Mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents

BACKGROUND: We investigated the mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents. METHODS: Crescentic glomerulonephritis was induced by i.v. injection of rabbit anti-rat glomerular basement membrane antiserum in WKY rats. RESULTS: Cellular crescents (83.5% of glomeruli) were first observed at day 7 after disease induction. Immunostaining of alpha-smooth muscle actin (alpha-SMA), as a marker for the myofibroblast phenotype, was found in some periglomerular regions as early as day 3, when it was also seen in parietal epithelial cells (PEC) of Bowman's capsule at day 5 and in crescent formation at day 7. Proliferation marker Ki67-positive PEC was found at day 3, and double Ki67- and alpha-SMA-positive PEC could be seen at day 5. The migratory figure of PEC with the expression of alpha-SMA was found by immunoelectron microscopy. At day 7, some crescent cells were stained positive for PEC marker, protein gene product 9.5, in association with alpha-SMA or Ki67. Expression of transforming growth factor (TGF)-beta receptor types I and II, as well as platelet-derived growth factor (PDGF) receptor beta and PDGF-B increased in PEC as early as day 3. At day 5 marked deposition of cellular and common fibronectin, but not other extracellular matrix components examined was found in Bowman's spaces where ED 1-positive macrophages infiltrated. CONCLUSIONS: PEC may be stimulated to proliferate and/or transdifferentiate into myofibroblast phenotype possibly by action of TGF-beta and PDGF and/or binding of fibronectin to PEC, then migrate and/or proliferate, participating in glomerular crescents.     

Roles of and correlation between alpha-smooth muscle actin, CD44, hyaluronic acid and osteopontin in crescent formation in human glomerulonephritis

BACKGROUND: alpha-Smooth muscle actin (SMA), CD44, hyaluronic acid (HA) and osteopontin (OPN) are involved in crescent formation; however, the correlation between these molecules during the formation and progression of the crescents in human glomerulonephritis (GN) has not been fully evaluated. METHODS: To investigate the expression of alpha-SMA, CD44, HA, OPN and CD68 renal biopsy specimens from 14 patients with crescentic GN were examined by immunohistochemistry. All crescents were separated into cellular, fibrocellular and fibrous. The extent of staining in each crescent was scored semiquantitatively. The change in the expression of each molecule and its correlation with other molecules during the formation and progression of the crescents were estimated statistically. RESULTS: The expression of alpha-SMA was significantly up-regulated in the fibrocellular crescents compared with that in the cellular and fibrous crescents. The expression of CD44, OPN and CD68 was significant in the cellular crescents compared with that in the fibrocellular and fibrous crescents. The deposition of HA in the three groups of crescents was high level. However, that of HA was not significant among three groups of crescent. The expression of CD44 in the cellular crescents correlated significantly with the expression of OPN and CD68, and the deposition of HA in the cellular crescents. The expression of OPN in the cellular crescents correlated with the deposition of HA and the expression of CD68 in the cellular crescents. The expression of alpha-SMA in the cellular and fibrocellular crescents correlated with the deposition of HA in the cellular and fibrocellular crescents. CONCLUSION: The expression of CD44, HA, OPN and CD68 was up-regulated at the early stage of the crescent formation in human crescentic GN. Moreover, myofibroblasts and cell-matrix interactions mediated by the CD44-OPN and CD44-HA receptor-ligand pairs may play important roles in the formation and progression of the crescents.     

Expression of gremlin, a bone morphogenetic protein antagonist, in glomerular crescents of pauci-immune glomerulonephritis.

BACKGROUND: Recent evidence in vitro and in vivo suggests that gremlin, a bone morphogenetic protein antagonist, is participating in tubular epithelial mesenchymal transition (EMT) in diabetic nephropathy as a downstream mediator of TGF-beta. Since EMT also occurs in parietal epithelial glomerular cells (PECs) leading to crescent formation, we hypothesized that gremlin could participate in this process. With this aim we studied its expression in 30 renal biopsies of patients with pauci-immune crescentic nephritis. METHODS: Gremlin was detected by in situ hybridization (ISH) and immunohistochemistry (IMH) and TGF-beta by ISH and Smads by southwestern histochemistry (SWH). Phosphorylated Smad2, CTGF, BMP-7, PCNA, alpha-SMA, synaptopodin, CD-68, and phenotypic markers of PECs (cytokeratin, E-cadherin), were detected by IMH. In cultured human monocytes, gremlin and CTGF induction by TGF-beta was studied by western blot. RESULTS: We observed strong expression of gremlin mRNA and protein in cellular and fibrocellular crescents corresponding to proliferating PECs and monocytes, in co-localization with TGF-beta. A marked over-expression of gremlin was also observed in tubular and infiltrating interstitial cells, correlating with tubulointerstitial fibrosis (r=0.59; P<0.01). A nuclear Smad activation in the same tubular cells, that are expressing TGF-beta and gremlin, was detected. In human cultured monocytes, TGF-beta induced gremlin production while CTGF expression was not detected. CONCLUSION: We postulate that gremlin may play a role in the fibrous process in crescentic nephritis, both in glomerular crescentic and tubular epithelial cells. The co-localization of gremlin and TGF-beta expression found in glomeruli and tubular cells suggest that gremlin may be important in mediating some of the pathological effects of TGF-beta.     

Glomerular epithelial-myofibroblast transdifferentiation in the evolution of glomerular crescent formation.

BACKGROUND:Glomerular cellular crescents consist of epithelial cells and macrophages, which can undergo an irreversible process of fibrous organization. However, the origin of the fibroblast-type cells that mediate this fibrous organization is unclear. METHODS: This study examined glomerular epithelial- myofibroblast transdifferentiation (GEMT) in the formation and evolution of glomerular crescents in two distinct rat models of glomerulonephritis: 5/6 nephrectomy and antiglomerular basement membrane (GBM) disease. RESULTS: Early in the course of both disease models, and prior to crescent formation, immunohistochemistry staining and in-situ hybridization demonstrated de novo expression of alpha-smooth-muscle actin (alpha-SMA), a marker of smooth muscle cells and myofibroblasts, by glomerular parietal epithelial cells (GPEC). The expression of alpha-SMA by GPEC was accompanied by a loss of E-cadherin staining, a marker of epithelial cells. At this early stage of GEMT, ultrastructural studies identified the presence of characteristic actin microfilaments and dense bodies within GPEC which retained a normal epithelial morphology with apical-basal polarity and microvilli. A late stage of transdifferentiation was seen in fibrocellular crescents. In this case, GPEC attached to intact segments of the capsular basement membrane contained large bundles of actin microfilaments throughout the cell, and this was accompanied by a loss of polarity, microvilli, and tight junctions. There was a significant correlation between the presence of alpha-SMA(+) GPEC and glomerular crescent formation. Cellular crescents contained small numbers of alpha-SMA(+) myofibroblasts. These cells become the dominant population in fibrocellular crescents, which was associated with marked local proliferation. Relatively few alpha-SMA(+) myofibroblasts remained in fibrotic/organizing crescents. Most cells within cellular and fibrocellular crescents expressed transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (FGF-2), suggesting that these growth factors may regulate this GEMT process during the evolution of glomerular crescents. CONCLUSIONS: This study provides the first phenotypic and morphological evidence that glomerular epithelial-myofibroblast transdifferentiation participates in the formation and evolution of glomerular crescents.     

Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis

Although experimental crescentic glomerulonephritis starts with an endocapillary inflammation, the crescents themselves seem to originate from the proliferation of parietal epithelial cells (PEC). In this study, an attempt was made to disclose a link between the two processes by a morphologic analysis of early stages of the disease. Mice were immunized with rabbit IgG in complete Freund's adjuvant on day -6. At day 0, they received an intravenous injection of a rabbit antiglomerular basement membrane serum. On days 3, 6, and 10, the kidneys were fixed by vascular perfusion for examination by light and electron microscopy. On day 3, morphologic alterations affected mainly the endocapillary compartment; most podocytes appeared to be intact. On day 6, alterations of podocytes were widespread, including foot process effacement and prominent microvillous transformation, and some crescents were found. On day 10, crescents were found in 40% of glomeruli. The most surprising finding was podocytes that adhered to both the glomerular basement membrane and the parietal basement membrane, thus forming bridges between the tuft and Bowman's capsule. Those podocyte bridges were sparse on day 3 but were regularly encountered on days 6 and 10 in glomeruli without crescents and also as a component of crescents. They were interposed between PEC and later between the cells of a crescent without formation of junctional connection with these cells. It is proposed that the spreading of podocytes on the parietal basement membrane represents a lesion of the parietal epithelium and that this process initiates the proliferation of PEC to form a crescent.     

PINCH-1 promotes tubular epithelial-to-mesenchymal transition by interacting with integrin-linked kinase

PINCH-1 is an adaptor protein that binds to the integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays a critical role in mediating tubular epithelial-to-mesenchymal transition (EMT). To determine whether PINCH-1 is also involved in the EMT process, we investigated its regulation and function during TGF-beta1-stimulated EMT. TGF-beta1 induced PINCH-1 mRNA and protein expression in human proximal tubular epithelial cells in a time-dependent fashion, an effect that was largely dependent on intracellular Smad signaling. Overexpression of PINCH-1 suppressed epithelial markers E-cadherin and ZO-1 and increased fibronectin expression and extracellular assembly, whereas knockdown of PINCH-1 via small interfering RNA reduced TGF-beta1-mediated fibronectin expression and partially restored E-cadherin. PINCH-1 formed a ternary complex with ILK at the focal adhesion sites of tubular epithelial cells. Treatment with an ILK inhibitor or disruption of the ILK/PINCH-1 interaction by overexpressing a dominant-negative N-terminal ankyrin domain of ILK resulted in reduced fibronectin deposition, indicating that the ability of PINCH-1 to stimulate EMT is ILK-dependent. In a mouse model of obstructive nephropathy, PINCH-1 expression increased in a time-dependent manner, suggesting that it may play a role in EMT and renal fibrosis in vivo. We conclude that PINCH-1, through its interaction with ILK, plays an important role in regulating TGF-beta1-mediated EMT and could be a potential future therapeutic target to prevent progression of renal disease.     

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.     

Tissue factor pathway inhibitor expression in human crescentic glomerulonephritis

BACKGROUND: Tissue factor (TF) pathway inhibitor (TFPI), the major endogenous inhibitor of extrinsic coagulation pathway activation, protects renal function in experimental crescentic glomerulonephritis (GN). Its glomerular expression and relationship to TF expression and fibrin deposition in human crescentic GN have not been reported. METHODS: Glomerular TFPI, TF, and fibrin-related antigen (FRA) expression were correlated in renal biopsies from 11 patients with crescentic GN. Biopsies from 11 patients with thin basement membrane disease and two normal kidneys were used as controls. RESULTS: TFPI was undetectable in control glomeruli but was detectable in interstitial microvessels. In crescentic biopsies, TFPI was detected in cellular crescents and was more prominent in fibrous/fibrocellular crescents, indicating a correlation with the chronicity of crescentic lesions. TFPI appeared to be associated with macrophages but not endothelial or epithelial cells. TFPI was generally undetectable in regions of the glomerular tuft with minimal damage. In contrast, TF and FRA were strongly expressed in regions of minimal injury, as well as in more advanced proliferative and necrotizing lesions. Despite prominent TF expression, FRA was less prominent in fibrous/fibrocellular crescents in which TFPI expression was maximal. CONCLUSIONS: These data suggest that TFPI is strongly expressed in the later stages of crescent formation and is inversely correlated with the presence of FRA in human crescentic GN. This late induction of TFPI may inhibit TF activity and favor reduced fibrin deposition in the chronic stages of crescent formation.     

Urinary FSP1 is a biomarker of crescentic GN

Fibroblast-specific protein 1 (FSP1)-expressing cells accumulate in damaged kidneys, but whether urinary FSP1 could serve as a biomarker of active renal injury is unknown. We measured urinary FSP1 in 147 patients with various types of glomerular disease using ELISA. Patients with crescentic GN, with or without antinuclear cytoplasmic antibody-associated GN, exhibited elevated levels of urinary FSP1. This assay had a sensitivity of 91.7% and a specificity of 90.2% for crescentic GN in this sample of patients. Moreover, we found that urinary FSP1 became undetectable after successful treatment, suggesting the possible use of FSP1 levels to monitor disease activity over time. Urinary FSP1 levels correlated positively with the number of FSP1-positive glomerular cells, predominantly podocytes and cellular crescents, the likely source of urinary FSP1. Even in patients without crescent formation, patients with high levels of urinary FSP1 had large numbers of FSP1-positive podocytes. Taken together, these data suggest the potential use of urinary FSP1 to screen for active and ongoing glomerular damage, such as the formation of cellular crescents.     

Crescent-forming mechanism in an irreversible Thy-1 model in rats.

BACKGROUND: The crescent-forming mechanism has not yet been fully clarified and a cell which constitutes a crescent still remains controversial. This study was undertaken to analyze the crescent-forming mechanism in an irreversible Thy-1 model by applying a new marker-recognizing monoclonal antibody (mAb) OS-3. METHODS: An irreversible Thy-1 model was induced by an intravenous injection of 500 microg of anti-Thy-1 mAb 1-22-3 to unilaterally nephrectomized Wistar rats. Seven rats were sacrificed 3, 7 and 14 days after the mAb injection respectively and the renal tissues were examined histologically and immunohistochemically. RESULTS: Inflammatory cells were demonstrated mostly in the interstitium, but they were located within advanced cellular crescents in later stages. OS-3, which stained parietal glomerular epithelial cell (PGEC) only partly in a normal rat kidney section, reacted to PGEC more extensively at day 3 and also with cellular crescents at day 7. During the course of this model the podocytes lost their characteristic to be stained by anti-podocalyxcin Ab and obtained a new marker of a diseased state, i.e. to be positively stained by OS-3. CONCLUSION: Glomerular epithelial cells, but not inflammatory cells, are suggested to directly participate in the crescent formation in early stages, and podocytes with phenotypic changes might be partly involved in the formation of the crescents.     

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.     

Angiogenic protein Cyr61 is expressed by podocytes in anti-Thy-1 glomerulonephritis

Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.     

Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney

Parietal epithelial cells (PEC) of Bowman's capsules cover the inner aspect of Bowman's capsules and are believed to contribute to extracapillary lesions of glomerulonephritis such as crescent formation. In glomerular research including cell culture experiments and pathology, differentiation between PEC and podocytes has frequently been a major problem. Immunohistochemistry of the adult rat kidney for protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in PEC. At the urinary pole of the glomerulus, immunoreactive PEC were clearly differentiated from proximal tubular cells that were negative for PGP 9.5. In the subcapsular nephrogenic zone of newborn rat kidney, immunoreactivity was observed in almost all cells in the commashaped body and early S-shaped body and selectively in PEC in the late S-shaped body and capillary-stage glomerulus. In rat glomerular disease models (Masugi-nephritis and puromycin aminonucleoside nephrosis), cells that consisted of cellular crescents or adhered to glomerular tufts were positive for PGP 9.5. The selective localization of PGP 9.5 in PEC in rat kidney provides a new cytochemical marker for identifying the cells. Development expression of the protein suggests that PGP 9. 5 is involved in the processes of nephrogenesis of rat kidney.     

Glomerular epithelial-mesenchymal transdifferentiation in pauci-immune crescentic glomerulonephritis.

BACKGROUND: Among the cellular changes occurring in renal fibrosis, epithelial-mesenchymal cell transdifferentiation or transition (EMT) is a phenomenon characterized in epithelial cells by loss of epithelial markers and acquisition of mesenchymal phenotype and of fibrosing properties. METHODS: To test the hypothesis that EMT is involved in human pauci-immune crescentic glomerulonephritis (PICGN), we studied 17 renal biopsies from 11 PICGN patients for: (i) proliferating cell nuclear antigen (PCNA) and cell cycle inhibitors (cyclin-dependent kinase inhibitors) p27 and p57; (ii) cell lineage phenotype markers: podocalyxin, synaptopodin and GLEPP-1 for podocytes; CD68 for macrophagic epitope; CD3 for T lymphocytes; alpha-smooth muscle actin (alpha-SMA) for myofibroblasts; vimentin for mesenchymal cells; and cytokeratins (CKs) for parietal epithelial cells (PECs); (iii) glomerular fibrosis by labelling collagens I, III and IV, and heat-shock protein 47 (HSP47), a marker of collagen-synthesizing cells; and (iv) co-localization of alpha-SMA, CK and HSP47 using confocal laser microscopy. RESULTS: The crescent cells proliferated greatly. They did not express p27 and p57. Different cell lineage markers could be identified in crescents: the major component was made of 'dysregulated' PECs negative for CK, followed by PECs positive for CK, macrophagic cells and myofibroblasts. Furthermore, some cells co-expressed CK and alpha-SMA. This latter co-expression suggests a transitional phase in the dynamic phenomenon of EMT. Therefore, proliferative and dysregulated glomerular epithelial cells could be a possible cellular source of myofibroblasts via EMT. In addition, HSP47 labelled many crescent cells and frequently co-localized in CK-positive epithelial cells and in alpha-SMA-positive myofibroblasts, indicating that these cells were involved in glomerular accumulation of collagens. CONCLUSION: EMT is a transient cellular phenomenon present in glomeruli in human PICGN contributing to the formation of myofibroblasts from epithelial cells and to glomerular fibrosis.     

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.     

Cellular and non-cellular compositions of crescents in human glomerulonephritis

The composition of glomerular crescents was examined on the frozen kidney sections obtained from 10 patients (5 patients with IgA nephropathy, two with Henoch-Sch?nlein purpura nephritis and three with glomerulonephritis due to undetermined etiology) using well-defined monoclonal and polyclonal antibodies to coagulation proteins, extracellular matrices, intermediate filament proteins and immune cells. Fibrinogen/fibrin related antigens (FRA), which were stained with anti-fibrinogen serum, were positive in the crescents of all the patients, but monoclonal antibody to crosslinked fibrin or von Willebrand factor (factor VIII related) antigen did not bind to the crescents. This suggests that the FRA deposited in the crescents is fibrinogen or its degradation products rather than fibrin. Staining for intrinsic components of renal basement membrane, including type IV and V collagens, laminin and fibronectin, were consistently positive in all stages of the crescents. Cytokeratin, showing cytoplasmic staining of the glomerular parietal epithelium and tubular epithelium in the normal kidney, was demonstrated in three patients with cellular crescents. Vimentin, which is normally distributed in parietal and visceral epithelial cells in the glomeruli and interstitial cells, was found at all stages of the crescents. These findings suggest that in the early stage of crescent formation, glomerular epithelial cells play an important role, and that the accumulation of intrinsic basement membrane constituents is associated with the formation and progression of the crescents. None of the crescent cells reacted with either of two monoclonal antibodies (Mo2 and FMC 32) to monocytes/macrophages or with nonspecific esterase staining. It seems that, at least in our patients, monocytes are a minor factor contributing to the formation of glomerular crescents.     

Podocyte involvement in human immune crescentic glomerulonephritis

BACKGROUND: The role of podocytes in human crescentic glomerulonephritis (GN) has been underestimated. This may be due to the confounding fact that "dysregulated" podocytes are able to proliferate, lose their markers, and acquire new epitopes. Moreover, in experimental anti-glomerular basement membrane (GBM) crescentic GN, podocytes participate in the crescent formation. The aim of this study was to investigate the involvement of podocytes in human immune crescentic GN. METHODS: Renal biopsies from 12 patients with anti-GBM disease and 14 with class IV lupus GN were studied by immunohistochemistry for the following markers: (1) synaptopodin, GLEPP1, podocalyxin, podocin, alpha-actinin-4, and vimentin for podocyte identification; (2) PCNA, Ki-67, and p57 for cell cycle assessment; (3) cytokeratins for identifying epithelial cells but not normal podocytes; (4) CD68 for tagging a macrophagic epitope; (5) alpha-smooth-muscle actin (alpha-SMA), a phenotypic marker of myofibroblasts. RESULTS: "True" (capsular) crescents lining Bowman's capsule and (tuft) "pseudocrescents" covering the glomerular tuft with a persistent patent urinary space were present in the 2 types of crescentic GN in similar percentages. Several features indicated that podocytes were involved in the formation of the both crescent types. Identifiable podocytes expressed proliferation markers. Podocyte cytoplasmic expansions and racket-like podocytes bridged between the tuft and Bowman's capsule. True and pseudocrescents contained labeled podocytes. In addition, podocytes located outside of the crescents had often lost their markers (dedifferentiation) and acquired new epitopes (cytokeratins and CD68). CONCLUSION: In human immune crescentic GN, podocytes undergo proliferation and dysregulation that are indicative of a podocytopathy. Podocytes contribute to crescent formation.