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Genetic Podocyte Lineage Reveals Progressive Podocytopenia with Parietal Cell Hyperplasia in a Murine Model of Cellular/Collapsing Focal Segmental Glomerulosclerosis

2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2009.080789

1525-2191

Taisei Suzuki, Taiji Matsusaka, Makiko Nakayama, Takako Asano, Teruo Watanabe, Iekuni Ichikawa, Michio Nagata,

Cell Adhesion Molecules Research

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle. Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle. Glomerulosclerosis is a common pathological process that promotes functional deterioration of the kidney irrespective of the cause of the disease. In addition to mesangial proliferation and matrix accumulation pathways that underlie glomerulosclerosis,1Striker LJ Doi T Elliot S Striker GE The contribution of glomerular mesangial cells to progressive glomerulosclerosis.Semin Nephrol. 1989; 9: 318-328PubMed Google Scholar, 2Fogo AB Mesangial matrix modulation and glomerulosclerosis.Exp Nephrol. 1999; 7: 147-159Crossref PubMed Scopus (92) Google Scholar recent studies suggest that alterations in resident epithelial cells are pivotal to the progression of glomerulosclerosis, and such alterations are typical in focal segmental glomerulosclerosis (FSGS).3Kriz W Gretz N Lemley KV Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Crossref PubMed Scopus (510) Google Scholar, 4D'Agati VD Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Crossref PubMed Scopus (118) Google ScholarThe glomerulus consists of two resident epithelial cell types, ie, podocytes and parietal cells (PECs). Both cell types share the proliferative phenotype during glomerulogenesis. At the capillary loop stage, podocytes express specific markers of differentiation accompanied with cell cycle inactivation.5Pavenstädt H Kriz W Kretzler M Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307PubMed Google Scholar, 6Combs HL Shankland SJ Setzer SV Hudkins KL Alpers CE Expression of the cyclin kinase inhibitor, p27kip1, in developing and mature human kidney.Kidney Int. 1998; 53: 892-896Crossref PubMed Scopus (64) Google Scholar, 7Nagata M Nakayama K Terada Y Hoshi S Watanabe T Cell cycle regulation and differentiation in the human podocyte lineage.Am J Pathol. 1998; 153: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Likewise, loss of podocytes has been shown to cause segmental glomerulosclerosis in postadaptive FSGS, and the amount of urinary podocytes is correlated with disease progression in human FSGS and experimental renal diseases. 8Nagata M Kriz W Glomerular damage after uninephrectomy in young rats.II Mechanical stress on podocytes as a pathway to sclerosis Kidney Int. 1992; 42: 148-160PubMed Scopus (224) Google Scholar, 9Hara M Yanagihara T Kihara I Urinary podocytes in primary focal segmental glomerulosclerosis.Nephron. 2001; 89: 342-347Crossref PubMed Scopus (126) Google Scholar, 10Yu D Petermann A Kunter U Rong S Shankland SJ Floege J Urinary podocyte loss is a more specific marker of ongoing glomerular damage than proteinuria.J Am Soc Nephrol. 2005; 16: 1733-1741Crossref PubMed Scopus (220) Google Scholar In contrast, epithelial cell hyperplasia is involved in cellular/collapsing FSGS.4D'Agati VD Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Crossref PubMed Scopus (118) Google Scholar, 11Barisoni L Schnaper HW Kopp JB A proposed taxonomy for the podocytopathies: a reassessment of the primary nephrotic diseases.Clin J Am Soc Nephrol. 2007; 2: 529-542Crossref PubMed Scopus (195) Google Scholar, 12Churg J Habib R White RH Pathology of the nephrotic syndrome in children: a report for the International Study of Kidney Disease in Children.Lancet. 1970; 760: 1299-1302Abstract PubMed Scopus (442) Google Scholar, 13Schwartz MM Lewis EJ Focal segmental glomerular sclerosis: the cellular lesion.Kidney Int. 1985; 28: 968-974Crossref PubMed Scopus (129) Google ScholarLesions in cellular/collapsing FSGS reveal unique features, and the proliferating epithelial cells typically overlie the collapsed tuft showing visceral cell hyperplasia.11Barisoni L Schnaper HW Kopp JB A proposed taxonomy for the podocytopathies: a reassessment of the primary nephrotic diseases.Clin J Am Soc Nephrol. 2007; 2: 529-542Crossref PubMed Scopus (195) Google Scholar, 13Schwartz MM Lewis EJ Focal segmental glomerular sclerosis: the cellular lesion.Kidney Int. 1985; 28: 968-974Crossref PubMed Scopus (129) Google Scholar, 14D'Agati VD Fogo AB Bruijn JA Jennette JC Pathologic classification of focal segmental glomerulosclerosis: a working proposal.Am J Kidney Dis. 2004; 43: 368-382Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar The origin of proliferating epithelial cells in cellular/collapsing FSGS has been investigated by immunostaining with specific cell markers in conjunction with proliferation markers. From the loss of podocyte markers, Barisoni et al concluded that hyperplastic epithelial cells in cellular/collapsing FSGS are de-differentiated podocytes.15Barisoni L Kriz W Mundel P D'Agati V The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy.J Am Soc Nephrol. 1999; 10: 51-61Crossref PubMed Scopus (5) Google Scholar By contrast, we and another group have observed the expression of the PEC markers cytokeratin and Pax-2 in these lesions.16Nagata M Hattori M Hamano Y Ito K Saitoh K Watanabe T Origin and phenotypic features of hyperplastic epithelial cells in collapsing glomerulopathy.Am J Kid Dis. 1998; 32: 962-968Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 17Ohtaka A Ootaka T Sato H Soma J Sato T Saito T Ito S Significance of early phenotypic change of glomerular podocytes detected by Pax2 in primary focal segmental glomerulosclerosis.Am J Kidney Dis. 2002; 39: 475-485Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Because immunostaining studies to identify cell markers are insufficient to determine cellular origin, whether visceral cell hyperplasia is the result of podocytes that have lost their podocyte markers and are expressing PEC markers de novo, or proliferating PECs that persistently express their own markers remains unclear. The interactions between two resident epithelial cells that mimic glomerulogenesis (ie, cell proliferation and phenotypic alterations) may play a key role in the progression of FSGS. Nevertheless, the molecular mechanisms orchestrating these epithelial interactions have not been elucidated.The cell cycle inhibitor p21 has been suggested to play unique roles in glomerular injury. Pippin et al showed that up-regulation of p21 is associated with sublytic C5b-9-induced podocyte injury, suggesting that p21 limits podocyte re-entry into the cell cycle in the setting of cell injury.18Pippin JW Durvasula R Petermann A Hiromura K Couser WG Shankland SJ DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes.J Clin Invest. 2003; 111: 877-885Crossref PubMed Scopus (125) Google Scholar Conversely, de novo expression of p21 was detected in hyperplastic epithelial cells in human glomerular diseases.19Shankland 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: 674-683Crossref PubMed Scopus (173) Google Scholar These studies suggest a role for p21 in glomerular diseases; however, its function remains unclear. Interestingly, p21-deficient mice with glomerulonephritis present a pathology that is characteristic of visceral epithelial hyperplasia leading to FSGS. Furthermore, these cells express ezrin, a podocyte marker; the authors concluded that the lack of p21 promoted podocyte mitogenicity, resulting in collapsing FSGS.20Kim YG Alpers CE Brugarolas J Johnson RJ Couser WG Shankland SJ The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis.Kidney Int. 1999; 55: 2349-2361Crossref PubMed Scopus (72) Google Scholar This model may provide an opportunity to study in situ interactions of epithelial cells that participate in cellular/collapsing FSGS.Recently, genetic tagging has enabled us to trace the lineage of individual cells, even after the cells have become phenotypically transformed by proliferation or injury. Specifically, we and another group have established podocyte-specific Cre-expressing mice crossed with ROSA26 mice.21Moeller MJ Sanden SK Soofi A Wiggins RC Holzman LB Podocyte-specific expression of Cre recombinase in transgenic.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (246) Google Scholar, 22Asano T Niimura F Pastan I Fogo AB Ichikawa I Matsusaka T Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis.J Am Soc Nephrol. 2005; 16: 2257-2262Crossref PubMed Scopus (70) Google ScholarHere, we investigated podocyte lineage in a previously established cellular/collapsing FSGS model in p21-deficient mice to characterize the cellular origin and phenotypic alterations by genetic tagging. Our results suggest that progressive podocytopenia with PEC hyperplasia forms the basis of FSGS in this model. In addition, a novel function of p21 as a cell cycle regulator for PECs was identified.Materials and MethodsTransgenic Mice and Experimental FSGSNphs1-Cre/ROSA-loxP mice and p21−/− mice were mated, and Nphs1-Cre/ROSA-loxP/p21−/− mice were generated.22Asano T Niimura F Pastan I Fogo AB Ichikawa I Matsusaka T Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis.J Am Soc Nephrol. 2005; 16: 2257-2262Crossref PubMed Scopus (70) Google ScholarNphs1-Cre/ROSA-loxP/p21+/+ mice were used as controls. To induce FSGS, duck anti-rabbit kidney antibody was injected intraperitoneally into mice.23Watanabe T Hattori F Tanaka K An experimental study on the origin of foam cells in glomerulonephritis.Acta Pathol Jpn. 1982; 32: 371-383PubMed Google Scholar The antibody was injected into 10- to 12-week-old male mice on 2 consecutive days, and they were sacrificed on day 5 or 14.20Kim YG Alpers CE Brugarolas J Johnson RJ Couser WG Shankland SJ The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis.Kidney Int. 1999; 55: 2349-2361Crossref PubMed Scopus (72) Google Scholar Urine was collected before sacrifice. The kidneys were perfused with phosphate-buffered saline. Tissue blocks were fixed either in 10% formalin or 2% glutaraldehyde for histological examination. In addition, blocks were embedded in OCT compound for cryosectioning. For X-gal staining, the kidneys were perfused with phosphate-buffered saline, followed by perfusion with fixative solution consisted of 10% formaldehyde (Wako, Osaka, Japan), 0.2% glutaraldehyde, and 10% polyoxyethylene (9) octylphenyl (Wako) in phosphate-buffered saline. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the University of Tsukuba (Ibaraki, Japan), and the study was approved by the Institutional Review Board of the University of Tsukuba.Staining and Morphological AnalysisParaffin sections were processed for periodic acid-Schiff staining, a periodic acid-silver methenamine staining, and Masson's trichrome staining. LacZ expression was visualized by X-gal staining of frozen sections 3 μm thick. Briefly, the sections were incubated at 37°C overnight in X-gal solution. Paraffin sections 2 μm thick were stained with anti-synaptopodin antibody (mouse monoclonal antibody, 1:1 dilution; PROGEN Biotechnik, Heidelberg, Germany) as a podocyte-specific marker using a Vector M.O.M. Peroxidase Kit (Vector Laboratories, Burlingame, CA) and an Avidin/Biotin Blocking Kit (Vector Laboratories) in accordance with the manufacturers' instructions. Anti-WT1 antibody (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-nestin antibody (mouse monoclonal antibody, 1:50 dilution; Chemicon, Temecula, CA) were also used as podocyte-specific markers. Anti-laminin antibody (mouse monoclonal antibody, 1:60 dilution; Sigma-Aldrich, St. Louis, MO) was used to outline the glomerular basement membrane (GBM). Anti-cytokeratin antibody (rabbit polyclonal antibody, 1:50 dilution; Dako, Glostrup, Denmark), anti-Pax-2 antibody (rabbit polyclonal antibody, 1:50 dilution; Zymed Laboratories Inc., San Francisco, CA) and anti-claudin-1 antibody (rabbit polyclonal antibody, 1:300 dilution; Invitrogen, Carlsbad, CA) were used as PEC markers. Incubation of all rabbit polyclonal antibodies was followed by EnVision+ Single Reagent (Dako) reaction. To avoid mouse-on-mouse nonspecific reactions, a modification of the ARK (Dako) detection procedure was used. Mac-3 antibody (rat monoclonal, 1:50 dilution; BD Biosciences, San Jose, CA) was used to stain macrophages. Anti-Ki-67 antibody (rat monoclonal, 1:100 dilution; Dako) was used to detect cell cycle activity. Biotinylated rabbit anti-rat IgG (1:100 dilution; Chemicon) was used as a secondary antibody, followed by reaction with peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan). Peroxidase activity was visualized using Liquid DAB+ Substrate Chromogen System (Dako). Glomerular binding of duck IgG (1:20 dilution; Nordic Immunology, Tilberg, The Netherlands) or mouse IgG (1:80 dilution; MP Biomedical, Aurora, OH) was quantified via immunofluorescence. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (Apoptosis in Situ Detection Kit; Wako) was performed to detect apoptotic cells.Urinary Protein and CreatinineThe urinary concentrations of protein and creatinine were measured using a Hitachi 7170 autoanalyzer (Hitachi High-Technologies, Tokyo, Japan).Histological AnalysisGlomerular pathology was assessed by semiquantitative grading using extracapillary hyperplastic and sclerosis indices. The severity of hyperplasia and sclerosis for each glomerulus was graded from 0 to 4+ as follows: 0, no lesion; 1+, extracapillary hyperplasia or sclerosis of 75% of the glomerulus, respectively. The whole-kidney average sclerosis index was obtained by averaging scores from all glomeruli on one section.24Ma LJ Marcantoni C Linton MF Fazio S Fogo AB Peroxisome proliferator-activated receptor-γ agonist troglitazone protects against nondiabetic glomerulosclerosis in rats.Kidney Int. 2001; 59: 1899-1910Crossref PubMed Scopus (177) Google Scholar TUNEL-positive cells in glomeruli (over 50 per mouse), including Bowman's space and capsule, were counted, and the means were calculated.Statistical AnalysisStudent's t-test was used for urinary protein analysis, and the two-sided Mann-Whitney U-test was used for statistical analyses of differences in extracapillary hyperplastic indices, glomerular sclerosis indices, and TUNEL analyses.ResultsProteinuria and Glomerular PathologyUrinalysis indicated that the protein to creatinine ratio was significantly greater in p21−/− mice than in p21+/+ mice, both of which exhibited nephropathy, on day 5 (P < 0.01), whereas the protein to creatinine ratio was similar between groups on day 14 (Figure 1A). Figure 2 shows the sequential glomerular lesions in p21−/− mice with nephropathy. In p21−/− mice on day 5, the glomerular lesions showed focal or global tuft collapse with extracapillary hyperplasia, which was frequently observed in both mono-layered and multilayered visceral cells. In addition, we observed prominent tubular microcystic dilatation with proteinaceous casts in p21−/− mice, while no such changes were apparent in p21+/+ mice. Tuft necrosis with fibrin exudate, mesangial proliferation, inflammatory infiltrates, and Bowman's capsular destruction were not observed. Hyperplastic epithelial cells occasionally had cytoplasmic vacuoles or reabsorption droplets. Careful inspection of serial sections occasionally revealed bridges connecting visceral hyperplastic cells and parietal epithelial cells. Glomeruli with segmental sclerosis on day 14 included “tip” lesions and tuft collapse with matrix accumulation and hyalinosis. The extracapillary hyperplastic index on day 5 was significantly higher in p21−/− than in p21+/+ mice (P < 0.05, Figure 1B). Extracapillary cellular lesions included hypercellular and/or monolayer lesions; both lesion types were localized to the visceral side of the collapsed tuft segment. The glomerular sclerosis index was higher in p21−/− than in p21+/+ mice (Figure 1C). Immunohistochemistry revealed that hypercellular and monolayer extracapillary lesions frequently expressed cytokeratin and Pax-2, but not synaptopodin or macrophage markers (Figure 3, A and B; data not shown in Pax-2 and Mac-3). Immunofluorescence analysis revealed glomerular binding of anti-mouse, but not anti-duck, IgG on the GBM (data not shown).Figure 2Representative micrographs showing cellular/collapsing FSGS in Nephs1-Cre/ ROSA26-loxP/p21−/− mice. Low-magnification view of the renal cortex in p21+/+ mice (A) and p21−/− mice (B) both with nephropathy on day 5. p21−/− mice with nephropathy showed microcystic tubules with protein casts. Glomerular profiles in p21−/− mice with nephropathy on day 5 (C and D) and day 14 (E and F). c: The markedly collapsed glomerular tuft is surrounded by hyperplastic epithelial cells with cytoplasmic vacuolization and occasional protein droplets. D: The collapsed glomerular tuft is surrounded by hyperplastic visceral epithelial cells. Note the presence of a visceral-parietal epithelial bridge. E: A segmentally collapsed tuft formed a synechia to the Bowman's capsule in the tip domain. F: Segmental glomerulosclerosis and hyalinosis were noted. Epithelial hyperplasia was rarely observed in p21−/− mice with nephropathy on day 14. A and B: Masson's trichrome stain; C–F: periodic acid silver methenamine. Scale bar = 200 μm (A, B) and 20 μm (C–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunohistochemistry and X-gal staining of glomeruli in p21−/− mice with nephropathy. A: Synaptopodin staining was absent in visceral epithelial cells on the collapsed tuft (arrows), whereas preserved tufts were covered with synaptopodin-positive cells (arrowhead). B: Bowman's capsule and a collapsed glomerular tuft were entirely covered with keratin-positive epithelial cells. Visceral-parietal epithelial bridges expressed keratin. C: Double staining for laminin and X-gal showed a few X-gal-negative cells on the glomerular basement membrane, which was delineated by laminin staining (arrowheads). D: WT1 and LacZ were co-expressed in the right half of this glomerular surface, whereas hyperplastic epithelial cells in the left portion were negative for both WT1 and X-gal (arrowheads). E: Ki-67 and X-gal double-positive cells were rarely observed in glomeruli. Ki-67 staining was sometimes observed among parietal epithelial cells. F: Ki-67-positive cells in the hyperplastic lesion were virtually white. Scale bar = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Genetic Identification of Podocytes by X-gal StainingIn all cells of the podocyte lineage in Nphs1-Cre/ROSA26 mice, the lacZ marker gene is irreversibly activated after Cre-mediated excision by nephrin promoter activation. Thus, podocyte lineage cells are theoretically stably labeled with lacZ, even when they are injured and lose podocyte markers. The result reveals that blue X-gal staining was exclusively limited to podocytes under normal conditions (Figure 4). All surface cells of normal glomeruli were X-gal-positive, whereas those of segmentally sclerotic glomeruli were composed of both X-gal-positive and -negative cells. Visceral cell hyperplasia ultimately consisted of purely unstained cells, with no admixture of blue-stained cells. In addition, no blue cells are presented beneath the non-tagged hyperplastic epithelial cells. Monolayer lesions on areas of tuft collapse/sclerosis were also unstained. We occasionally found an unstained bridge connecting parietal and visceral aspects of the epithelium but rarely isolated blue cells on the parietal side (parietal podocytes). With advancing sclerosis, glomeruli showed a reduction in the number of blue cells, indicating progressive podocytopenia with sclerosis.Figure 4X-gal staining under differential interface contrast microscopy. A: A p21+/+ mouse (control, without nephropathy). Tagged cells covered the outermost portion of the glomerular tuft; however, they were not observed in any other portion of the nephron. B–E: p21−/− mice with nephropathy on day 5. B: An isolated monolayer of non-tagged cells covered a portion of the glomerular tuft (arrowhead). C: A monolayer of white cells (arrowheads) covered a portion of the collapsed tuft with continuity to parietal cells via a visceral-parietal bridge (arrow). Note that the bridging cells were white. D: Most of the collapsed tuft surface was covered with a monolayer of non-tagged cells. E: Marked hyperplastic epithelial cells (arrowhead) were white. F: Very few blue cells remained in glomeruli with advanced sclerosis on day 14. Scale bars = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Double staining with X-gal and laminin revealed that the few cells attached to the GBM were X-gal-negative and were not connected to the parietal cell layer in this section. Hyperplastic epithelial cells were ultimately WT1-negative, whereas X-gal-positive cells in the same glomeruli strongly expressed WT1. Ki-67-positive blue cells were rarely observed; they were isolated and not found within the hyperplastic epithelial lesions. Ki-67-positive cells were sometimes present on Bowman's capsule but were X-gal-negative. Visceral epithelial hyperplasia occasionally ( 0.05, n = 5 per group). Scale bar = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThe present study, which examined a unique murine model of FSGS exhibiting visceral epithelial hyperplasia,20Kim YG Alpers CE Brugarolas J Johnson RJ Couser WG Shankland SJ The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in exper