubulointerstitial damage in human benign nephrosclerosis. J Int Med Res 1995;23:480–486. Razzaque MS, Koji T, Kawano H, Harada T, Nakane PK, Taguchi T: Glomerular expression of type III and type IV collagens in benign nephrosclerosis: Immunohistochemical and in situ hybridization study. Pathol Res Pract 1994;190:493–499. Derhaschnig U, Shehata M, Herkner H, Bur A, Woisetschlager C, Laggner AN, Hirschl MM: Increased levels of transforming growth factor-␤1 in essential hypertension. Am J Hypertens 2002;15:207–211. Dahly AJ, Hoagland KM, Flasch AK, Jha S, Ledbetter SR, Roman RJ: Antihypertensive effects of chronic anti-TGF-␤ antibody therapy in Dahl S rats. Am J Physiol 2002;283:R757–R767. Laviades C, Varo N, Diez J: Transforming growth factor-␤ in hypertensives with cardiorenal damage. Hypertension 2000;36:517–522. Cerwenka A, Swain SL: TGF-␤1: Immunosuppressant and viability factor for T lymphocytes. Microbes Infect 1999;1:1291–1296. Reinhold D, Wrenger S, Kahne T, Ansorge S: HIV-1 Tat: Immunosuppression via TGF-␤1 induction. Immunol Today 1999;20:384–385. Yokoi H, Mukoyama M, Sugawara A, Mori K, Nagae T, Makino H, Suganami T, Yahata K, Fujinaga Y, Tanaka I, Nakao K: Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol 2002;282:F933–F942.

Razzaque/Azouz/Shinagawa/Taguchi

184

41

42

43 44 45

46

47 48

49

50

51

52

53

54

55

56 57

58 59

Razzaque MS, Foster CS, Ahmed AR: Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003; 44:1998–2003. Finckenberg P, Lassila M, Inkinen K, Pere AK, Krogerus L, Lindgren L, Mervaala E, Vapaatalo H, Nurminen ML, Ahonen J: Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet. Transplantation 2001;71:951–958. Razzaque MS, Taguchi T: Collagen-binding heat-shock protein 47 expression in anti-thymocyte serum-induced glomerulonephritis. J Pathol 1997;183:24–29. Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heatshock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183–1188. Razzaque MS, Ahmed AR: Collagens, collagen-binding heat-shock protein 47 and transforming growth factor-␤1 are induced in cicatricial pemphigoid: Possible role(s) in dermal fibrosis. Cytokine 2002;17:311–316. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat-shock protein 47. Cell Tissue Res 1998;293:471–478. Razzaque MS, Foster CS, Ahmed AR: Role of heat-shock protein 47 and TGF-␤1 in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:1616–1621. Razzaque MS, Hossain MA, Kohno S, Taguchi T: Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat-shock protein 47. Virchows Arch 1998;432:455–460. Razzaque MS, Kumatori A, Harada T, Taguchi T: Coexpression of collagens and collagen-binding heat-shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80:434–443. Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14: 1199–1212. Tikkanen I, Uhlenius N, Tikkanen T, Miettinen A, Tornroth T, Fyhrquist F, Holthofer H: Increased renal expression of cytokines and growth factors induced by DOCA-NaCl treatment in Heymann nephritis. Nephrol Dial Transplant 1995;10:2192–2198. Shindo T, Kurihara H, Maemura K, Kurihara Y, Ueda O, Suzuki H, Kuwaki T, Ju KH, Wang Y, Ebihara A, Nishimatsu H, Moriyama N, Fukuda M, Akimoto Y, Hirano H, Morita H, Kumada M, Yazaki Y, Nagai R, Kimura K: Renal damage and salt-dependent hypertension in aged transgenic mice overexpressing endothelin-1. J Mol Med 2002;80:105–116. Saleh D, Furukawa K, Tsao MS, Maghazachi A, Corrin B, Yanagisawa M, Barnes PJ, Giaid A: Elevated expression of endothelin-1 and endothelin-converting enzyme-1 in idiopathic pulmonary fibrosis: Possible involvement of proinflammatory cytokines. Am J Respir Cell Mol Biol 1997;16:187–193. Park SH, Saleh D, Giaid A, Michel RP: Increased endothelin-1 in bleomycin-induced pulmonary fibrosis and the effect of an endothelin receptor antagonist. Am J Respir Crit Care Med 1997;156: 600–608. Tharaux PL, Chatziantoniou C, Casellas D, Fouassier L, Ardaillou R, Dussaule JC: Vascular endothelin-1 gene expression and synthesis and effect on renal type I collagen synthesis and nephroangiosclerosis during nitric oxide synthase inhibition in rats. Circulation 1999;99:2185–2191. Antoniades HN, Bravo MA, Avila RE, Galanopoulos T, Neville-Golden J, Maxwell M, Selman M: Platelet-derived growth factor in idiopathic pulmonary fibrosis. J Clin Invest 1990;86:1055–1064. Yoshida M, Sakuma J, Hayashi S, Kinya A, Saito I, Harada S, Sakatani M, Yamamoto S, Matsumoto N, Kaneda Y, Kishimoto T: A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin-6, transforming growth factor-␤1, or platelet-derived growth factor B gene. Proc Natl Acad Sci USA 1995;92:9570–9574. Dobrian A, Wade SS, Prewitt RL: PDGF-A expression correlates with blood pressure and remodeling in 1K1C hypertensive rat arteries. Am J Physiol 1999;276:H2159–H2167. Kishioka H, Fukuda N, Wen-Yang H, Nakayama M, Watanabe Y, Kanmatsuse K: Effects of PDGF A-chain antisense oligodeoxynucleotides on growth of cardiovascular organs in strokeprone spontaneously hypertensive rats. Am J Hypertens 2001;14:439–445.

Hypertensive Nephrosclerosis

185

60

61

62

63 64 65

Yaguchi T, Fukuda Y, Ishizaki M, Yamanaka N: Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis. Pathol Int 1998; 48:954–963. Madtes DK, Elston AL, Kaback LA, Clark JG: Selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2001; 24:599–607. Selman M, Ruiz V, Cabrera S, Segura L, Ramirez R, Barrios R, Pardo A: TIMP-1, -2, -3 and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? Am J Physiol 2000;279:L562–L574. Lindsay MM, Maxwell P, Dunn FG: TIMP-1: A marker of left ventricular diastolic dysfunction and fibrosis in hypertension. Hypertension 2002;40:136–141. Fogo AB: Mechanisms in nephrosclerosis and hypertension-beyond hemodynamics. J Nephrol 2001;4:S63–S69. Loskutoff DJ, Quigley JP: PAI-1, fibrosis and the elusive provisional fibrin matrix. J Clin Invest 2000;106:1441–1443.

Mohammed S. Razzaque, MBBS, PhD, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (USA) Fax ⫹1 425 732 1965, E-Mail [email protected]

Razzaque/Azouz/Shinagawa/Taguchi

186

Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger, 2003, vol 139, pp 187–204

Pathogenesis and Molecular Mechanisms of Chronic Allograft Nephropathy Nasimul Ahsana, Joseph Y. Cheungb a

University of Medicine and Dentistry of New Jersey – Robert Wood Johnson Medical School, New Brunswick, N.J. and bWeis Center for Research, Geisinger Medical Center, Danville, Pa., USA

Introduction

Today, renal transplantation is the treatment of choice for most patients with end-stage renal disease. Over the last two decades, a better understanding of the transplant immunology, tissue typing, organ preservation, and introduction of more potent immunotherapies has resulted in a steady improvement in the shortterm outcome of renal allografts. During a 13-year period (1988–2000), according to the Registry of the United Network of Organ Sharing, the 1-year renal allograft survival rates have improved to 89% for cadaveric and 94% for living related transplants. During the same period of time, the half-life of cadaveric renal allograft has improved from 7.8 to 11.6 years. From the same registry, it has been reported that approximately 44% of grafts will develop progressive dysfunction and will fail within a decade necessitating renal replacement therapy [1]. The single most common cause of long-term allograft failure is chronic allograft nephropathy (CAN). Both immune and non-immune factors are involved in its pathogenesis and several risk factors including donor age, ischemia-reperfusion injury, delayed graft function, and episodes of acute rejections have been implicated in its pathogenesis. In addition to proteinuria and hypertension, CAN is characterized by a slow but variable loss of renal allograft function. The histopathologic findings of CAN include interstitial fibrosis, extracellular matrix (ECM) accumulation, tubular atrophy, and glomerular and

Table 1. Different phases of CAN: induction, matrix synthesis and post-inflammatory phase. Following numerous insults sustained during transplantation, the allograft kidney responds by initiating repair mechanisms that lead to an infiltration of macrophages, proliferation of fibroblasts, and deposition of ECM A. Induction phase Chemokine release by tubular epithelial cells Mononuclear cell infiltration Release of pro-fibrogenetic cytokines Activation and proliferation of fibroblasts B. Matrix synthesis Matrix synthesis and deposition Ongoing release of pro-fibrogenetic cytokines C. Post-inflammatory phase Cessation of inflammatory stimulus More secretion of cytokines Proliferation of myofibroblasts Epithelial-mesenchymal transformation

vascular lesions. Recent studies suggest that secretion of cytokines (e.g., tumor necrosis factor- (TNF-)) and growth factors (e.g., transforming growth factor- (TGF-)), and up-regulation of the tissue inhibitors of matrix metalloproteinase (TIMP) are associated in the fibrogenetic process. In this section, the molecular mechanisms of CAN are reviewed and current hypotheses regarding its pathogenesis are highlighted. Response to Injury

In response to numerous insults received during transplantation, the allograft kidney responds by initiating repair mechanisms that lead to an infiltration of macrophages, proliferation of fibroblasts, and deposition of ECM (table 1). The origin of interstitial fibroblasts remains unclear but studies have suggested that fibroblast or fibroblast-like cells originate from resident cells in the interstitium. Under the stimulation of cytokines and growth factors, fibroblasts become myofibroblasts. Myofibroblasts, expressing -smooth muscle actin (-SMA) becomes the main cellular component of the interstitium [2–4]. Up-regulation of tubular collagen IV and TGF--stimulated synthesis of proteoglycan and fibronectin have also been demonstrated. In addition, tubular epithelial cells undergo phenotypic changes in response to pro-fibrotic cytokines and growth factors and play a role in the production of ECM [5–7].

Ahsan/Cheung

188

Clinical Features of CAN

CAN is characterized by a relatively slow but progressive deterioration in renal allograft function, which clinically presents in one of two ways, as a finding in patients undergoing biopsy either (i) for an acute rise in serum creatinine or proteinuria, or (ii) for gradually declining renal function or proteinuria. In the majority (80%) of these patients with histological evidence of CAN, a linear relationship usually exists between the time of diagnosis of CAN and the deterioration of renal allograft function [8]. CAN is frequently associated with de novo or accelerated hypertension and is the most common cause for posttransplant nephrotic syndrome [9, 10]. These clinical manifestations however, are non-specific, and other causes of graft dysfunction such as acute rejection, drug toxicity and recurrent diseases must be excluded prior to making the diagnosis of CAN. Pathologic Findings

In 1953, David Humes and co-workers described the pathologic findings from a patient with a successful cadaveric renal transplantation [11]: The tubular degeneration, casts, focal cellular infiltration, and interstitial edema were felt to be consistent with the picture of ischemic nephrosis. There were striking changes in the intrinsic blood vessels. A severe degree of arteriosclerosis had developed. There was marked thickening of the intima with narrowing of the lumen, in some vessels almost to the point of occlusion. The inner portion of the intima showed dense sclerosis, and the outer portion, just within the inner elastic lamina, contained numerous lipid-laden macrophages.

Similar to the description above, the histopathology of CAN consists of atherosclerosis, glomerular lesions and glomerulosclerosis, interstitial fibrosis, and tubular atrophy. Graft atherosclerosis consists of concentric intimal hyperplasia resulting from the migration of myofibroblasts from media and is accompanied by infiltration of the vessel wall with mononuclear cells and T lymphocytes [12–14]. The underlying pathological process however, is an excessive fibrosis due to enhanced accumulation of ECM proteins. The degree of fibrosis seems to provide the best morphological correlation with renal function and prognosis. The glomerular lesions of CAN include wrinkling and collapse of the glomerular tuft, glomerular hypertrophy, mesangial matrix expansion, and focal glomerulosclerosis [15, 16] (fig. 1a). Immunofluorescent studies show non-specific linear immunoglobulin G (IgG) deposits along the glomerular basement membrane or granular deposits of IgG or IgA in peripheral capillary loops [17] (fig. 1b). Ultrastructural studies demonstrate multilayering of the peritubular capillary basement membranes [18] (fig. 1c). Distributed in the glomeruli, the vessel wall

Pathogenesis and Molecular Mechanisms of CAN

189

a

b

c Fig 1. a–c Histopathological findings of CAN. a Glomerular lesions of CAN showing wrinkling and collapse of the glomerular tuft, glomerular hypertrophy, mesangial matrix expansion and focal glomerulosclerosis (hematoxylin and eosin stain). b Immunofluorescence study showing non-specific linear IgG deposits along the glomerular basement membrane and granular deposits of IgG in peripheral capillary loops. c Ultrastructural study prepared from a biopsy with CAN showing multilayering of the peritubular capillary basement membranes. No immune complex deposits are seen.

and the interstitium are various ECM proteins consisting of collagen type IV, laminin and heparan sulfate, as well as decorin and collagen type III [19].

Risk Factors

In the development of CAN, both immune and alloantigen-dependent, and nonimmune or alloantigen-independent factors are involved (table 2). Alloantigen-Dependent Several epidemiological studies have demonstrated that there is a strong association between acute rejection and CAN. An acute rejection episode that occurs late (after the first 3 months post-transplant) is more predictive of CAN

Ahsan/Cheung

190

Table 2. Risk factors associated with CAN. In the development of CAN, both immune (alloantigen-dependent) and nonimmune (alloantigen-independent) factors are involved

A. Alloantigen-dependent factors Acute rejection Histocompatibility mismatch Prior sensitization Suboptimal immunosuppression Medical non-compliance B. Alloantigen-independent factors Ischemic injury and delayed graft function Older donor age Donor and recipient size mismatch Calcineurin inhibitor nephrotoxicity Hyperlipidemia Hypertension Cigarette smoking Hyperhomocystinemia Oxygen free radicals Proteinuria

than those that occur during the first 3 months [20]. In addition, multiple acute rejections and severity of acute rejection, either by histological grade or by the degree of increase of serum creatinine, are strong predictors for development of CAN [21]. Unfortunately, while introduction of cyclosporine led to dramatic (30%) reductions in the rate of early acute rejection, the nephrotoxicity of cyclosporine can theoretically cancel any benefit that a reduced incidence of acute rejection from cyclosporine may have on CAN. The contribution of calcineurin inhibitors to development of CAN, however, remains controversial. Mismatch of major histocompatibility complex (MHC) between recipient and donor is also associated with CAN and late allograft failure [22]. Clearly, cadaveric kidney transplants that have zero MHC mismatches have the best long-term allograft survival. An association has also been found between the preformed antibody loads present in recipients at the time of transplantation and subsequent CAN [23]. Usually measured as the percentage of panel reactive antibodies (PRA), recipients with high PRA are more likely to develop CAN and poor long-term allograft survival [24]. Several studies have shown that poor adherence to medications increases the likelihood of late graft failure, presumably from CAN, suggesting the hypothesis that CAN is mediated by alloantigen-dependent factors. These patients are likely to be non-compliant with other medications, e.g. anti-hypertensive agents (an alloantigenic-independent factor), and follow-up visits hindering early diagnosis and treatment of allograft dysfunction.

Pathogenesis and Molecular Mechanisms of CAN

191

Alloantigen-Independent A risk factor analysis of CAN has showed that patients with prolonged ischemic exposure and delayed graft function (DGF) have a higher rate of late allograft failure. In these patients, serum creatinine at the time of discharge has been shown to be determinants of late graft loss [25]. It has been hypothesized that both ischemic insult and DGF result in a reduced number of functioning nephrons leading to CAN and late graft failure. Others have demonstrated a strong effect of donor age on long-term outcome and graft half-life [26]. In these circumstances, a relative deficiency in the number of nephrons may create a physiologic response that ultimately leads to graft failure. The nephrotoxicity of calcineurin inhibitors (CI), e.g. cyclosporine, tacrolimus has been well described. Many of the histological changes of chronic CI-induced nephrotoxicity resemble of those of CAN. The extent of interstitial fibrosis which is a feature of CAN and CI toxicity has been correlated to the expression of TGF- mRNA [27–30]. Multivariate analyses have found that recipients with hypercholesterolemia are at increased risk for developing CAN. Graft biopsies from patients with elevated cholesterol levels frequently display more severe chronic damage and fibrointimal hyperplasia of the arteries resembling accelerated atherosclerosis [31, 32]. Recipient hypertension and cigarette smoking are additional risk factors that have negative effects on graft vasculopathy and may contribute to CAN when compared with non-smokers and patients with a normal blood pressure. Compared to control, transplant recipients have been found to increased levels of plasma homocysteine [33]. Although homocysteine may be injurious to vascular endothelial cells, there is no data suggesting that reducing homocysteine improves graft function. Similar to the pathogenesis of systemic atherosclerosis and interstitial inflammation, oxygen free radicals could also play a theoretical role in vasculopathy and fibrosis of CAN [34]. Most epidemiological studies suggest that about two-thirds of the patients with persistent proteinuria will have CAN on biopsy [35] and a number of studies have identified proteinuria as an important risk factor for graft loss to CAN. Proteinuria has been shown to cause interstitial nephritis in experimental animals. In humans, the degree of proteinuria predicts progression of renal disease [36]. Thus it is possible that proteinuria could cause tubulointerstitial damage and contribute to renal injury in CAN.

Pathogenesis

The pathologic hallmark of CAN is chronic allograft fibrosis. In the physiological state a balance exists between ECM synthesis and degradation, which

Ahsan/Cheung

192

in the case of renal fibrosis is shifted towards increased matrix synthesis and decreased degradation. Renal injury results in functional impairment and structural damage, followed by an inflammatory response consisting of an influx of mononuclear cells, proliferation of tissue fibroblasts, deposition of ECM protein, scar formation, and ultimately tissue restoration. Whereas ischemic damage and acute rejection episodes resolve more or less completely in most instances, irreversible fibrosis sometimes ensues. A host of mediators, including pro-inflammatory cytokines, enzymes and growth factors such as TGF-, platelet-derived growth factor (PDGF), interleukin (IL)-1, TNF-, angiotensin II (ANG-II) and endothelin, is involved in various stages of the inflammatory and tissue restoration responses [37]. Interstitial Fibroblasts Interstitial fibroblasts are still believed to be the main effector cells in renal fibrogenesis. These cells become activated by certain cytokines such as TGF-, PDGF and fibroblast growth factor type 2 (FGF-2). The later is a strong mitogen of renal fibroblasts and increased up-regulation of FGF-2 has been recognized in biopsies from human kidneys with chronic disease [37, 38]. These cytokines are localized mainly in the interstitial fibroblast and play important role in fibroblast stimulation and formation of ECM (fig. 2). Extracellular Matrix ECM proteins are divided into collagen or non-collagenous classes. In the kidney, the most important collagen proteins are types I and III, which are fibrillar proteins, and type IV, which is a non-fibrillar protein. The tubulointerstitium contains all three of these collagens, but the glomeruli contain only type III and IV collagen. Non-collagenous matrix proteins include the glycoproteins: laminin, fibronectin and tenascin, and the proteoglycans: decorin and biglycan [39]. Recent studies have shown that there exists an association between increased glycoproteins and the overexpression of numerous cytokines. In pathological states an increase in synthesis, a decrease in breakdown, or a combination of both results in an accumulation of matrix, leading to fibrosis. Four families of proteolytic enzymes are involved in the degradation of matrix proteins of which, the plasminogen activation system and the matrix metalloproteinase (MMPs) appear to be the most important. Plasminogen Activation System The plasminogen/plasmin system was first recognized for its fibrindegrading activity. The plasminogen activator inhibitor type 1 (PAI-1), the main inhibitor of plasminogen activation, regulates fibrinolysis and plasminmediated MMP activation. PAI-1 is also a component of the ECM. PAI-1 is not

Pathogenesis and Molecular Mechanisms of CAN

193

Glomerulus

T lymphocytes

TGF- PDGF

Macrophages

Tubular epithelial cells

TGF- PDGF

TGF- PDGF

TGF- PDGF

FGF-2, IL-1 Fibroblasts

ECM synthesis

Proliferation

Fig 2. Inflammatory and post-inflammatory phases of fibrogenesis. Interstitial fibroblasts are believed to be the main effector cells in renal fibrogenesis. These cells become activated by certain cytokines such as TGF-, PDGF and FGF-2. The latter is a strong mitogen of renal fibroblasts and increased up-regulation of FGF-2 has been recognized in biopsies from human kidneys with chronic disease. These cytokines are localized mainly in the interstitial fibroblast and play important role in fibroblast stimulation and formation of ECM.

expressed in the normal human kidney but is strongly induced in various forms for inflammatory processes, leading to renal fibrosis [40, 41]. Thrombin, ANG-II and TGF- are potent in vitro and in vivo agonists in increasing PAI-1 synthesis [42, 43] (fig. 3). Matrix Metalloproteinase The MMPs and their inhibitors (TIMP-1, TIMP-2 and TIMP-3) exist in a number of different metalloproteinases. These contain several conserved motifs and a zinc-binding site, which is required for full enzymatic activation. In health, the activity of MMPs is strictly controlled to maintain normal tissue integrity. This control occurs at three levels: transcription, extracellular activation and inhibition [44, 45]. At least 20 MMPs have now been identified. With the exception of MMP-9, MMPs are produced constitutively in physiological states and the majority of them are secreted in an inactive form because of the binding of an unpaired cysteine to zinc at the active site. Disruption of the cysteine-zinc bond either by a conformational change or by a limited proteolysis,

Ahsan/Cheung

194

TGF- – ANG-II

Soluble or bound PAI-1

u-PA activity

MMP activation

Plasmin formation ECM degradation Fibrinolysis

Fibroblast invasion

ECM protein accumulation

Myofibroblast formation

Collagen deposition

Fibrosis

Fig 3. Role of PAI-1 in fibrogenesis and CAN. The plasminogen activator inhibitor type 1 (PAI-1), the main inhibitor of plasminogen activation, regulates fibrinolysis and plasmin-mediated MMP activation. PAI-1 is also a component of the ECM. PAI-1 is strongly induced in various forms for inflammatory kidney diseases, leading to renal fibrosis.

e.g. by plasmin, leads to the turning on of the switch. In diseased conditions in vivo, human mesangial cells express PAI-1, and this up-regulation may be induced by pro-fibrotic cytokines such as TNF- and IL-1, and growth factors such as TGF- and PDGF [46, 47]. Once activated, members of the MMP family can activate other MMPs [48]. MMPs are inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). TIMPs form a 1:1 stoichiometric complex with MMPs, inhibiting both the latent and activated enzyme [49, 50]. Heat-Shock Protein-47 (HSP-47) and a-SMA HSP-47 is a 47-kDa heat-shock protein, first described as a collagen-binding stress protein and acts like a molecular chaperone during the processing and secretion of procollagens [51]. Several studies have shown that expression of

Pathogenesis and Molecular Mechanisms of CAN

195

HSP is closely related to collagen synthesis. In their study with renal tissue from CAN, Abe et al. [52] demonstrated localization of HSP-47 in interstitial fibroblasts and renal tubular epithelial cells. In addition, the majority of HSP-47-positive cells in the interstitium were also positive for -SMA. -SMA is regarded as a marker for activated fibroblasts (myofibroblasts) and it appeared that the transformation of fibroblasts into myofibroblasts is a key step in the increased collagen synthesis in CAN [53]. Apoptosis Apoptosis is an important regulatory process that controls cell numbers during physiologic and various pathological conditions. Regulation of apoptotic mechanism is complex and involves pro- and anti-apoptotic factors. Upon activation by their ligands (FasL), members of the TNF superfamily receptors including Fas, initiate an intracellular cascade resulting in cleavage of key proteins, activation of caspases, and eventual apoptotic cell death [54, 55]. Proinflammatory cytokines produced during tissue injury, e.g., acute rejection and ischemia reperfusion (IR) injury can stimulate Fas expression in renal tubular cells [56]. The expression of such a receptor together with FasL expression in neighboring tubular cells may induce a cycle of apoptosis that may result in tubular atrophy and interstitial fibrosis seen in CAN [57, 58]. Pro-Fibrotic Stimulus Numerous cytokines, adhesion molecules and growth hormones with fibrogenetic potentials are released into the circulation in response to injuries sustained during or after renal transplantation (table 3). Of these, TGF- and ANG-II appear to be the most potent pro-fibrotic mediators and have been the most extensively studied. Transforming Growth Factor TGF- is the most potent pro-fibrotic cytokine. It exists structurally as a holodimer, with each monomer having a molecular mass of 12.5 kDa. A deficiency of TGF- is incompatible with life. There are three isoforms of TGF-, which are released as inactive precursors-complex bound with latency-associated peptides [59, 60]. Various factors can activate TGF-, which may be inhibited by many local factors. TGF- also stimulates and inhibits cell growth and proliferation and serves as a regulatory molecule, restoring balance after deviations from normal [61, 62]. There is a vast body of evidence supporting the role of TGF- in renal fibrogenesis and increased renal allograft expression of TGF- occurs in clinical settings. Both in vitro and in vivo models have been used to demonstrate the fibrogenic consequences of prolonged TGF- stimulation [63–65]. For example,

Ahsan/Cheung

196

Table 3. Pro-fibrotic signals playing critical roles in CAN. Numerous cytokines, adhesion molecules and growth hormones with fibrogenetic potentials are released into the circulation in response to injuries sustained during or after renal transplantation. Of these, TGF- and ANG-II appear to be the most potent pro-fibrotic mediators Mediators

Functions

TNF- Interleukin-1

Stimulates endothelial cell production of adhesion molecules Up-regulates adhesion molecules on endothelial cells; induces smooth muscle cells, fibroblasts and PDGF Mitogen for smooth muscle cells and mesenchymes Mitogen and smooth muscle chemoattractant Vascular smooth muscle chemoattractant Stimulates ECM production Increases ECM deposition via TGF- Stimulates proliferation of renal fibroblasts

Interleukin-6 PDGF FGF-2 TGF- ANG-II Endothelin-1

TNF- Tumor necrosis factor-; PDGF platelet derived growth factor; FGF-2 fibroblast growth factor-2; TGF- transforming growth factor-; ANG-II angiotensinogen II; ECM extracellular matrix.

intravenous administration of recombinant TGF- produces rapid glomerulosclerosis in rats and rabbits [66]. Recent studies have demonstrated that TGF- may also induce proliferation by induction of the mitogens, namely connective tissue growth factor (CTGF) or basic FGF-2. CTGF contributes to renal fibrogenesis via control of cAMP levels [67] and TGF- up-regulates the expression of FGF-2 in renal interstitial fibroblasts [68, 69]. Several other properties of TGF- suggest that it may have a prominent role in allograft fibrosis: (1) stimulation of the production of ECM proteins; (2) promotion of the production of protease inhibitors, such as PAI-1; (3) up-regulation of integrin expression, and (4) reduction in the activity of matrixdegrading proteins [70]. Risk Factors for CAN and Role of TGF-b (fig. 4) Acute and Chronic Rejection There are several immune and non-immune mediated risk factors for development of CAN (see above) all of which can influence the expression of TGF-, supporting its role in the development of CAN. In a rat model using a strain combination in which acute rejection developed almost immediately, Nagano et al. [71] demonstrated a significant rise in TGF- mRNA levels in the kidney. In a mouse model of renal transplantation that develops changes similar to CAN, TGF- expression was found to be increased by both immunohistochemistry

Pathogenesis and Molecular Mechanisms of CAN

197

Ischemia Reperfusion

Rejection

Drugs

Allograft injury/repair

TGF-

Cytokines e.g. PDGF

Cellular proliferation

Endothelin-1

ECM/fibrosis

Hypertension

CAN

Fig 4. Risk factors for TGF- formation and pathogenesis of CAN. TGF- is the most potent pro-fibrogenetic cytokine. Increased renal allograft expression of TGF- occurs in various clinical settings including rejection, ischemia-reperfusion and drug toxicity. TGF- may also induce cellular proliferation by induction of the mitogens, namely CTGF or basic FGF-2.

and Northern blotting [72]. Another group also used Northern blotting to measure TGF- mRNA in the F344-to-Lewis rat kidney transplant model and showed that during the time points between 4 and 42 weeks after transplantation, TGF- levels were significantly elevated compared to isograft controls [73]. This was associated with marked fibrosis suggesting that TGF- has a causative role in ECM expansion that occurs in CAN. IR Injury Injury caused during IR involves activation of endothelial cells and the release of several inflammatory mediators including oxygen free radicals [74]. The latter is strong stimulus for release and activation of TGF-. This has been shown by studies that demonstrate the up-regulation of TGF- mRNA and protein levels in rats undergoing transient renal pedicle clamping [75]. In the long term, rats with renal IR injury develop marked fibrosis similar to that found in CAN, which is associated with persistent TGF- up-regulation [76]. Drug Toxicity Long-term administration of the cyclosporine and tacrolimus is associated with histological changes similar to those of CAN [77]. In addition, these drugs

Ahsan/Cheung

198

also induce hyperlipidemia, hypertension and diabetes; all of these are established risk factors for CAN. Of these two drugs, recent studies have suggested that cyclosporine induces greater degree of TGF- production. When compared in a randomized trial, in the cyclosporine group, there was a significant increase in the incidence and severity of acute rejection episodes and enhanced expression of TGF- in the biopsy specimens [78]. In rodent model of IR injury, Jain et al. [79] showed significantly more proteinuria and higher serum creatinine levels in animals treated with cyclosporine. Both TGF- and TIMP-1 expression were significantly increased in the cyclosporine-treated group. Finally, in protocol biopsy specimens from clinical trials, which compared tacrolimus and cyclosporine, a significantly higher level of collagen III and TIMP-1 expression was also seen in cyclosporine arm [80]. Long-term outcome of the differential expressions of TGF-, collagen III and TIMP-1 in patients treated with these agents remains to be seen. The data from all these studies are thus suggestive of the notion that a significant correlation exists between the expression of TGF- and interstitial fibrosis and CAN.

Angiotensin II

Intraglomerular hypertension may result from an increase in systemic pressure associated with acute rejection episodes and chronic cyclosporine nephrotoxicity and can lead to early allograft failure [81]. Beyond its role in the regulation of glomerular hemodynamics, the intrarenal renin-angiotensin system (RAS) has been recognized as a growth promotor [82–84]. In vitro data indicates that ANG-II increases synthesis and decreases degradation of pathological ECM, which is independent of blood pressure [85, 86]. It has been reported that the addition of ANG-II to cultured vascular smooth muscle cells leads to an increased secretion of TGF- and chronic angiotensin infusion in rats results in interstitial fibrosis. In many models of renal fibrosis and in a number of human renal diseases, blockade of ANG-II retards disease progression. Evidence is emerging that the renin-angiotensin system may mediate chronic cyclosporine nephrotoxicity [87]. Using a salt-depleted rat model of chronic cyclosporine nephrotoxicity, Shihab et al. [88] showed that cyclosporine-induced interstitial fibrosis could be reversed by angiotensin-converting enzymes inhibitor (ACE-I) enalapril or the ANG-II type I receptor (AT1) blocker losartan. Finally, angiotensin has been shown to induce PAI-1, which is also elevated in fibrotic disease processes [89, 90] and a number of laboratories have recently shown that ANG-II added to cultured cells causes marked increases in PAI-1 resulting in the accumulation of ECM proteins [91, 92].

Pathogenesis and Molecular Mechanisms of CAN

199

Conclusion

CAN remains the leading cause of graft failure after the first year following transplantation. Both alloantigen-dependent and alloantigen-independent factors are involved in the initiation and progression of CAN. Histologically, fibrosis due to increased production of ECM is the hallmark of CAN. Many pro-inflammatory cytokines are up-regulated during the process: current evidence strongly implicates TGF- to be the major pro-fibrogenetic stimulus. At present no effective therapy is available to inhibit or reverse the process of CAN. However, a better understanding of the molecular mechanisms involved offers a number of potential new strategies. Partial inhibition of TIMP-1 and TGF- may prove effective in reducing fibrosis and prolonging allograft survival. Larger randomized studies are required to assess whether or not ACE-I and AT1 antagonist therapies exert beneficial effects after kidney transplantation.

References 1 2 3 4 5 6 7 8

9 10 11

12 13 14

Cecka JM: The UNOS Renal Transplant Registry; in Cecka, Terasaki (eds): Clinical Transplants 2001. Los Angeles, UCLA Immunogenetics Center, 2001, pp 1–18. Weber KT: Fibrosis, a common pathway to organ failure: Angiotensin II and tissue repair. Semin Nephrol 1997;17:467–491. Paul LC: Chronic allograft nephropathy – A model of impaired repair from injury? Nephrol Dial Transplant 2000;15:149–151. Strutz F: Novel aspects of renal fibrogenesis. Nephrol Dial Transplant 1995;10:1526–1532. Abo-Zenah H, Katsoudas S, Wild G, De Takats D, Shortland J, Brown CB, El Nahas AM: Early human renal allograft fibrosis: Cellular mediators. Nephron 2002;91:112–119. Waller JR, Nicholson ML: Molecular mechanisms of renal allograft fibrosis.Br J Surg 2001;88:1429–1441. Jain S, Furness PN, Nicholson ML: The role of transforming growth factor- in chronic renal allograft nephropathy. Transplantation 2000;69:1759–1766. Riggio RR, Haschemeyer R, Suthanthiran M, Cheigh J, Tapia L, Stubenbord W, Miller I, Stenzel KH: Predictability of renal allograft failure time in long-term survivors: A hypothesis. Transplant Proc 1985;17:2311–2313. Massy ZA, Guijarro C, Weiderkehr MR, Ma JZ, Kasiske BL: Chronic renal allograft rejection: Immunologic and nonimmunologic risk factors. Kidney Int 1996;49:518–524. Raine AEG: Does antihypertensive therapy modify chronic allograft failure? Kidney Int 1995; 48(suppl):107–111. Kassiske BL: Long-term post-transplantation management and complications; in Danovitch GM (ed): Handbook of Kidney Transplantation. Philadelphia, Lippincott Williams & Wilkins, 2001, pp 182–220. Kasiske BL, Kabil RSN, Lee HS, Rao KV: Histopathological findings associated with a chronic, progressive decline in renal allograft function. Kidney Int 1991;80:514–524. Sanfilippo F: Renal transplantation; in Sale GE (ed): The Pathology of Organ Transplantation. Boston, Butterworths, 1990, pp 51–101. Mason DW, Morris PJ: Effector mechanisms in allograft rejection. Annu Rev Immunol 1986;4: 119–145.

Ahsan/Cheung

200

15

16 17 18

19

20 21 22 23 24 25 26

27 28

29

30

31

32 33 34 35 36 37

Barrientos A, Portoles J, Herrero JA, Torralbo A, Prats D, Gutierrez-Millet V, Blanco J: Glomerular hyperfiltration as a nonimmunologic mechanism of progression of chronic renal rejection. Transplantation 1994;57:753–756. Cheigh JS, Mouradian J, Soliman M, Tapia L, Riggio RR, Stenzel KH, Rubin AL: Focal segmental glomerulosclerosis in renal transplants. Am J Kidney Dis 1983;2:449–455. Habib R, Antigua C, Hinglais N, Gagnadoux MF, Broyer M: Glomerular lesions in the transplanted kidney in children. Am J Kidney Dis 1987;10:198–207. Monga G, Mazzucco G, Messina M, Motta M, Quaranta S, Novara R: Intertubular capillary changes in kidney allografts: A morphologic investigation on 61 renal specimens. Mod Pathol 1992;5:125–130. Vleming LJ, Baelde JJ, Westendorp RGJ, Daha MR, van Es LA, Bruijn JA: Progression of chronic renal disease in humans is associated with the deposition of basement membrane components and decorin in the interstitial extracellular matrix. Clin Nephrol 1995;44:211–219. Matas AJ, Gillingham KJ, Payne WD, Najarian JS: The impact of an acute rejection episode on long-term renal allograft survival (t1/2). Transplantation 1994;57:857–859. Almond PS, Matas A, Gillingham KJ, Dunn DL, Payne WD, Gores P, Gruessner R, Najarian JS: Risk factors for chronic rejection in renal allograft recipients. Transplantation 1993;55:752–757. Takemoto S, Terasaki PI, Cecka JM, Cho YM, Gjertson DW: Survival of nationally shared HLAmatched kidney transplants from cadaveric donors. N Engl J Med 1992;327:834–939. Tullius SG, Tinley NL: Both alloantigen-dependent and -independent factors influence chronic allograft rejection. Transplantation 1995;59:313–318. Morris PJ: Factors influencing outcome in cadaveric renal transplantation. Br J Surg 1994;81: 635–636. Shoskes D, Cecka M: Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation 1998;55:1697–1701. Alexander JW, Bennet LE, Beren TJ: Effects of donor age on outcome of kidney transplantation: A two-year analysis of transplants reported to the United Network for Organ Sharing Registry. Transplantation 1994;57:1491–1499. Burke JF, Pirsch JD, Ramos EL, Salomon DR, Stablein DM, Van Buren DH, West JC: Long-term efficacy and safety of cyclosporine in renal-transplant patients. N Engl J Med 1994;331:358–363. Randhawa PS, Tsamandas AC, Magnone M, Jordan M, Shapiro R, Starzl TE, Demetris AJ: Microvascular changes in renal allografts associated with FK506 (Tacrolimus) therapy. Am J Surg Pathol 1996;20:306–312. Connolly JO, Gane E, Higgins RM, O’Donnell PJ, Devlin J, Scoble JE, Williams R: Renal arteriopathy associated with FK506 therapy following liver transplantation. Nephrol Dial Transplant 1994;9:834–836. Cuhaci B, Kumar MSA, Bloom RD, Prati B, Haussman G, Laskow DA, Alidoost M, Grotkowski C, Cahill K, Butani L, Sturgill BC, Pankewycz OG: Transforming growth factor- levels in human allograft chronic fibrosis correlates with rate of decline in renal function. Transplantation 1999;68:785–790. Wissing K, Abramowcs D, Broeders N, Vereerstraeten P: Hypercholesterolemia is associated with increased kidney graft loss caused by chronic rejection in male with previous acute rejection. Transplantation 2000;70:464–472. Brazy PC, Pirsch JD, Belzer FO: Factors affecting renal allograft function in long-term recipients. Am J Kidney Dis 1992;19:558–566. Bostrom AG, Gohl RY, Morrissy P: Hyperhomocysteinemia in chronic renal transplant recipients. Graft 2000;3:195–204. Norman JT, Clark IM, Garcia PL: Hypoxia promotes fibrogenesis in human renal fibroblast. Kidney Int 2000;58:2351–2366. Remuzzi G, Ruggenenti P, Benigni A: Understanding the nature of renal disease progression. Kidney Int 1997;51:2–15. Eddy AA: Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 1996;7:2494–2508. Kuncio GS, Neilson EG, Haverty T: Mechanisms of tubulointerstitial fibrosis. Kidney Int 1991;39:550–556.

Pathogenesis and Molecular Mechanisms of CAN

201

38

39 40 41

42

43 44 45 46

47 48 49

50 51 52

53 54 55 56 57 58 59 60 61 62

63

Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border WA: Expression of transforming growth factor- isoforms in human glomerular diseases. Kidney Int 1996;49: 461–469 Furness PN: Extracellular matrix and the kidney. J Clin Pathol 1996;49:355–359. Rerolle JP, Hertig A, Nguyen G, Sraer JD, Rondeau EP: Plasminogen activator inhibitor type 1 is a potential target in renal fibrogenesis. Kidney Int 2000;58:1841–1850. Grandaliano G, Di Paolo S, Monno R, Stallone G, Ranieri E, Pontrelli P, Gesualdo L, Schena FP: Protease-activated receptor 1 and plasminogen activator inhibitor 1 expression in chronic allograft nephropathy: The role of coagulation and fibrinolysis in renal graft fibrosis. Transplantation 2001;72:1437–1443. Wilson HM, Reid FJ, Brown PA, Power DA, Haites NE, Booth NA: Effect of transforming growth factor-1 on plasminogen activators and plasminogen activator inhibitor-1 in renal glomerular cells. Exp Nephrol 1993;1:343–350. Kanalas JJ, Hopfer U: Effect of TGF-1 and TNF- on the plasminogen system of rat proximal tubular epithelial cells. J Am Soc Nephrol 1997;8:184–192. Nagase H, Woessner JF: Matrix metalloproteinases. J Biol Chem 1999;274:491–494. Ye S, Humphries S, Henney A: Matrix metalloproteinases: Implication in vascular matrix remodeling during atherogenesis. Clin Sci 1998;94:103–110. Salo T, Turpeeniemi-Hujanen R, Tryggvason K: Tumor-promoting esters and cell proliferation stimulate secretion of basement membrane (type IV) collagen-degrading metalloproteinase by human fibroblasts. J Biol Chem 1985;260:8526–8531. Martin J, Knowlden J, Davies M, Williams JD: Identification and dependent regulation of human mesangial cell metalloproteinases. Kidney Int 1994;46:877–885. Nagase H: Activation mechanisms of matrix metalloproteinases. J Biol Chem 1997;378:151–160. Miyazaki M, Nishino T, Abe K, Furusu A, Koji T, Kohno S: Regulation of renal extracellular matrix metabolism; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 141–155. Murphy G, Willenbrock F: Tissue inhibitors of matrix metalloendopeptidases. Methods Enzymol 1995;248:496–510. Nakai A, Satoh M, Hirayoshi K, Nagata K: Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol 1992;117:903–914. Abe K, Ozono Y, Miyazaki M, Koji T, Shioshita K, Furusu A, Tsukasaki S, Matsuya F, Hosokawa N, Harada T, Taguchi T, Nagata K, Kohno S: Interstitial expression of heat-shock protein-47 and -smooth muscle actin in renal allograft failure. Nephrol Dial Transplant 2000;15:529–535. Boukhalfa G, Desmouliere A, Rondeau E, Gabbiani G, Sraer JD: Relationship between -smooth muscle actin expression and fibrotic changes in human kidney. Exp Nephrol 1996;4:241–247. Cohen GM: Caspases: The executioners of apoptosis. Biochem J 1997;326:1–16. Faubion WA, Gores GJ: Death receptors in liver biology and pathobiology. Hepatology 1999;29:1–4. Razzaque MS, Ahsan N, Teguchi T: Role of apoptosis in fibrogenesis. Nephron 2002;90:365–372. Hishikawa K, Nakaki T, Fujii T: Transforming growth factor- induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells. Eur J Pharmacol 1999;385:287–290. Coleman AB, Momand J, Kane SE: Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin. Mod Pharmacol 2000;57:324–333. Sporn MB, Roberts AB: Transforming growth factor-: Recent progress and new challenges. J Cell Biol 1992;119:1017–1021. Waltenberger J, Miyazono K, Funa K, Wanders A, Fellstrom B, Heldin CH: Transforming growth factor- and organ transplantation. Transplant Proc 1993;25:2038–2040. Ignotz RA, Endo T, Massague J: Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-. J Biol Chem 1987;262:6443–6446. Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJ, Angel P et al: Transforming growth factor- modulates the expression of collagenase and metallo-proteinase inhibitor. EMBO J 1987;6:1899–1904. Border WA, Okuda S, Languino LR, Ruoslahti E: Transforming growth factor- regulates production of proteoglycans by mesangial cells. Kidney Int 1990;37:689–695.

Ahsan/Cheung

202

64 65 66 67

68

69

70 71 72 73 74 75

76

77 78 79 80

81 82 83 84 85

86 87

Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 1993;44:313–321. Paul LC, Saito K, Davidoff A, Benediktsson H: Growth factor transcripts in rat renal transplants. Am J Kidney Dis 1996;28:441–450. Terrell TG, Working PK, Chow CP, Green JD: Pathology of recombinant human transforming growth factor- in rats and rabbits. Int Rev Exp Pathol 1993;34:43–67. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, Grotendorst GR: Connective tissue growth factor mediates transforming growth factor--induced collagen synthesis: Down-regulation by cAMP. FASEB J 1999;13:1774–1786. Abraham DJ, Shiwen X, Black CM, Sa S, Xu Y, Leask A: Tumor necrosis factor- suppresses the induction of connective tissue growth factor by transforming growth factor- in normal and scleroderma fibroblasts. J Biol Chem 2000;275:15220–15225. Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, van Kooten C, Muller G: TGF-1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor-2. Kidney Int 2001;59:579–592. Sharma VK, Bologa RM, Xu G-P, Li B, Mouradian J, Wang J et al: Intra-graft TGF-1 mRNA: A correlate of interstitial fibrosis and chronic allograft nephropathy. Kidney Int 1996;49:1297–1303. Nagano H, Nadeau KC, Takada M, Kusaka M, Tilney NL: Sequential cellular and molecular kinetics in acutely rejecting renal allografts in rats. Transplantation 1997;63:1101–1108. Mannon RB, Kopp JB, Ruiz P, Griffiths R, Bustos M, Platt JL, Klotman PE, Coffman TM: Chronic rejection of mouse kidney allografts. Kidney Int 1999;55:1935–1944. Shihab FS, Tanner AM, Shao Y, Weffer MI: Expression of TGF-1 and matrix proteins is elevated in rats with chronic rejection. Kidney Int 1996;50:1904–1913. Weight SC, Bell PR, Nicholson ML: Renal ischaemia-reperfusion injury. Br J Surg 1996;83:162–170. Basile DP, Rovak JM, Martin DR, Hammerman MR: Increased transforming growth factor-1 expression in regenerating rat renal tubules following ischemic injury. Am J Physiol 1996;270:F500–F509. Azuma H, Nadeau K, Takada M, Mackenzie HS, Tilney NL: Cellular and molecular predictors of chronic renal dysfunction after initial ischemia/reperfusion injury of a single kidney. Transplantation 1997;64:190–197. Paul LC: Adverse effects of immunosuppressive drugs on long-term graft outcome. Transplant Proc 1998;30:4001–4004. Baboolal K, Jones GA, Janezic A, Griffiths DR, Jurewicz WA: Molecular and structural consequences of early renal allograft injury. Kidney Int 2002;61:686–696. Jain S, Bicknell GR, Nicholson ML: Tacrolimus has less fibrogenic potential than cyclosporine A in a model of renal ischemia-reperfusion injury. Br J Surg 2000;87:1563–1568. Bicknell GR, Williams ST, Shaw JA, Pringle JH, Furness PN, Nicholson ML: Differential effects of cyclosporine and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants. Br J Surg 2000;87:1569–1575. Curtis JJ, Luke RG, Whelchel JD, Diethelm AG, Jones P, Dustan HP: Inhibition of angiotensinconverting enzyme in renal transplant recipients with hypertension. N Engl J Med 1983;308:377–381. Dzau VJ: Cell biology and genetics of angiotensin in cardiovascular disease. J Hypertens 1994;12(suppl 4):3–10. Unger T, Gohlke P, Paul M, Rettig R: Tissue renin-angiotensin systems: Fact or fiction? J Cardiovasc Pharmacol 1991;18(suppl 2):20–25. Wolf G, Neilson EG: Angiotensin II as a renal growth factor. J Am Soc Nephrol 1993;3:1531–1540. Razzaque MS, Azouz A, Shinagawa T, Taguchi T: Factors regulating the progression of hypertensive nephrosclerosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 173–186. Kakinuma Y, Kawamura T, Bills T, Yoshioka T, Ichikawa I, Fogo A: Blood pressure-independent effect of angiotensin inhibition on vascular lesions of chronic renal failure. Kidney Int 1992;42:46–55. Becker BN, Jacobson LM, Hullett DA, Radke NA, Oberley TD, Brazy PC, Kirk AD: Type 2 angiotensin II receptor expression in human renal allografts: An association with chronic allograft nephropathy. Clin Nephrol 2002;57:19–26.

Pathogenesis and Molecular Mechanisms of CAN

203

88 89 90 91 92

Shihab FS, Bennett WM, Tanner AM, Andoh TF: Angiotensin II blockade decreases TGF-1 and matrix proteins in cyclosporine nephropathy. Kidney Int 1997;52:660–673. Border WA, Okuda S, Languino LR, Ruoslahti E: Transforming growth factor- regulates production of proteoglycans by mesangial cells. Kidney Int 1990;37:689–695. Tamaki K, Okuda S: Role of TGF- in the progression of renal fibrosis; Razzaque MS, Taguchi T (eds): Renal Fibrosis. Contrib Nephrol. Basel, Karger 2003, vol 139, pp 44–65. Kerins DM, Hao Q, Vaughan DE: Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV. J Clin Invest 1995;96:2515–2520. Vaughan DE, Lazos SA, Tong K: Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells. A potential link between the renin-angiotensin system and thrombosis. J Clin Invest 1995;95:995–1001.

Nasimul Ahsan, MD, Division of Nephrology and Transplantation, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, One Robert Wood Johnson Place, MEB 412, New Brunswick, NJ 08903 (USA) Tel. 1 732 2356955, Fax 1 732 2356431, E-Mail [email protected]

Ahsan/Cheung

204

Author Index

Abe, K. 141 Ahsan, N. 187 Azouz, A. 173 Cheung, J.Y. 187 Cochrane, A.L. 102 Furuichi, K. 66 Furusu, A. 141 Imai, E. 120 Inan, M.S. 90 Kashihara, N. 156 Kohno, S. 141 Koji, T. 141

Maeshima, Y. 32 Makino, H. 32, 156 Manchang, L. 20 Maruyama, S. 20 Matsuo, S. 20 Miyazaki, M. 141 Morioka, T. 12 Morita, Y. 20 Moriyama, T. 120 Nishino, T. 141 Oite, T. 12 Okuda, S. 44

Ricardo, S.D. 102 Shinagawa, T. 173 Sugiyama, H. 156 Taguchi, T. 1, 90, 173 Tamaki, K. 44 Wada, T. 66 Yao, J. 12 Yokoyama, H. 66 Yuzawa, Y. 20

Razzaque, M.S. 1, 90, 173

205

Subject Index

Actin isoforms 127 ␣-smooth muscle actin, myofibroblast expression 48, 49, 121, 125–127 structure 127 Adamalysins, functions 8 Angiotensin II chemokine modulation 72 chronic allograft nephropathy role 199 diabetic nephropathy role 52, 53 nuclear factor-␬B modulation and therapeutic targeting 39, 97 polymorphisms in associated genes 174 reactive oxygen species induction of production 110, 111 transforming growth factor-␤ modulation 51, 52, 54, 55, 72, 73 Antisense knockdown, see specific genes Apoptosis chronic allograft nephropathy 196 cyclosporin A induction 168 definition 156 glomerular disease pathology overview 156, 157 glomerulosclerosis human disease 159, 160 progression mechanisms 169, 170 subtotal nephrectomized rat 157–159 mesangial cells extracellular matrix interactions 162–164 mitochondrial pathway 167, 168

reactive oxygen species induction 160–162 mitochondria apoptosis mechanisms 164, 165 oxidative modifications of DNA 165–167 nitric oxide induction 111, 112 nuclear factor-␬B modulation 93, 94 Bone marrow-derived cells, fibrosis role 150 Chemokines, see also specific chemokines angiotensin II modulation 72 anti-inflammatory agent inhibition of production 71, 72 classification 67, 68 ex vivo gene transfer and cell-mediated renal injury 76 functional overview 69, 70 human renal disease roles crescentic glomerulonephritis 78, 79 eotaxin 80, 81 fractalkine 81 interleukin-8 77 MCP-1 77–80 MIP-1␣ 78, 79 knockout mouse studies 74, 76 leukocyte trafficking role 67, 68 mesangial cell expression and response 70, 71

206

proximal tubular epithelial cell expression and response 72, 73 receptors cell distribution 66, 70 types 67, 68 renal disease animal model studies of expression and effects 73–75 therapeutic targeting glucocorticoids 71, 84 mitogen-activated protein kinase signaling inhibition 83, 84 neutralizing antibody studies anti-GBM antibody nephritis 82 anti-Thy-1.1 antibody nephritis 82, 83 immune complex-mediated glomerulonephritis 82 receptor antagonists 83 strategies 81 Chronic allograft nephropathy angiotensin II role 199 apoptosis 196 clinical features 187–189 extracellular matrix metabolism matrix metalloproteinases 194, 195 plasminogen-plasmin pathway 193, 194 fibrosis 192, 193, 196, 197 graft survival rate 187 heat shock protein 47 role 195, 196 interstitial fibroblasts 193 management 200 pathologic findings 189, 190 phases 188 risk factors alloantigen-dependent factors 190, 191 alloantigen-independent factors 192 transforming growth factor-␤ drug induction 198, 199 fibrosis induction 196, 197 ischemia-reperfusion injury 198 rejection prognosis 198 Collagen, see Extracellular matrix Complement, activation in tubulointerstitial injury with proteinuria 24–26 Connective tissue growth factor

Subject Index

fibrosis role 4, 6 hypertensive nephrosclerosis role 177, 178 myofibroblast expression 123, 124 Crescentic glomerulonephritis, chemokine role 78, 79 Cyclin-dependent kinase, expression in renal disease 35, 36 Cyclosporin A apoptosis induction 168 nephrotoxicity 191, 192 Diabetic nephropathy collagen production 142, 143 macrophage infiltration 104 oxidative stress 108, 109 transforming growth factor-␤ role 52, 53 Duffy antigen receptor for chemokines, renal disease role 69, 70 E2F antisense knockdown studies in glomerulonephritis 37, 38 mesangial cell expression 36, 37 target genes 36 Endothelin, hypertensive nephrosclerosis role 180, 181 Eotaxin, human renal disease role 80, 81 Epithelial-mesenchymal transdifferentiation definition 133 renal fibrogenesis role 133 steps 134, 135 Extracellular matrix cell sources 142 chronic allograft nephropathy and metabolism matrix metalloproteinases 194, 195 plasminogen-plasmin pathway 193, 194 collagen production in diabetic nephropathy 142, 143 components 141, 142 degradation, see Matrix metalloproteinases deposition in fibrosis 4, 44 growth factors in production 142

207

Extracellular matrix (continued) mesangial cell interactions and apoptosis 162–164 plasminogen-plasmin pathway in metabolism 149, 150 remodeling 7, 8, 146, 147

nuclear factor-␬B regulation 96 Interleukin-8 human renal disease role 77 wound healing role 2 Iron, tubulointerstitial fibrosis pathogenesis 26, 27

Fibroblast, see Myofibroblast Fibrosis, see also specific conditions causes 3 cytokines and growth factors 3, 4, 6, 7 stages 3, 7 FK506, see Tacrolimus Fractalkine, human renal disease role 81

Leukocyte, trafficking 67, 68 Lipoprotein, tubulointerstitial fibrosis role 27

Glucocorticoids, chemokine inhibition 71, 84 Heat shock protein 47 chronic allograft nephropathy role 195, 196 collagen interactions 6 hypertensive nephrosclerosis role 178, 179 Hepatic growth factor, extracellular matrix remodeling 146, 147 Hypertensive nephrosclerosis adhesion molecules 176 connective tissue growth factor role 177, 178 endothelin role 180, 181 end-stage renal disease association 173, 174 epidemiology 173, 174 heat shock protein 47 role 178, 179 matrix metalloproteinase role 182 MCP-1 role 176 microenvironments 175 pathogenesis 174, 175 platelet-derived growth factor role 181, 182 prospects for study 182, 183 transforming growth factor-␤ role 176, 177 Interleukin-1 inflammation role in kidney disease 95, 96

Subject Index

Macrophage infiltration in renal disorders chemoattractants 104–106 overview 103, 104 tubulointerstitial injury 102, 103 Matrix metalloproteinases chronic allograft nephropathy 194, 195 diseased kidney expression animal models 147, 148 human disease 148 Ets-1 in transcription 7, 8 hypertensive nephrosclerosis role 182 inhibitors 148, 149 membranous type metalloproteinases 145, 146 regulation of expression 146, 147 substrates 144, 145 tissue inhibitors, see Tissue inhibitors of metalloproteinases types and sources 7, 144–146 MCP-1 expression regulation 106 hypertensive nephrosclerosis role 176 macrophage infiltration role in renal disease 104–106 renal disease role 70, 72–74, 76–80, 82, 83 MCP-3, renal disease role 73 Mesangial cell, see also Myofibroblast activation markers 125, 126 molecular mechanisms 127 apoptosis extracellular matrix interactions 162–164 mitochondrial pathway 167, 168

208

reactive oxygen species induction 160–162 behavior modulation by matrix or cell-cell interactions 12–14 chemokine expression and response 70, 71 contractility 124, 125 cytokine and growth factor regulation 32–36, 126 E2F expression and inhibition studies 36–38 extracellular matrix component expression 13, 125 gap junction intercellular communication 14 glomerular abundance 124 progressive glomerulosclerosis role 17, 18 MIP-1␣, renal disease role 78, 79, 82 Mitochondria, apoptosis role mechanisms 164, 165 mesangial cell apoptosis pathway 167, 168 oxidative modifications of DNA 165–167 Mitogen-activated protein kinase, chemokine signaling and inhibition 83, 84 Myofibroblast, see also Mesangial cell actin expression 48, 49, 121 cytokine and growth factor production 123, 124 definition 121 development role 122 differentiation factors 123 epithelial-mesenchymal transdifferentiation 133–135 morphology 121 oxidative stress response 132, 133 renal disease distribution 120, 121 renal origins 122, 123 tubulointerstitial fibrosis role 128, 129 types 121 wound healing role 122 Nephrosclerosis, see Hypertensive nephrosclerosis

Subject Index

Nitric oxide apoptosis induction 111, 112 functions 111 peroxynitrite formation and oxidative stress 112, 113 synthesis 111 Nuclear factor-␬B activation in kidney 96, 97 angiotensin II modulation and therapeutic targeting 39, 97 antisense knockdown studies in glomerulonephritis 38, 39 apoptosis modulation 93, 94 discovery 90 fibrosis role 98 functional overview 90, 99 inducers and activation 93 inflammation role 94–96 inhibitory subunit degradation 92, 93 protein family 91 regulation of expression 93 modulators 91–93 prospects for study 99 structure 90, 91 target genes 38, 96 tubulointerstitial injury role 22 Osteopontin, macrophage infiltration role in renal disease 105 Oxidative stress antioxidants enzymes 106, 108 supplements 107 diabetic nephropathy 108, 109 peroxynitrite formation and oxidative stress 112, 113 reactive oxygen species angiotensin II induction of production 110, 111 apoptosis induction 160–162 fibrosis induction mechanisms 109, 110 signaling 108, 109 types 106 tubulointerstitial injury 131–133 unilateral ureteral obstruction 103, 106, 108, 113, 114, 132, 133

209

p38, see Mitogen-activated protein kinase Peroxynitrite, formation and oxidative stress 112, 113 Plasminogen-plasmin pathway, extracellular matrix metabolism 149, 150, 193, 194 Platelet-derived growth factor fibrosis role 4 hypertensive nephrosclerosis role 181, 182 mesangial cell effects 32, 33, 126 wound healing role 2 Progressive glomerulosclerosis animal models 15–17 mesangial cell function 17, 18 vascular regeneration impairment in progression 17 Proliferating cell nuclear antigen, antisense knockdown studies in glomerulonephritis 35 Proteinuria chronic allograft nephropathy 192 prognostic value 130 reabsorption and degradation of urinary protein 22, 23 tubulointerstitial injury albumin in pathogenesis 23, 24 animal models 21, 22 complement activation 24–26, 131 cytokines and growth factors 27 incidence 21 iron in pathogenesis 26, 27 lipoprotein roles 27 management 21, 28 mechanisms of injury 22, 23, 130, 131 Proximal tubular epithelial cells chemokine expression and response 72, 73 epithelial-mesenchymal transdifferentiation 133–135 tubulointerstitial injury with proteinuria albumin in pathogenesis 23, 24 animal models 21, 22 complement activation 24–26 cytokines and growth factors 27 iron in pathogenesis 26, 27 lipoprotein roles 27 management 21, 28 mechanisms of injury 22, 23

Subject Index

RANTES macrophage infiltration role in renal disease 105, 106 renal disease role 70–72, 74, 76, 83 Reactive oxygen species, see Oxidative stress Renal transplantation, see Chronic allograft nephropathy Scarring, see Fibrosis Smads, signaling 47, 48 ␣-Smooth muscle actin, see Actin Tacrolimus (FK506), nephrotoxicity mechanisms 97, 98, 192 Tissue inhibitors of metalloproteinases diseased kidney expression animal models 147, 148 human disease 148 knockout mouse 149 regulation of expression 146, 147 specificity 146 types 146 Transforming growth factor-␤ angiotensin II modulation 51, 52, 72, 73 antisense knockdown studies in glomerulonephritis 34, 35 biological activity 4, 5, 33 chronic allograft nephropathy drug induction 198, 199 fibrosis induction 196, 197 ischemia-reperfusion injury 198 rejection prognosis 198 diabetic nephropathy role 52, 53 diagnostic and prognostic value 57 fibrosis role 4, 44, 45, 48–50, 114 hypertensive nephrosclerosis role 176, 177 latent form activation 50, 51 production 46, 47 structure 46 mesangial cell effects 33–35, 126 myofibroblast expression 124, 130 polymorphisms 57 receptor and Smad signaling 47, 48, 57

210

therapeutic targeting activation inhibition 55 angiotensin inhibition 54, 55 neutralizers 55, 56 rationale 53, 54 receptor blocking 56 signaling inhibition 56 wound healing role 2, 49, 58 Tubulointerstitial fibrosis macrophage infiltration 102, 103 myofibroblast role 128, 129 nuclear factor-␬B role 22 oxidative stress 131–133 prognostic value 20, 21 proteinuria albumin in pathogenesis 23, 24 animal models 21, 22 complement activation 24–26, 131 cytokines and growth factors 27 incidence 21 iron in pathogenesis 26, 27 lipoprotein roles 27

Subject Index

management 21, 28 mechanisms of injury 22, 23, 130, 131 Tumor necrosis factor-␣ inflammation role in kidney disease 95, 96 nuclear factor-␬B regulation 96 Unilateral ureteral obstruction myofibroblast role 128, 129 oxidative stress 103, 106, 108, 113, 114, 132, 133 Vascular endothelial growth factor, wound healing role 2 Wound healing cytokine regulation 2 growth factors 2 myofibroblast role 122 overview 1 transforming growth factor-␤ role 2, 49, 58

211

Renal Fibrosis (Contributions to Nephrology)

Cellular Stress Responses In Renal Diseases (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Acute Kidney Injury (Contributions to Nephrology, 156)

Obesity And the Kidney (Contributions to Nephrology)

Kidney Transplantation: Strategies to Prevent Organ Rejection (Contributions to Nephrology)

Cardiorenal Syndromes in Critical Care (Contributions to Nephrology, Vol. 165)

Nutrition and Kidney Disease: A New Era (Contributions to Nephrology)

Acute Blood Purification (Contributions to Nephrology, Vol. 166)

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Peritoneal Dialysis: A Clinical Update (Contributions to Nephrology)

Fluid Overload: Diagnosis and Management (Contributions to Nephrology, Vol. 164)

Hemodialysis Vascular Access and Peritoneal Dialysis Access (Contributions to Nephrology)

Introduction to Renal Therapeutics

A Practical Manual of Renal Medicine: Nephrology, Dialysis and Transplantation

Vital Nephrology

Cystic Fibrosis

Clinical Nephrology

Peritoneal Dialysis: From Basic Concepts to Clinical Excellence (Contributions to Nephrology Vol 163)

Regenerative Nephrology

Regenerative Nephrology

Sepsis, Kidney And Multiple Organ Dysfunction: 3rd International Course on Critical Care Nephrology, Vicenza, June 2004 (Contributions to Nephrology)

Renal Physiology

Clinical Nephrology

Cardiovascular Disorders in Hemodialysis: 14th International Course on Hemodialysis, Vicenza, May 2005 (Contributions to Nephrology)

Nephrology in 30 Days

Intensive Care in Nephrology

Understanding Cystic Fibrosis

Manual of Nephrology

Idiopathic Pulmonary Fibrosis Market

Renal Fibrosis (Contributions to Nephrology)

Cellular Stress Responses In Renal Diseases (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Acute Kidney Injury (Contributions to Nephrology, 156)

Obesity And the Kidney (Contributions to Nephrology)

Kidney Transplantation: Strategies to Prevent Organ Rejection (Contributions to Nephrology)

Cardiorenal Syndromes in Critical Care (Contributions to Nephrology, Vol. 165)

Nutrition and Kidney Disease: A New Era (Contributions to Nephrology)

Acute Blood Purification (Contributions to Nephrology, Vol. 166)

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Peritoneal Dialysis: A Clinical Update (Contributions to Nephrology)

Fluid Overload: Diagnosis and Management (Contributions to Nephrology, Vol. 164)

Hemodialysis Vascular Access and Peritoneal Dialysis Access (Contributions to Nephrology)

Introduction to Renal Therapeutics

A Practical Manual of Renal Medicine: Nephrology, Dialysis and Transplantation

Vital Nephrology

Cystic Fibrosis

Clinical Nephrology

Peritoneal Dialysis: From Basic Concepts to Clinical Excellence (Contributions to Nephrology Vol 163)

Regenerative Nephrology

Regenerative Nephrology

Sepsis, Kidney And Multiple Organ Dysfunction: 3rd International Course on Critical Care Nephrology, Vicenza, June 2004 (Contributions to Nephrology)

Renal Physiology

Clinical Nephrology

Cardiovascular Disorders in Hemodialysis: 14th International Course on Hemodialysis, Vicenza, May 2005 (Contributions to Nephrology)

Nephrology in 30 Days

Intensive Care in Nephrology

Understanding Cystic Fibrosis

Manual of Nephrology

Idiopathic Pulmonary Fibrosis Market

Recommend Documents