Specialized collagen mRNA and secreted collagens in human glomerular epithelial, mesangial, and tubular cells.

Isolated glomeruli and cultured cells were examined under nonmitogenic conditions by Northern hybridization of steady-state mRNA levels for procollagens alpha 1(I), alpha 1(III), alpha 1(IV), alpha 2(IV), beta-actin, and fibronectin. Procollagens concurrently secreted from these cells were characterized after limited pepsin digestion. Poly(A)-mRNA from freshly isolated whole porcine glomeruli was primarily type IV. For cultured glomerular and tubular epithelial cells, the collagen mRNA species were almost exclusively alpha 1(IV) and alpha 2(IV). Correspondingly, the secreted collagen was almost entirely type IV. The mRNA signals for collagens in glomerular mesangial cells included alpha 1(I), alpha 1(IV), alpha 2(IV), and less alpha 1(III). The secreted collagens were also types I and IV, with less types III and V. There were similar patterns of mRNA signal levels for the two type IV collagens and similar patterns of expression of alpha 1(I) and alpha 1(III). In situ hybridization showed the fibroblast and epithelial cell populations homogeneous in expressing the same mRNA signals seen by Northern hybridization. These findings establish the correlation of collagen mRNA and protein expression of collagens in differentiated glomerular cells in culture, under resting nonmitotic conditions.     

A pitfall of glomerular sieving: profibrotic and matrix proteins derive from the Bowman's capsule and not the glomerular tuft in rats with renovascular hypertension.

BACKGROUND: The glomeruli in the non-clipped kidney of rats with 2-kidney, 1-clip hypertension are a classical model for studying the mechanisms of glomerular injury. METHODS: In the present study, we compared the glomerular expression of PAI-1 and collagen I alpha1 mRNA from glomeruli isolated by the classic technique of sieving with the recently developed technique of tissue laser microdissection. For quantification of mRNA from both methods, real-time PCR was used. RESULTS: Real-time PCR revealed a 9.0 +/- 1.3- and a 7.1 +/- 0.2-fold induction of PAI-1 and collagen I alpha 1, respectively, in the glomeruli from hypertensive rats isolated by sieving. However, in situ hybridization and microdissection revealed that expression of both mRNAs was mainly from the Bowman's capsule and not from the glomerular tuft (10.7 +/- 1.3- and 7.2 +/- 0.6-fold higher induction in whole glomeruli compared with tuft alone). CONCLUSION: This emphasizes that studies focusing on processes in the mesangium, endothelial cells or podocytes should not rely on glomeruli obtained by sieving. Rather, a technique like the laser microdissection or in situ hybridization should be applied which allows the clear separation of different glomerular and periglomerular compartments.     

Growth factor expression in a murine model of cryoglobulinemia

BACKGROUND: Increased expression of growth factors including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to play pivotal roles during mesangial expansion and glomerulosclerosis. Thymic stromal lymphopoietin (TSLP) transgenic mice develop mixed cryoglobulinemia and a membranoproliferative glomerulonephritis (MPGN). Here we describe the renal expression of isoforms of PDGF and TGF-beta in relation to changes in extracellular matrix (ECM) components and markers of cell proliferation and activation in this model. METHODS: A total of 123 mice, including 61 TSLP transgenic mice and 62 wild-type controls, were sacrificed at defined intervals. PDGF-A chain, -B chain, PDGF alpha- and beta-receptor (beta-R) and TGF-beta1 mRNA were analyzed by in situ hybridization. Expression of alpha smooth muscle actin (alphaSMA), collagen type I, collagen type IV, laminin, and a marker of proliferating cells (PCNA) were assessed by immunohistochemistry. Slides also were studied by combined immunohistochemistry and in situ hybridization with an antibody that recognizes monocytes/macrophage and with riboprobes that detect PDGF B-chain, PDGF beta-R or TGF-beta1 mRNA. RESULTS: Increased numbers of proliferating glomerular cells appeared early in the disease course, associated with de novo expression of alphaSMA. Expression of PDGF B-chain and beta-R mRNA was increased in the mesangium and in parietal epithelial cells of TSLP transgenic mice and correlated with the number of PCNA positive cells. Increased TGF-beta1 mRNA expression paralleled the deposition of type IV collagen. A significant proportion of Mac-2 positive macrophages expressed TGF-beta1 mRNA, while only a small percentage of glomerular macrophages expressed PDGF B-chain mRNA. No PDGF beta-R mRNA expression by macrophages was detected. CONCLUSION: TSLP transgenic mice develop a membranoproliferative glomerulonephritis in which glomerular cell proliferation and matrix deposition are associated with an increased expression of PDGF B-chain, PDGF beta-R and TGF-beta1. These findings extend the paradigms covering these growth factors established in the rat Thy 1 model of mesangiolysis and repairs to a murine model of progressive glomerulonephritis closely resembling human MPGN.     

Osteopontin expression in human crescentic glomerulonephritis

Osteopontin expression in human crescentic glomerulonephritis. BACKGROUND: Osteopontin is a molecule with diverse biological functions, including cell adhesion, migration, and signaling. The expression of osteopontin has been demonstrated in a number of models of renal injury in association with accumulations of monocyte/macrophages, including recent reports of osteopontin expression in glomerular crescents in a rat model of anti-glomerular basement membrane glomerulonephritis. METHODS: Glomerular expression of osteopontin in biopsies of human crescentic glomerulonephritis (N = 25), IgA nephropathy with crescents (N = 2), and diffuse proliferative lupus glomerulonephropathy with crescents (N = 1) was studied by immunohistochemistry, in situ hybridization, and combined immunohistochemistry/in situ hybridization. Additionally, antibodies to cell-specific phenotypic markers were used to identify cellular components of the glomerular crescent, which express osteopontin protein and mRNA. RESULTS: All of the crescents present in the biopsies studied contained a significant number of cells that expressed osteopontin protein and mRNA, demonstrated by immunohistochemistry and in situ hybridization, respectively. Using replicate tissue sections and combined immunohistochemistry/in situ hybridization, we showed that the majority of the strongly osteopontin-positive cells are monocyte/macrophages. In addition to the very strong and cell-associated localization, a weaker and more diffuse pattern of osteopontin protein and mRNA expression could be seen in a number of crescents. None of the osteopontin mRNA-expressing cells could be identified as parietal epithelial cells, CD3-positive T cells, or alpha-smooth muscle actin-positive myofibroblasts. Interstitial monocyte/macrophages did not express osteopontin, except when located in a periglomerular inflammatory infiltrate. CONCLUSIONS: Macrophages present in the human glomerular crescent express osteopontin protein and mRNA at a high level. This expression supports a role for osteopontin in the formation and progression of the crescentic lesion via chemotactic and signaling properties of the molecule.     

Anti-perforin antibody treatment ameliorates experimental crescentic glomerulonephritis in WKY rats

The depletion of CD8+ cells has been shown to prevent the initiation and progression of antiglomerular basement membrane (GBM) crescentic glomerulonephritis (GN) in Wistar-Kyoto (WKY) rats. In this study, we asked whether CD8+ cells produce their effects by perforin/granzyme-mediated or by Fas ligand (FasL)-mediated pathways. The glomerular mRNA expression of perforin and granzyme B corresponded with the number of CD8+ cells, whereas that of granzyme A, Fas, and FasL did not. The enhanced mRNA level of perforin and granzyme B was not evident in CD8+-depleted rats. The number of apoptotic cells in the glomeruli was significantly increased at day 3. Perforin mRNA was found in cells infiltrating the glomerulus by in situ hybridization and by using dual-staining immunohistochemistry perforin protein was found in glomerular CD8+ cells. We found that perforin was readily visualized at the inner surface of the glomerular capillaries by immunoelectron microscopy. Based on these results, we treated animals with a perforin antibody in vivo and found that it significantly reduced the amount of proteinuria, frequency of crescentic glomeruli, and the number of glomerular monocytes and macrophages, although the number of glomerular CD8+ cells was not changed. Our results suggest that CD8+ cells play a role in glomerular injury as effector cells in part through a perforin/granzyme-mediated pathway in the anti-GBM WKY rat model of crescentic GN.     

TGF-beta type II receptor deficiency prevents renal injury via decrease in ERK activity in crescentic glomerulonephritis

The role of transforming growth factor-beta (TGF-beta) receptor complex in the pathogenesis of crescentic glomerulonephritis (GN) is not clear. To test the hypothesis that TGF-beta signaling plays a crucial role in the development and progression of crescentic GN by inducing the activation of extracellular signal-regulated kinase (ERK) and expression of its target genes, anti-glomerular basement membrane (GBM) GN was induced in TGF-beta type II receptor (TGF-betaIIR) gene heterozygous (TGF-betaIIR(+/-)) C57BL/6J mice and wild-type animals. GN was initiated in preimmunized mice by administration of rabbit anti-mouse GBM serum. TGF-betaIIR deficiency was significantly associated with decreased renal damage at days 14, 21, and 28 after induction of GN: renal function impairment, proteinuria, proportion of crescents, glomerular accumulation of periodic acid-Schiff-positive material, relative cortical interstitial volume, as well as renal cortical phosphorylation of ERK and plasminogen activator inhibitor type I (PAI-1) and alpha2(I) collagen mRNA levels were significantly decreased in TGF-betaIIR(+/-) mice compared with wild-type animals. These results provide the first direct evidence that TGF-betaIIR deficiency protects against renal injury in crescentic GN, possibly by inhibiting the sustained activation of ERK and PAI-1 and alpha2(I) collagen gene expression. Thus, TGF-beta signaling appears to play an important role in the development and progression of crescentic GN by inducing the ERK activity, and PAI-1 and alpha2(I) mRNA expression.     

Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease

BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases.     

Glomerular expression of C-C chemokines in different types of human crescentic glomerulonephritis.

BACKGROUND: Crescentic glomerulonephritis (CGN) presents a rapidly progressive glomerulonephritis clinically, in which macrophages play a crucial role in the pathogenesis. However, the precise molecular mechanism of macrophage recruitment and activation has not been fully elucidated. C-C chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha and beta (MIP-1alpha and MIP-1beta), are major chemoattractants for macrophages. We attempted to study the expression of C-C chemokines and their correlation with CD68-positive macrophages in crescentic glomeruli to investigate further their possible roles in crescent formation and progression to fibrosis in different types of human CGN. METHODS: The expression of MCP-1, MIP-1alpha, MIP-1beta and CD68 was detected in glomeruli with different forms of crescents (cellular, fibrocellular and fibrous crescents) by immunohistochemistry in serial sections of renal biopsies taken from 32 patients with biopsy-proven CGN including eight patients with anti-glomerular basement membrane (GBM) disease (type I CGN), 12 patients with immune complex-mediated CGN (type II CGN) and another 12 patients with pauci-immune CGN (type III CGN) enrolled in this study. Eight normal human kidneys were obtained from cadaveric renal transplant donors whose kidneys were technically unsuitable for transplantation, serving as controls. RESULTS: MCP-1, MIP-1alpha, MIP-1beta and CD68 were undetectable in glomeruli of normal kidney. In crescentic biopsies, MCP-1, MIP-1alpha, MIP-1beta and CD68 were detected in fibrocellular crescents and were even more prominent in cellular crescents, but were undetectable in fibrous crescents. Using consecutive sections for staining, it was demonstrated that a high proportion of infiltrating CD68-positive macrophages, mainly localized to the area of the expression of chemokines, were MCP-1, MIP-1alpha and MIP-1beta positive in crescents. Chemokines were expressed mainly by CD68-positive macrophages and parietal epithelial cells in crescents. The number of MCP-1- and MIP-1alpha-positive cells in glomeruli with cellular crescents was positively correlated with the number of CD68-positive cells (r = 0.568 and 0.749, respectively, both P < 0.01). The number of MCP-1- and MIP-1alpha-positive cells and the incidence of Bowman's capsule rupture in glomeruli of patients with type I CGN were higher than those of type II and type III CGN. CONCLUSIONS: These observations suggest that the expressed C-C chemokines, MCP-1, MIP-1alpha and MIP-1beta, may mediate the inflammatory process of crescent formation and progression to fibrosis. The strong correlation of MCP-1 and MIP-1alpha with infiltrating macrophages within glomeruli with cellular crescents suggested that these chemokines might be of particular importance for macrophage recruitment to this site. MCP-1 and MIP-1alpha were correlated to type I CGN with its more severe inflammatory course and worse prognosis. The variance of glomerular expression of C-C chemokines may contribute to the difference in histopathological features and prognosis in these three types of CGN.     

Intrarenal synthesis of IL-6 in IgA nephropathy

Summary: Recent in vitro studies have shown the synthesis of interleukin-6 (IL-6) in glomerular mesangial and epithelial cells, and suggested the involvement of IL-6 in mesangial proliferative glomerulonephritis. However, the expression site of IL-6 mRNA in renal tissue of IgA nephropathy (IgAN), the most common chronic mesangial proliferative glomerulonephritis, remains obscure. to localize IL-6 mRNA in renal biopsy specimens of IgAN, we used nonradioactive in situ hybridization (ISH) developed in our laboratory, sensitive in detecting individual cells positive for a specific mRNA. In some sections, periodic acid-Schiff staining was performed after ISH in order to identify the topographical relation between IL-6 mRNA positive cells and glomerular basement membrane and mesangial area. In situ hybridization for IL-6 mRNA and immunohistochemistry for CD3 and CD68, markers for lymphocytes and monocytes, respectively, were also performed on serial sections to examine the contribution of infiltrated mononuclear cells to cells positive for IL-6 mRNA in glomeruli. Glomerular resident cells, including glomerular mesangial and epithelial cells and cells of Bowman's capsule, as well as tubular epithelial cells and infiltrated mononuclear cells expressed IL-6 mRNA. We also compared the localization of IL-6 mRNA and protein and showed different distribution between the gene product and protein. the expression of IL-6 mRNA correlated with the degree of mesangial cell proliferation and tubulointerstitial changes. Our results indicate that IL-6 is synthesized in renal tissues of IgAN and suggest that the increased IL-6 expression may be important in the pathogenesis of IgAN.     

Proteinase-3 mRNA expressed by glomerular epithelial cells correlates with crescent formation in Wegener's granulomatosis

BACKGROUND: Wegener's granulomatosis (WG) is characterized by systemic vasculitis with crescentic glomerulonephritis (CGN) and circulating autoantibodies directed against neutrophil cytoplasmic antigens (ANCA). Proteinase 3 (PR-3), a neutral serine proteinase in neutrophils implicated in the growth control of myeloid cells, has been identified as the target antigen for ANCA in WG. Since the kidneys are frequently involved in WG, we studied the in situ expression of PR-3 by renal parenchymal cells. METHODS: We assessed the expression of PR-3 in kidney biopsies of 15 patients with WG by immunohistochemistry (IHC) and in situ hybridization (ISH). Normal kidney tissue served as the control. RESULTS: We detected PR-3 mRNA and PR-3 protein in distal tubular epithelial cells (TECs) and glomerular epithelial cells (GECs) in normal kidney tissue and in CGN. Furthermore, a strong glomerular PR-3mRNA expression restricted to the site of cellular crescents was detected in patients with WG. The analysis of 144 glomeruli with cellular or sclerotic crescents revealed a positive correlation of glomerular PR-3mRNA expression with the percentage of cellular crescents per glomerulus. The capability of human TECs and GECs to synthesize PR-3 was confirmed by Northern blot and ISH on cultured cells. CONCLUSION: These data provide evidence that nonhematopoetic renal parenchymal cells express PR-3 and that glomerular expression of PR-3 is associated with crescent formation in WG. Our findings suggest that renal parenchymal cells may directly be involved in the pathogenesis of CGN in WG.     

Immunohistochemical analysis of extracellular components in the glomerular sclerosis of patients with glomerulonephritis

Immunofluorescence and immunoperoxidase staining was carried out to determine correlations between the progression of glomerular sclerosis and changes in the amount or distribution of glomerular extracellular components, including type I, III, IV, and VI collagens, laminin and fibronectin, in patients with IgA nephropathy, membranoproliferative glomerulonephritis and rapidly progressive glomerulonephritis. Staining of type I and III collagens was not observed in glomeruli from normal individuals or patients with mild glomerulonephritis. In the advanced stages of glomerulonephritis, the staining of type IV and VI collagens, laminin and fibronectin was marked in the glomerular mesangium, and the distribution of fibronectin extended to the glomerular capillary walls in the sclerotic lesions of glomeruli. However, the staining intensity of type IV collagen, laminin and fibronectin was gradually decreased during the progression of glomerular sclerosis. On the other hand, the staining of type I and III collagens was observed focally in sclerotic or hyalinotic glomeruli and around such glomeruli in those patients. Light microscopic examination revealed that patients who showed marked staining of type I and III collagens by immunofluorescence had severe damage of Bowman's capsules. These results suggest that the hyperproduction and/or invasion of interstitial collagens, i.e., types I and III, are closely linked to the progression of glomerular sclerosis and hyalinosis in patients with various types of glomerulonephritis.     

Activation of mRNA expression of collagen,collagenase and its inhibitor on renal biopsy specimens in patients with IgA nephropathy

Summary: In situ hybridization of mRNA for collagen IV, collagen VI, stromelysin (MMP-3) and TIMP1 was examined in renal biopsy specimens from patients with IgA nephropathy (IgAN) or diabetic nephropathy with various degrees of tissue damage. The majority of cells in the glomeruli expressed these mRNA almost simultaneously, but a few cells demonstrated positive expression for only one of these probes. There was a parallel relationship between the degree of tissue damage and that of mRNA expressions of these probes in patients with IgAN, while patients with diabetic nephropathy showed a reverse relationship between these two parameters. It is concluded that patients with mesangial proliferative glomerulonephritis expressed mRNA for collagen collagenase and its inhibitor in the glomeruli in parallel with the progress of tissue damage. In contrast, glomerular samples from patients with diabetic nephropathy showed that there was an inverse relationship between tissue damage and expression of mRNA. It is concluded that expression of collagen, collagenase and its inhibitor parallels the progression of glomerular changes in IgAN, but such parallel expression was not observed in patients with diabetic nephropathy.     

Angiopoietin correlates with glomerular capillary loss in anti-glomerular basement membrane glomerulonephritis

BACKGROUND: Emerging evidence suggests that endothelial turnover occurs in several glomerular diseases and correlates with resolution or progression of glomerular lesions. We hypothesized that the growth factors modulating embryonic kidney endothelial cell survival and capillary morphogenesis may be implicated in capillary loss that occurs in immune-mediated glomerulonephritis (GN). METHODS: GN was induced in C57BL/6 mice by intravenous administration of sheep anti-mouse glomerular basement membrane (GBM) globulin and assessed with markers of vascularity in glomerular lesions, correlating these with expression of specific vascular growth factors. RESULTS: As assessed by periodic acid Schiff staining, 14 +/- 4% (mean +/- SD) glomeruli were affected by sclerosis at 14 days after globulin administration, and 33 +/- 5% were affected at 21 days. By 21 days, a significant increase of plasma creatinine and urinary protein occurred. P-selectin expression was increased in glomerular capillaries 14 days after disease induction, and capillary loss, as assessed by immunohistochemistry for platelet-endothelial cell adhesion molecule, vascular endothelial growth factor (VEGF) receptor 2 and the angiopoietin (Ang) receptor Tie-2, was recorded at 14 and 21 days in glomeruli affected by proliferative crescents and/or sclerosis. VEGF-A immunostaining, evident in control glomeruli, was qualitatively diminished in glomeruli with lesions. Ang-1 immunostaining was detected in control glomeruli and was diminished at 14 days after administration of anti-mouse GBM globulin; instead, Ang-1 was immunolocalized to distal tubules. In contrast, Ang-2 immunostaining was barely detectable in control glomeruli but was prominent in disease glomeruli. In GN mice, rare apoptotic glomerular endothelia were detected by electron microscopy and in situ end-labeling, but such cells were not seen in controls. CONCLUSIONS: Loss of glomerular capillaries during the course of anti-GBM GN in mice was temporally associated with decreases in endothelial survival molecules VEGF-A and Ang-1, and with up-regulation of Ang-2, an antagonist of Ang-1. A changing balance of these growth factors may contribute to decreased glomerular vascularity in crescentic GN.     

Important role for macrophages in induction of crescentic anti-GBM glomerulonephritis in WKY rats.

BACKGROUND: A crucial role for CD8(+) cells in induction of crescentic anti-glomerular basement membrane (GBM) glomerulonephritis (GN) in WKY rats was demonstrated in studies showing that depletion of CD8(+) cells completely suppressed glomerular accumulation of monocytes/macrophages (Mo/Mphi), crescent formation and proteinuria. Because these studies did not definitively identify CD8(+) cells as the cause of tissue injury, we examined the roles of Mo/Mphi in the development of anti-GBM GN. METHODS: We examined correlations between the amount of urinary protein and the numbers of glomerular CD8(+) cells or Mo/Mphi in rats after administrating different doses of anti-GBM antibody (5.0, 7.5, 10.0 and 25.0 microl/100 g body weight). The roles of Mo/Mphi in induction of GN were examined in animals by depleting Mo/Mphi in the glomerulus. To do this, rats were injected intravenously with liposome-encapsulated dichloromethylene diphosphonate (liposome-MDP) from day 3 to day 7 after anti-GBM antibody injection and they were then sacrificed at day 8. RESULTS: Liposome-MDP treatment significantly reduced the number of ED-1(+) Mo/Mphi accumulated in glomeruli from 32.1 +/- 1.2 to 1.4 +/- 0.3/glomerular cross-section (mean +/- SD, P < 0.01), and the amount of urinary protein from 103.8 +/- 19.8 to 31.8 +/- 15.9 mg/day (P < 0.01), as well as the incidence of crescentic glomeruli from 91.3 +/- 2.7 to 23.3 +/- 7.6% (P < 0.01) at day 8. This treatment also reduced the number of CD8(+) cells accumulating in the glomeruli from 5.4 +/- 0.7 to 0.5 +/- 0.1/glomerular cross-section (P < 0.01). Upregulation of glomerular intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was suppressed by Mo/Mphi depletion. CONCLUSION: These results indicate that Mo/Mphi play an important role in the induction of crescentic anti-GBM GN and glomerular injury.     

Leukocyte analysis using monoclonal antibodies in human glomerulonephritis

The leukocyte subpopulations were analyzed within both the glomeruli and the interstitium in renal biopsies from 145 patients with various forms of glomerulonephritis. Cells were identified by monoclonal antibodies to leukocyte cell-surface antigens and immunoperoxidase labelling. Leukocytes, as defined by a monoclonal antibody to the leukocyte common antigen (PHM1), were present in normal, human renal tissue in both glomeruli (2.8 +/- 0.6 cells/glom. cross section) and interstitium (102 +/- 18 cells/mm2). Monocytes constituted the predominant infiltrating cell type in normal glomeruli (1.3 +/- 0.2) and T cells were rarely found (0.3: range 0 to 0.8), whereas both monocytes (34 +/- 10/mm2) and T lymphocytes (33 +/- 14/mm2) were found in the normal interstitium. In the non-proliferative forms of glomerulonephritis there was no significant increase in the number of glomerular inflammatory cells when compared with normal glomeruli. However, significantly increased numbers of T lymphocytes were seen in the interstitium of biopsies with minor non-specific changes (67 +/- 15/mm2), membranous nephropathy (134 +/- 30/mm2), focal glomerulosclerosis (207 +/- 53/mm2), and diabetic nephropathy (198 +/- 81/mm2). In the proliferative forms of glomerulonephritis only crescentic GN and post-infectious GN demonstrated significantly-increased glomerular monocytes and granulocytes. There was no significant increase in the number of glomerular T cells when compared with normal glomeruli. However, there was a significant increase in the number of interstitial T lymphocytes in all forms of proliferative glomerulonephritis when compared with the normal interstitial cell population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical analysis of extracellular components on the glomerular sclerosis in patients with glomerulonephritis and diabetic nephropathy

Immunofluorescence and immunoperoxidase staining were carried out to determine the correlations between the progression of glomerular sclerosis and changes in the amount and distribution of glomerular extracellular components, such as Type I, III, IV, V, VI collagen, laminin (LN) and fibronectin (FN) in patients with various types of glomerulonephritis and diabetic nephropathy. Six patients with IgA nephropathy, four patients with membrano-proliferative glomerulonephritis, four patients with rapidly progressive glomerulonephritis and six patients with diabetic nephropathy were examined. The intensity and distribution of Type IV collagen, LN and FN were similar between the glomeruli from normal individuals and patients with mild stages of glomerulonephritis and diabetic nephropathy. However, staining of Type I, III or V collagen was not observed in the glomeruli from normal individuals and such patients. In more advanced stages of glomerulonephritis and diabetic nephropathy, the amounts of Types IV and VI collagen, LN and FN were increased markedly in the mesangium, and their distribution extended along the glomerular capillary walls. The intensity of Type IV collagen, LN or FN in the nodular sclerotic lesions of glomeruli was decreased significantly in patients with glomerulonephritis and diabetic nephropathy. On the other hand, staining of Types I, III and V collagen was observed focally in the sclerotic or hyalinotic glomeruli and around such glomeruli in these patients. In light microscopic examinations, the patients who had marked staining of Type I, III or V collagen by immunofluorescence showed severe damage of the basement membrane in Bowman's capsules. It is concluded that hyperproduction and/or infiltration of interstitial collagens, i.e. Types I, III and V collagen, is closely linked to the progression of glomerular sclerosis and hyalinosis in patients with various types of glomerulonephritis and diabetic nephropathy.