Mechanisms of Progressive Renal Disease in Glomerulonephritis

Mechanisms of Progressive Renal Disease in Glomerulonephritis William G. Couser, MD, and Richard J. Johnson, MD • Progressive renal disease in glomerulonephritis (GN) involves both glomerular and interstitial processes. In the glomerulus, sclerosis occurs witlt progressive accumulation of extracellular matrix components that reduce filtration surface area. In the interstitium, early inflammatory changes accompany GN with later development of fibrosis and tubular atrophy. Our studies have focused on the role of early cellular events in the development of glomerular and interstitial fibrosis. In the antithymocyte serum (ATS) model of mesangial proliferative GN, mesangial cell prOliferation is initiated by processes involving complement and platelets and may involve basic fibroblast growth factor (bFGF). Mesangial cell proliferation is maintained by an autocrine mechanism involving upregulation of mesangial cell PDGF and PDGF receptors. Mesangial cells also change phenotype with expression of a-smooth muscle actin and production of type I collagen. These early changes precede upregulation of genes for the production of extracellular matrix components and the development of mesangial matrix expansion and sclerosis. Matrix expansion is reduced by factors that block cell proliferation, including platelet and complement depletion, heparin, and antibody to PDGF. A similar sequence of early platelet infiltration, increased expression of PDGF, and mesangial cell proliferation occurs early in the development of glomerulosclerosis in the remnant kidney model, and mesangial cell proliferation is a prominent early feature of experimental diabetic nephropathy. We believe these early glomerular cellular changes are linked to the later development of sclerosis. In the interstitium, acute GN is accompanied by upregulation of mRNA and protein for osteopontin, a macrophage chemotactic/adhesive factor expressed by cortical tubules following several types of glomerular injury. Interstitial macrophage localization occurs in areas of osteopontin expression followed by the development of interstitial fibrosis. These studies of early cellular events in the development of both glomerular and interstitial fibrosis may provide important clues to the mechanisms that underlie progressive renal disease in GN. © 1994 by the National Kidney Foundation, Inc. INDEX WORDS: Glomerulonephritis; interstitial nephritiS; growth factors; osteopontin.

4 PPROXIMA TELY 30% of patients with .ft end-stage renal disease in the United States

have lost kidney function as a consequence of glomerulonephritis (GN). J This percentage is higher in other parts of the world. In a minority of cases, GN causes end-stage renal disease by an acute, rapidly progressive process that results in diffuse glomerular necrosis or thrombosis without a chronic progressive phase. 2 However, in most types of progressive GN, including IgA nephropathy, lupus nephritis, membranoproliferative GN, etc, loss of renal function occurs more slowly and is accompanied by decreasing kidney size with the development of glomerulosclerosis and interstitial fibrosis. A similar process probably accounts for progression in non nephritic glomerular diseases, such as diabetes and focal sclerosis. Bricker et al were among the first to suggest that the mechanisms that lead from an initial glomerular insult to eventual renal failure are probably ones that are common to many diseases and are likely different from the processes that mediate the initial phase of glomerular injury.3 Much has been learned about progressive glomerular disease over the ensuing three decades since Bricker et aI's suggestion, and several mechanisms have been shown to participate in this process. Many of these were reviewed during

this symposium. These include systemic and glomerular hypertension, factors related to glomerular hypertrophy, consequences of prolonged hyperlipidemia, immune events, etc (reviewed in ref 4). We review recent data from our laboratory suggesting that previously unappreciated early cellular events in the glomerulus may be central to the pathogenesis of progressive glomerular disease. These data recently have been reviewed in more detail elsewhere. 5-8 PROGRESSION IN GLOMERULONEPHRITIS: GLOMERULAR CELLULAR EVENTS

Experimental Studies Central to the development of chronic glomerulosclerosis is the accumulation of excess matrix components that constitute the sclerotic From the Department o/Medicine. Division o/Nephrology. University o/ If,'ashingtoll. Seallie. IVA. R eceived AI/gust 19. 1993; accepted in revised/arm August 30. 1993. Supported by research grants /rom the Unit ed States Public Health Service (DK 34198. DK 43422. DK 02142. and DK 0746 7) and the Northwest Kidne}' Foundation. Address reprint request t o William G. Couser. M D.Division a/Nephrology. RM-/l. University a/ Washington. Seattle. WA 98195. © 1994 by the National Kidney Foundation. Inc. 0272-6386/94/ 2302-000 7$3.00/ 0

American Journal of Kidney Diseases, Vol 23, No 2 (February), 1994: pp 193-198

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lesion (reviewed in ref8). Since the synthesis and turnover of these components are functions of resident glomerular cells, an understanding of the response of these cells to various inflammatory and hemodynamic stimuli is essential. Much progress has been made in the past decade in defining the spectrum of responses that normal glomerular cells may exhibit in response to mediators of inflammation in vitro (reviewed in refs 9 and 10). In our laboratory, we have focused on attempting to define which of these in vitro responses participate in the development of progressive glomerular disease in vivo. Our studies implicate cell proliferation as a primary and central event which precedes upregulation of genes that result in an overproduction of extracellular matrix components in several models of progressive glomerular disease. Anti- Thy 1 (ATS) model of mesangial proliferative glomerulonephritis. In most progressive glomerular diseases, matrix expansion and sclerosis begin with the expansion of mesangial matrix, which is synthesized primarily by resident mesangial cells. An immunologic model that closely simulates diseases of mesangial matrix expansion secondary to GN is the ATS model. In this model, the injection of antibody to the Thy 1.1 antigen on the mesangial cell membrane induces an initial phase of mesangiolysis accompanied by a glomerular platelet infiltrate. II These events are followed in 2 to 4 days by a phase of prominent mesangial cell proliferation that can be quantitated by staining for the proliferating cell nuclear antigen, which is actively expressed by proliferating cells during the late G 1 to S phase of the cell cycle. In seve~al studies of this model, we have established that mesangial cell proliferation is mediated by platelets that localize by complement-dependent mechanisms and presumably release factors that initiate mesangial cell proliferation. 12 One candidate for such a factor is bFGF, which is released by platelets and lysed mesangial cells and can stimulate mesangial cell proliferation in vivo and in vitro. 13 When mesangial cell activation and proliferation occur, there is a phenotypic change characterized by de novo expression of (X-smooth muscle actin l4 and type I collagen synthesis, 15 indicating that proliferating mesangial cells have acquired the characteristics of myofibroblasts. 16 Proliferating mesangial cells also exhibit an increase in platelet-

derived growth factor (PDGF) B-chain mRNA and protein and a similar increase in mRNA and protein for PDGF t1-receptor, thereby establishing an autocrine mechanism that apparently maintains mesangial cell proliferation. 17 There also is a prominent macrophage infiltrate in glomeruli early in this lesion l4 ,17 as well as an associated increase in transforming growth factor-t1 (TGFmRNA and protein. 18 Whether the TGF-t1 is derived from macrophages or mesangial cells has not been determined. Accompanying these cellular events is an increase in both mRNA and protein for several extracellular matrix components, including types I and IV collagen, laminin, fibronectin, nidogenentactin, and heparan sulfate proteoglycan. 15 The suggestion that mesangial cell proliferation is linked to overproduction of matrix components is supported by the observations that agents which reduce proliferation, including complement depletion,12 heparin administration,19 and administration of antibody to PDGF,2o all reduce mesangial matrix expansion as well. A role for TGF-t1 in mediating the matrix expansion that follows mesangial cell proliferation is suggested by studies which demonstrate that administration of antibody to TGF-t121 or natural inhibitors such as decorin 22 also reduce matrix expansion in the ATS model. Thus, in the ATS model, the increase in gene expression and the production of extracellular matrix components that eventually lead to sclerosis appear to accompany and be tightly linked to an earlier phase of mesangial cell proliferation. Remnant kidney model. The remnant kidney model induced by 5/6 nephrectomy is probably the most extensively studied model of progressive glomerular disease. In this model, typical glomerulosclerosis develops in weeks following subtotal nephrectomy and is believed to be mediated by hemodynamic factors, including glomerular hypertension. 4 In this model, a phenotypic change to express (X-smooth muscle actin is seen in mesangial cells within 3 days of nephrectomy, and a phase of mesangial cell proliferation ensues by day 5 and lasts for several weeks. 23 This mesangial proliferative response is associated with an increase in mesangial staining for PDGF B-chain as well as with increased glomerular expression ofPDGF mRNA by in situ hybridization. 24 Similar changes occur in gene expression and protein

m

PROGRESSION IN GLOMERULONEPHRITIS

for PDGF ,6-receptor. 24 These mesangial proliferative changes are preceded by a mild platelet infiltration. 24 As in the ATS model, a prominent glomerular macrophage infiltrate occurs (maximal at week 10) and represents the majority of proliferating glomerular cells by week 10. 24 This macrophage influx correlated with both proteinuria and glomerulosclerosis, which were first demonstrated by weeks 2 to 3 in the remnant kidney mode1. 24 Other investigators have noted a glomerular macrophage infiltrate in the remnant kidney model as well. 25 Thus, cellular events, including mesangial cell proliferation and macrophage infiltration, may well have an important role in the development of glomerulosclerosis in the remnant kidney model. Experimental diabetes. Numerous theories for the pathogenesis of progressive diabetic nephropathy have been put forth that implicate hemodynamic factors, metabolic changes, and hormonal events in this process. Much of these data are reviewed by Dr Hostetter elsewhere in this symposium. Our studies have again focused on the early cellular events in diabetic nephropathy. In sequential studies of the glomerulus in rats with diabetic nephropathy induced by streptozotocin, we have again documented an early phase of mesangial cell proliferation that peaks at day 3 but persists throughout the first 30 days of this disease. 26 In contrast to the ATS and remnant kidney models of progressive glomerular disease, we have not been able to demonstrate any striking increase in glomerular platelet localization. However, we have documented some increase in mRNA for PDGF B-chain as well increased mesangial staiping ofbFGF, observations that suggest a potential role for growth factors in the pathogenesis of diabetic nephropathy as well. As in the other models, there also is a prominent glomerular macrophage infiltrate in streptozotocin diabetes that is evident by day 6 and increases thereafter. Glomerular localization of macrophages is associated with an increase in glomerular mRNA for TGF-,6.27 All these changes can be prevented by insulin therapy, suggesting that they occur in response to the diabetic milieu rather than as a direct consequence of the administration of streptozotocin or other nondiabetic factors. 26 ,27 These previously unrecognized cellular events precede any detectable changes in glomerular gene expression or protein

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content of types I and IV collagen, laminin, and heparan sulfate proteoglycan. 26 They also precede any detectable evidence of diabetic glomerulosclerosis by light microscopy. Thus, as in the other models studied, cellular events including mesangial cell proliferation and macrophage infiltration appear to precede and be linked to the later development of diabetic sclerotic changes. Glomerular epithelial cell proliferation: experimental membranous nephropathy. In contrast to the disease models discussed above, in which glomerulosclerosis is preceded by mesangial cell proliferation and expansion ofmesangial matrix, in membranous nephropathy progressive glomerular disease is associated with thickening of the capillary wall, which also is due to an excess of matrix components. In experimental studies, the principal constituent of this capillary wall thickening is laminin,28 and in human disease both laminin and novel collagen chains (alpha 3 and alpha 4 chains of type IV collagen) appear to be involved. 29 The accumulation of excess matrix along the capillary wall presumably represents overproduction (or reduced degradation) of matrix components by the glomerular epithelial cell (GEC). In sequential studies of GEC changes in the passive Heymann nephritis model of membranous nephropathy (MN), we have documented recently that initial injury to GECs by antibody and C5b-9 induces an increase in GEC proliferation that is most prominent between days 5 and 10, coincident with the onset of proteinuria. 3o Proliferation is abolished by complement depletion, and C5b-9 is a known mitogen for cells in vitro. 3 ! Glomerular epithelial cell proliferation in experimental MN is preceded by an increase in staining and gene expression for PDGF B-chain, although the absence of PDGF receptors on GECs makes the significance of this observation uncertain. 30 Other known GEC mitogens that might participate include insulin, leukotrienes C4 and D4, thrombin, and interleukin-l,6. Interleukin-l,6 induces PDGF gene expression and also activates laminin synthesis and induction of an NFKB-like DNAbinding protein in GECs. 32 As in diseases of the mesangial cell, all these early cellular events precede any detectable increases in glomerular deposition of normal extracellular matrix components. 28 ,30 Although an increased appearance of type I collagen mRNA is seen in GECs at this

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stage of the membranous lesion, there does not appear to be increased translation of this protein or increased deposition in glomerular basement membrane. 28 . 33 Thus, in early MN, as in diseases localized initially to the mesanglum, a phase of cellular activation, growth factor expression, and proliferation precedes upregulation of genes that lead to overproduction of normal extracellular matrix and may be linked to the later development of capillary wall thickening and sclerosis.

Human Studies The hypothesis that overproduction of matrix components by either mesangial or epithelial cells may be linked to early cellular events including cell proliferation is consistent with observations in humans. Studies of a variety of different proliferative glomerular nephritides, including JgA nephropathy, diffuse proliferative lupus nephritis, and mesangial proliferative GN, have documented a prominent increase in proliferating glomerular cells that were not derived from the circulation and probably represented proliferating resident glomerular cells. 34 Increased expression of a-smooth muscle actin also was documented. 34 Similar to the experimental studies reviewed above, glomerular cell proliferation also was documented in several noninflammatory glomerular lesions, including focal sclerosis, diabetic . nephropathy, and amyloid. 34 PROGRESSION IN GLOMERULONEPHRITIS: INTERSTITIAL EVENTS

Although most research on progressive glomerular disease has focu~ed on changes in the glomerulus itself, it is generally accepted that interstitial changes, including cellular infiltration and fibrosis, correlate better with clinical parameters of progression than do any currently recognized glomerular changes. 35 .36 The mechanisms of interstitial disease in GN have not been well defined. In different circumstances, interstitial inflammation and fibrosis may result from the deposition of antibody or the formation of immune complexes on tubular basement membranes, cell-mediated immune reactions to interstitial antigens, inflammatory reactions to tubular constituents such as Tamm-Horsfall protein that become localized in the interstitium, and direct or indirect effects of various inflam-

COUSER AND JOHNSON

matory mediators derived directly from the glomerulus (reviewed in ref 36). We recently have become interested in the possible role of osteopontin (OPN) in the interstitial changes that accompany progressive glomerular disease. Osteopontin is an RGD-containing, secreted glycoprotein made constitutively by renal tubular epithelial cells37 that also exhibits chemotactic and adhesive properties for macrophages. 38.39 Systemic infusion of angiotensin II induces dramatic interstitial inflammation characterized by monocyte/macrophage infiltrates that localize in areas where tubules exhibit increased OPN mRNA and protein. 4o In studies of three different models of GN (the ATS model of mesangial proliferative GN, the passive heymann nephritis (PHN) model of membranous nephropathy, and amino nucleoside nephrosis) we have now documented significant tubulointerstitial injury occurring between 5 and 14 days after the onset of glomerular disease and characterized by mononuclear cell infiltrates, tubular dilatation and atrophy, and cast formation. 41 In all three models, upregulation of OPN mRNA by in situ hybridization and of OPN protein by immunostaining were demonstrated in cortical renal tubular epithelium. 41 Increased OPN expression preceded detectable macrophage infiltrates and evidence of interstitial inflammation. Osteopontin expression often was periglomerular early and more diffusely distributed throughout the cortex later. In all models studied, interstitial macrophage infiltrates exhibited a striking tendency to localize in areas of increased tubular OPN expression. 41 We also have demonstrated increased OPN expression associated with interstitial inflammation and fibrosis in a model of cyclosporine nephropathy.42 The known interactions between OPN and macrophages established in vitro, the fact that increased tubular OPN expression precedes interstitial monocyte infiltration, and our findings that monocytes localize exclusively in areas of increased OPN expression in several models of progressive glomerular disease suggest that OPN may be an important mediator of progressive interstitial fibrosis in GN. Osteopontin may be a link between various cytokines, growth factors, and humoral mediators, such as angiotensin II, that are released in glomerular disease and the subsequent development of interstitial inflammation and fibrosis

PROGRESSION IN GLOMERULONEPHRITIS

that appears to be the principal structural correlate of deteriorating renal function in progressive glomerular disease. Obviously, other chemotactic and adhesive factors are likely to be involved as well, particularly members of the intercrine family, such as monocyte chemotactic peptide 1 (MPC-l), IP-l 0, IL-8, and Rantes43 and various intercellular adhesion molecules (ICAM 1 and 2), vascular cell adhesion molecules (VCAM-l), and endothelial leukocyte adhesion molecules (ELAM _1).44 Studies of the interaction between events in the glomerulus and development of interstitial disease are now attracting increasing attention from investigators of glomerular disease, and seem likely to shed significant new light on the mechanisms that underlie progression in glomerular disease. ACKNOWLEDGMENT The authors are grateful to their many colleagues who participated in these studies. including Jiirgen Floege, MD, Charles Alpers, MD, Hiroyuki Iida. MD. Ashio Yoshimura. MD. Bessie Young. MD, and Raimund Pichler; to Kathy Gordon, Pam Pritzl, and Donna Lombardi for their expert technical assistance; and to Neal Bricker, MD. for his insight and inspiration in stimulating many of the ideas that eventually led to these studies.

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mRNA in glomeruli of rats with passive Heymann nephritis. Kidney Int 43:121-127, 1993 34. Alpers CE, Hudkins KL. Gown AM, Johnson RJ: Enhanced expression of "muscle-specific" actin in glomerulonephritis. Kidney Int 41: 1134-1144, 1992 35. Nath KA: Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 20: 1-17, 1992 36. Yee J, Kuncio GS, Neilson EG: Tubulointerstitial injury following glomerulonephritis. Semin Nephrol II :361-366, 1991 37. Butler WT: The nature and significance of osteopontin. Connect Tissue Res 23: 123-136, 1989 38. Patarca R, Freeman GJ, Singh P, Wei F-Y, Durfee T, Blattner F, Regnier DC, Kozak CA, Mock BA, Morse HC III, Jerrells TR, Cantor H: Structural and functional studies of the early T lymphocyte activation I (Eta-I) gene. J Exp Med 170:145-161,1989 39. Singh RP, Patarca R, Schwartz J, Singh P, Cantor H: Definition of a specific interaction between the early T lymphocyte activation I (Eta-I) protein and murine macrophages in vitro and its effect upon macrophages in vivo. J Exp Med 171:1931-1942,1990 40. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, Schwartz SM: Renal injury from angiotensin II-mediated hypertension. Hypertension 19:464-474, 1992 41. Pichler R, Giachelli CM, Lombardi D, Pippin J, Gordon K, Alpers CE, Schwartz S, Johnson RJ: Tubulointerstitial disease in glomerulonephritis: Potential role of osteopontin (uropontin). Am J Pathol (submitted for publication) 42. Young B, Burdmann E, Alpers C, Eng E, Ando HT, Johnson R, Bennett W, Couser W: Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporin nephrotoxicity. J Am Soc NephroI4:762, 1993 (abstr) 43. Oppenheim 11, Zachariae CO, Mukaida N, Matushima K: Properties of the novel proinflammatory supergene "intercrine" cytokine family. Ann Rev ImmunoI9:617-648, 1991 44. Main IW, Nikolic-Peterson DJ, Atkins RC: T cells and macrophages and their role in renal injury. Semin Nephrol 12:395-407, 1992