Activation of podocyte Notch mediates early Wt1 glomerulopathy
Authors: Rowan I. Asfahani, Mona M. Tahoun, Eve V. Miller-Hodges, Jack Bellerby, Alex K. Virasami, Robert D. Sampson, Dale Moulding, Neil J. Sebire, Peter Hohenstein, Peter J. Scambler, Aoife M. Waters
Journal: Kidney International (2018)

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis. The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis. Glomerulosclerosis accounts for 5% to 10% of pediatric and adult end-stage kidney disease and recurs in 15% to 30% of patients following kidney transplantation.1Saran R. Li Y. Robinson B. et al.US Renal Data System 2015 Annual Data Report: Epidemiology of Kidney Disease in the United States.Am J Kidney Dis. 2016; 67 (S1–S305): SviiPubMed Google Scholar Mutations in genes encoding transcription factors which regulate podocyte differentiation have been implicated in human glomerulosclerosis and include Wilms' tumor 1 (WT1).2Boyer O. Woerner S. Yang F. et al.LMX1B mutations cause hereditary FSGS without extrarenal involvement.J Am Soc Nephrol. 2013; 24: 1216-1222Crossref PubMed Scopus (63) Google Scholar, 3Rheault M.N. Gbadegesin R.A. 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Iatropoulos P. et al.Genotype-phenotype associations in WT1 glomerulopathy.Kidney Int. 2014; 85: 1169-1178Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar WT1 encodes a nuclear protein containing 4 zinc fingers that bind DNA and RNA and is highly expressed in mature podocytes.8Toska E. Roberts S.G. Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1).Biochem J. 2014; 461: 15-32Crossref PubMed Scopus (79) Google Scholar, 9Hastie N.D. Wilms' tumour 1 (WT1) in development, homeostasis and disease.Development. 2017; 144: 2862-2872Crossref PubMed Scopus (94) Google Scholar, 10Pelletier J. Schalling M. Buckler A.J. et al.Expression of the Wilms' tumor gene WT1 in the murine urogenital system.Genes Dev. 1991; 5: 1345-1356Crossref PubMed Scopus (384) Google Scholar, 11Bharathavikru R. Dudnakova T. 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Silverman B.L. et al.Germline intronic and exonic mutations in the Wilms' tumour gene (WT1) affecting urogenital development.Nat Genet. 1992; 1: 144-148Crossref PubMed Scopus (187) Google Scholar Mutations disrupting an alternative splice donor site in intron 9, results in Frasier syndrome, a disorder associated with focal segmental glomerulosclerosis (FSGS), predisposition to gonadoblastoma, and male pseudohermaphroditism.15Barbaux S. Niaudet P. Gubler M.C. et al.Donor splice-site mutations in WT1 are responsible for Frasier syndrome.Nat Genet. 1997; 17: 467-470Crossref PubMed Scopus (568) Google Scholar, 16Klamt B. Koziell A. Poulat F. et al.Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.Hum Mol Genet. 1998; 7: 709-714Crossref PubMed Scopus (285) Google Scholar Furthermore, mutations in WT1 have been reported in isolated primary steroid-resistant nephrotic syndrome, supporting a role for aberrant Wt1 function in the pathogenesis of glomerulosclerosis.5Chernin G. Vega-Warner V. Schoeb D.S. et al.Genotype/phenotype correlation in nephrotic syndrome caused by WT1 mutations.Clin J Am Soc Nephrol. 2010; 5: 1655-1662Crossref PubMed Scopus (78) Google Scholar, 7Lipska B.S. Ranchin B. Iatropoulos P. et al.Genotype-phenotype associations in WT1 glomerulopathy.Kidney Int. 2014; 85: 1169-1178Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Mechanisms underlying the development of WT1-related glomerulosclerosis are poorly understood with no effective treatments available. Recent studies employing chromatin immunoprecipitation sequencing, RNA sequencing, exon array, and bioinformatic analyses have shown a specific role for WT1 in regulating the podocyte-specific transcriptome. WT1 can bind to both promoters and enhancers of 18 known podocytopathy genes.17Lefebvre J. Clarkson M. Massa F. et al.Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.Kidney Int. 2015; 88: 321-331Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Kann M. Ettou S. Jung Y.L. et al.Genome-wide analysis of Wilms' tumor 1-controlled gene expression in podocytes reveals key regulatory mechanisms.J Am Soc Nephrol. 2015; 26: 2097-2104Crossref PubMed Scopus (79) Google Scholar Mutations in WT1 could lead to altered transcriptional regulation of these genes, which is necessary for podocyte differentiation and function, and contribute to the pathogenesis of glomerulosclerosis.17Lefebvre J. Clarkson M. Massa F. et al.Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.Kidney Int. 2015; 88: 321-331Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Kann M. Ettou S. Jung Y.L. et al.Genome-wide analysis of Wilms' tumor 1-controlled gene expression in podocytes reveals key regulatory mechanisms.J Am Soc Nephrol. 2015; 26: 2097-2104Crossref PubMed Scopus (79) Google Scholar Genes regulating podocyte differentiation have also been implicated in the pathogenesis of WT1-related disease. De novo expression of PAX2 protein and mRNA, a paired box transcription factor repressed by WT1 during nephrogenesis, has been observed in podocytes of Denys-Drash syndrome patient biopsies.19Yang Y. Jeanpierre C. Dressler G.R. et al.WT1 and PAX-2 podocyte expression in Denys-Drash syndrome and isolated diffuse mesangial sclerosis.Am J Pathol. 1999; 154: 181-192Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Furthermore, re-expression of Pax2 in podocytes, cell cycle re-entry, and reduced expression of podocyte proteins such as nephrin and α-actinin-4 have been reported in heterozygous Wt1tmT396/+ mice with glomerulosclerosis by 8 months of age.20Patek C.E. Fleming S. Miles C.G. et al.Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis.Hum Mol Genet. 2003; 12: 2379-2394Crossref PubMed Scopus (60) Google Scholar These findings suggested that Wt1tmT396/+ podocytes dedifferentiate and revert to an immature phenotype during disease progression.20Patek C.E. Fleming S. Miles C.G. et al.Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis.Hum Mol Genet. 2003; 12: 2379-2394Crossref PubMed Scopus (60) Google Scholar During glomerulogenesis, podocyte fate induction is regulated by Notch, a highly conserved and ubiquitous pathway that transduces short-range signals between neighboring cells.21Cheng H.T. Miner J.H. Lin M. et al.Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney.Development. 2003; 130: 5031-5042Crossref PubMed Scopus (179) Google Scholar, 22Cheng H.T. Kim M. Valerius M.T. et al.Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron.Development. 2007; 134: 801-811Crossref PubMed Scopus (285) Google Scholar, 23Liu Z. Chen S. Boyle S. et al.The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development.Dev Cell. 2013; 25: 585-598Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Chung E. Deacon P. Park J.S. Notch is required for the formation of all nephron segments and primes nephron progenitors for differentiation.Development. 2017; 144: 4530-4539Crossref PubMed Scopus (39) Google Scholar Ligand binding initiates regulated intramembrane proteolysis with subsequent nuclear translocation of the Notch intracellular domain (NICD) where it associates with RBPJ-κ, a DNA-binding protein, and promotes transcription of target genes (e.g., hairy enhancer of split [Hes]), which regulates tissue-specific differentiation. Notch1, Notch2, and downstream transcriptional targets Hes1 and Hey1 are expressed in podocyte precursors in the S-shaped body and are down-regulated during terminal differentiation.25Piscione T.D. Wu M.Y. Quaggin S.E. Expression of hairy/enhancer of split genes, Hes1 and Hes5, during murine nephron morphogenesis.Gene Expr Patterns. 2004; 4: 707-711Crossref PubMed Scopus (70) Google Scholar, 26Chen L. Al-Awqati Q. Segmental expression of Notch and Hairy genes in nephrogenesis.Am J Physiol Renal Physiol. 2005; 288: F939-F952Crossref PubMed Scopus (92) Google Scholar Ectopic podocyte Notch activation in differentiating and mature podocytes is associated with both diffuse mesangial sclerosis and FSGS, respectively, the latter being mediated by p53-induced podocyte apoptosis and the former associated with de novo Pax2 expression.27Waters A.M. Wu M.Y. Onay T. et al.Ectopic notch activation in developing podocytes causes glomerulosclerosis.J Am Soc Nephrol. 2008; 19: 1139-1157Crossref PubMed Scopus (100) Google Scholar, 28Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (323) Google Scholar Given the interplay between WT1 and Notch during glomerulogenesis and the fact that both diffuse mesangial sclerosis and FSGS phenotypes occur with mutations in WT1 as well as ectopic podocyte Notch activation, we hypothesized a role for podocyte Notch activation in the development of glomerulosclerosis related to loss of Wt1 function. In a model where Wt1 was deleted in mature podocytes, using a tamoxifen-based CRE-LoxP system, we observed increased podocyte loss during the development of glomerulosclerosis. At disease onset, we found upregulation of Notch pathway transcripts in mutant podocytes. At the same time point, we observed repression of FoxC2 and upregulation of Hey2 transcripts in primary mutant podocytes. Cleaved Notch1 and HES1 proteins were evident in mutant podocytes at disease manifestation. Induction of podocyte HES1 expression in vitro was associated with increased expression of Slug and Snail transcripts, genes implicated in epithelial to mesenchyme transition. Pharmacological inhibition of Notch with gamma-secretase inhibitors at disease onset rescued the severity of glomerulosclerosis and albuminuria. These data support a role for early Notch activation in the manifestation of Wt1 glomerulopathy, which may be mediated via repression of podocyte FoxC2. Wt1 deletion in mature podocytes results in glomerulosclerosis with compromised renal function by day 7 post tamoxifen induction in adult CAGG-CreERTM+/−;Wt1f/f transgenic mice.29Chau Y.Y. Brownstein D. Mjoseng H. et al.Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1.PLoS Genet. 2011; 7: e1002404Crossref PubMed Scopus (96) Google Scholar To investigate events leading to the induction of disease in these mice, we first determined the earliest point at which we could detect glomerulosclerosis after Wt1 deletion. Tamoxifen was administered for 3 consecutive days by i.p. injection to 5-week-old CAGG-CreERTM+/−;Wt1f/f transgenic mice and mice were nephrectomized at 4, 5, 6, and 12 days following injection. Successful Wt1 deletion was demonstrated by recombination polymerase chain reaction (PCR) and the reduction of Wt1 expression in glomeruli (Supplementary Figure S1). Following light microscopy analysis of periodic acid–Schiff (PAS)-stained kidney sections, we determined the severity of glomerulosclerosis by a semiquantitative analysis at each time point in CAGG-CreERTM+/−;Wt1f/f mutants, CAGG-CreERTM−/−;Wt1f/f controls, and heterozygous CAGG-CreERTM+/−;Wt1f/+ mice (Figure 1a–d). Heterozygous mice did not develop glomerulosclerosis by day (D) 12 postinduction (PI) (Supplementary Figure S2). At D4 PI, mutants exhibited early segmental glomerulosclerosis with focal foot process effacement and a trend toward higher levels of albuminuria (P = 0.21) (Figure 1a and a′; Supplementary Figures S3A and S4). By D5 PI, glomerular scarring was more extensive (≥ score 2) in mutants compared with control and heterozygous mice (*P < 0.05) (Figure 1b, Supplementary Figure S3B). Progression of disease was further supported by an increase in urine albumin-creatinine ratio in mutants compared with controls (*P = 0.01) (Figure 1b′). By D6 PI, extensive glomerulosclerosis with tubules containing protein casts were observed in mutants relative to controls with increased albuminuria (Figure 1c and c′, Supplementary Figure S3C). Late stage disease with global glomerulosclerosis was observed at D12 PI (Figure 1d and e, Supplementary Figure S3D), with peritubular cells expressing vascular smooth muscle actin, consistent with progression of tubulointerstitial disease (Supplementary Figure S5). We conclude that podocyte function appears compromised within 6 days of Wt1 deletion in mature podocytes. Therefore, investigations into the mechanisms underlying manifestation of disease should be undertaken within this time frame. Podocyte apoptosis has previously been implicated in the pathogenesis of glomerulosclerosis.30Schiffer M. Bitzer M. Roberts I.S. et al.Apoptosis in podocytes induced by TGF-beta and Smad7.J Clin Invest. 2001; 108: 807-816Crossref PubMed Scopus (544) Google Scholar Therefore, we next investigated whether apoptosis is evident during disease induction in CAGG-CreERTM+/−;Wt1f/f transgenic mice. Expression of Cleaved Caspase-3 protein was observed within mutant glomeruli as early as D4 PI but quantitatively, was not statistically significantly different compared with control glomeruli (Figure 2a) (P = 0.06, NS). By D5 PI, at the onset of early glomerulosclerosis, we observed an increased number of Cleaved Caspase-3/DAPI-positive cells in mutant glomeruli (Figure 2c, c′, and d) (***P < 0.0001), consistent with a temporal increase in apoptosis. Terminal deoxynucleotidyltransferase–mediated 2′-deoxyuridine 5′-triphosphate nick end-labeling (TUNEL)-positive mutant glomerular cells were evident at D5 PI but were absent in controls (Supplementary Figure S6). TUNEL staining of D6 PI primary mutant podocytes was also higher than for controls (Figure 2e, e′, and f) (*P = 0.04). At the same time point, we observed increased Caspase-3/7 positivity in primary mutant podocytes (Supplementary Figure S7) and an increased number of Annexin V/Sytox blue-positive primary mutant podocytes compared with controls (Supplementary Figure S8). TUNEL staining at D12 PI revealed clusters of TUNEL-positive cells in peripheral segments of mutant glomeruli not evident in the glomeruli of controls (Supplementary Figure S9). Together, these studies suggest that loss of Wt1 in mature podocytes is associated with podocyte apoptosis and development of glomerulosclerosis. Constitutive Notch activation in terminally differentiated podocytes results in podocyte apoptosis and glomerulosclerosis,27Waters A.M. Wu M.Y. Onay T. et al.Ectopic notch activation in developing podocytes causes glomerulosclerosis.J Am Soc Nephrol. 2008; 19: 1139-1157Crossref PubMed Scopus (100) Google Scholar, 28Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (323) Google Scholar supporting a role for Notch in podocyte injury. Following our observation of increased podocyte apoptosis in CAGG-CreERTM+/−;Wt1f/f transgenic mice, we hypothesized that podocyte Notch activation plays a role in early Wt1 glomerulopathy. At D4 PI before manifestation of disease, Notch pathway transcripts in primary podocytes of CAGG-CreERTM+/−;Wt1f/f transgenic mice were not statistically different to those of controls (Supplementary Figure S10). By D6 PI, concomitant with early glomerulosclerosis and albuminuria, we observed increases in canonical Notch pathway transcripts, Notch1, Nrarp, and Hey2. Increased levels of other Notch basic helix loop helix (bHLH) transcription factors Hes1, Hes3, Hes5, and HeyL were also observed in primary mutant podocytes but not in controls (Figure 3a), although these changes did not reach statistical significance. Using double immunofluorescence labeling, we observed cleaved Notch1 protein in nuclei of Nestin-positive podocytes as early as D4 PI in mutant kidney sections (Figure 3b and c). These findings were validated by Western blot analysis showing increased cleaved Notch1 protein in primary mutant podocytes relative to controls (Figure 3d). Increased podocyte Manic fringe (MFng) transcript was also observed in mutants compared with controls (Figure 3a). Manic Fringe is a glycosyltransferase that mediates glycosylation of the extracellular domain of Notch receptors during signal transduction.31Bruckner K. Perez L. Clausen H. Cohen S. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.Nature. 2000; 406: 411-415Crossref PubMed Scopus (592) Google Scholar, 32Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev Cell. 2017; 40: 193-201Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar We also found upregulation of Pofut1 protein in primary mutant podocytes at D6 PI (Figure 3e). Pofut1 is an O-fucosyltransferase 1 enzyme that mediates fucosylation of the extracellular domain of the Notch.33Okajima T. Xu A. Irvine K.D. Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe.J Biol Chem. 2003; 278: 42340-42345Crossref PubMed Scopus (169) Google Scholar Together, these data suggest that post-translational modification of Notch components are active in early Wt1 glomerulopathy. We next sought to determine whether the Notch ligand Jagged1 is expressed in CAGG-CreERTM+/−;Wt1 f/f transgenic mice owing to previous studies implicating podocyte Jagged1 in glomerulosclerosis.28Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (323) Google Scholar We observed a striking upregulation of Jagged1 protein in the parietal epithelium of mutants compared with controls (Figure 4a and b). Within segments of mutant glomeruli, foci of Podocin-positive cells expressing Jagged1 were also observed (Figure 4b′, high-power images). Immunoblotting of primary mutant and control podocyte lysates revealed induction of Jagged1 expression at D6 PI (Figure 4c). Together, these data suggest that Notch signaling is activated when glomerulosclerosis first manifests following deletion of Wt1 in mature podocytes. Given previous studies that showed that Wt1 and Foxc1a can inhibit NICD activation,34O'Brien L.L. Grimaldi M. Kostun Z. et al.Wt1a, Foxc1a, and the Notch mediator Rbpj physically interact and regulate the formation of podocytes in zebrafish.Dev Biol. 2011; 358: 318-330Crossref PubMed Scopus (66) Google Scholar we explored FoxC2 expression in this model. Using semiquantitative PCR analysis, we found decreased podocyte FoxC2 transcript in CAGG-CreERTM+/−;Wt1 f/f transgenic mice compared with their control littermates (*P = 0.04) (Figure 3a). This decrease was observed at the same time point when Notch components were upregulated and concordant with disease manifestation. These findings support the hypothesis that Notch activation in mature podocytes is normally repressed by FoxC2 and WT1 and loss of these proteins is associated with increased podocyte Notch activation. Following detection of podocyte Hes/Hey mRNA expression in primary mutant podocytes, we sought to validate the expression of HES1, a bHLH transcription factor in podocytes by triple immunofluorescence labeling. We observed clusters of HES1-expressing cells that were positive for the podocyte marker, Synaptopodin, at onset of glomerulosclerosis in mutants compared with controls (Figure 5). HES1-positive glomerular epithelial cells were observed in regions distinct from HES1-positive Lotus tetragonolobus lectin (LTL)-positive tubules (Figure 5b and b′). Clusters of Synaptopodin-positive HES1-podocytes were distinct from platelet–endothelial cell adhesion molecule–positive glomerular endothelial cells (Figure 5c, d, and d′). As HES1 has been previously implicated in epithelial to mesenchymal transition (EMT), we next determined that Snail and Slug transcript was upregulated in primary D6 PI mutant podocytes compared with controls (Figure 5e). To further understand the role of HES1 in podocytes, we transfected cultured primary Nphs2;rtTA transgenic murine podocytes with constructs expressing either TetOHes1 or green fluorescent protein (GFP) alone. HES1 was not expressed in untreated TetOHes1 nor doxycycline-treated GFP transfected Nphs2;rtTA podocytes. Treatment with doxycycline led to a dose-dependent increase in podocyte Hes1 mRNA and protein expression (Figure 5f, Supplementary Figure S11). Induction of Hes1 expression led to a 3-fold upregulation of Snail and Slug transcript compared with untreated TetOHes1 transfected and doxycycline-treated GFP transfected Nphs2;rtTA podocytes (Figure 5f). These results suggest that podocyte HES1 induction could mediate manifestation of glomerulosclerosis through regulation of EMT genes in podocytes. Following our observation of Notch activation in murine Wt1 glomerulopathy, we next tested biopsy samples from a human subject with FSGS associated with the WT1 c.1390G>T mutation. We found JAGGED1 expression in cells with focal nephrin staining but also found that expression was most marked in the parietal epithelium (Figure 6). The control biopsy from a nondiseased time 0 renal allograft biopsy revealed JAGGED1 expression in regions distal to nephrin staining suggesting endothelial JAGGED1 expression. JAGGED1 expression in the control parietal epithelium was much less than in mutant WT1 patient tissue. Expression of the Notch bHLH transcription factor, HES1, was also observed in nuclei of mutant WT1 glomeruli compared with in control biopsy tissue (Figure 6). We conclude that these data support a role for Notch activation in human WT1-mediated glomerular disease. As podocyte Notch activation is a feature of early disease, we hypothesized that pharmacological Notch inhibition at onset of glomerulosclerosis could influence severity of disease. CAGG-CreERTM+/−;Wt1f/f transgenic mice were treated by i.p. injection with the gamma secretase inhibitor GSI-IX, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), or dimethylsulfoxide (i.e., vehicle only) late D4 PI and early D5 PI (Figure 7a–d). In vehicle-treated mutants, we observed hyaline-filled tubules representing proteinaceous material and sclerotic glomeruli with mesangial proliferation (Figure 7b). These features were not evident in GSI-IX-treated mutants (Figure 7c). The proportion of vehicle-treated mutant glomeruli exhibiting extensive glomerulosclerosis was significantly higher than the proportion of GSI-IX-treated mutants (**P = 0.008) (Figure 7d). We validated inhibition of canonical Notch pathway transcripts by semiquantitative PCR (Figure 7e). Analysis of Wt1 transcript and protein expression did not reveal a significant difference between vehicle-treated or GSI-IX-treated mutant mice (Supplementary Figure S12). GSI-IX-treated mutants, compared with vehicle-treated mutants, showed an improvement in urine albumin-creatinine ratio at D5 PI (Figure 7f). Western blot analysis of urine albumin confirmed absence of albuminuria in GSI-IX-treated mice compared with in vehicle-treated mutants at D5 PI (Figure 7g). We conclude that the observed efficacy of GSI inhibition in amelioration of glomerulosclerosis supports the hypothesis that Notch is activated during disease development. We also tested the role of starting treatment in established disease by administering GSI-IX to CAGG-CreER TM+/−;Wt1f/f mutant mice on D7 PI. Following 2 doses of GSI-IX, mice were nephrectomized late on D8 PI, and kidney histology was analyzed for 2 mice per treatment. We did not observe a difference in degree of glomerulosclerosis, albuminuria, nor tubulointerstitial expression of vascular smooth muscle actin immunofluorescence (Supplementary Figure S13). We hypothesize that podocyte Notch activation plays a role in early Wt1 glomerulopathy. Given the upregulation of MFng and Rbpsuh transcripts at D6 PI, we sought to determine whether knockdown of Mfng or Rbpsuh would influence podocyte EMT gene or apoptosis gene expression. While we did observe evidence of Mfng and Rbpsuh down-regulation with repression of Hes1 and Hes3 transcripts, we did not observe significant repression of podocyte Snail and Slug transcript nor significant upregulation of podocyte-specific transcripts such as Nphs1 nor Nphs2 (Supplementary Figure S14). Reduced primary mutant podocyte viability precluded optimal transfection owing to slow growth and increased apoptosis. We conclude that genetic rescue of Wt1 glomerulopathy by Notch inhibition would be best validated with in vivo strategies. Using an inducible model of Wt1 deletion, we demonstrate a role for Notch activation in the pathogenesis of Wt1 glomerulopathy. The data provided in this study establish the utility of temporal semiquantitative analysis of glomerular scarring as a platform for the study of early pathological events in an inducible model of mature podocyte injury. We show that podocyte apoptosis is evident as early as the fourth day following tamoxifen administration to adult CAGG-CreERTM+/−;Wt1f/f transgenic mice before glomerulosclerosis is evident. This podocyte loss, secondary to Wt1 deletion, increases when overt albuminuria is evident. Furthermore, we show upregulation of Snail and Slug mRNA, genes that are implicat