Aberrant Notch1-dependent effects on glomerular parietal epithelial cells promotes collapsing focal segmental glomerulosclerosis with progressive podocyte loss
2013; Elsevier BV; Volume: 83; Issue: 6 Linguagem: Inglês
10.1038/ki.2013.48
ISSN1523-1755
AutoresToshiharu Ueno, Namiko Kobayashi, Makiko Nakayama, Yasutoshi Takashima, Takamoto Ohse, Ira Pastan, Jeffrey W. Pippin, Stuart J. Shankland, Noriko Uesugi, Taiji Matsusaka, Michio Nagata,
Tópico(s)Tuberous Sclerosis Complex Research
ResumoCollapsing focal segmental glomerulosclerosis (cFSGS) is a progressive kidney disease characterized by glomerular collapse with epithelial hyperplasia. Here we used a transgenic mouse model of cFSGS with immunotoxin-induced podocyte-specific injury to determine the role for Notch signaling in its pathogenesis. The mice exhibited progressive loss of podocytes and severe proteinuria concomitant with histological features of cFSGS. Hyperplastic epithelium was negative for genetic podocyte tags, but positive for the parietal epithelial cell marker claudin-1, and expressed Notch1, Jagged1, and Hes1 mRNA and protein. Enhanced Notch mRNA expression induced by transforming growth factor-β1 in cultured parietal epithelial cells was associated with mesenchymal markers (α-smooth muscle actin, vimentin, and Snail1). Notch inhibition in vitro suppressed these phenotypic transcripts and Notch-dependent cell migration. Moreover, Notch inhibition in vivo significantly decreased parietal epithelial cell lesions but worsened proteinuria and histopathology in our cFSGS model. Thus, aberrant Notch1-mediated parietal epithelial cell migration with phenotypic changes appears to underlie the pathogenesis of cFSGS. Parietal epithelial cell hyperplasia may also represent an adaptive response to compensate for a disrupted filtration barrier with progressive podocyte loss. Collapsing focal segmental glomerulosclerosis (cFSGS) is a progressive kidney disease characterized by glomerular collapse with epithelial hyperplasia. Here we used a transgenic mouse model of cFSGS with immunotoxin-induced podocyte-specific injury to determine the role for Notch signaling in its pathogenesis. The mice exhibited progressive loss of podocytes and severe proteinuria concomitant with histological features of cFSGS. Hyperplastic epithelium was negative for genetic podocyte tags, but positive for the parietal epithelial cell marker claudin-1, and expressed Notch1, Jagged1, and Hes1 mRNA and protein. Enhanced Notch mRNA expression induced by transforming growth factor-β1 in cultured parietal epithelial cells was associated with mesenchymal markers (α-smooth muscle actin, vimentin, and Snail1). Notch inhibition in vitro suppressed these phenotypic transcripts and Notch-dependent cell migration. Moreover, Notch inhibition in vivo significantly decreased parietal epithelial cell lesions but worsened proteinuria and histopathology in our cFSGS model. Thus, aberrant Notch1-mediated parietal epithelial cell migration with phenotypic changes appears to underlie the pathogenesis of cFSGS. Parietal epithelial cell hyperplasia may also represent an adaptive response to compensate for a disrupted filtration barrier with progressive podocyte loss. Focal segmental glomerulosclerosis (FSGS), a progressive kidney disease, is the major cause of end-stage renal failure.1.D'Agati V.D. Kaskel F.J. Falk R.J. Focal segmental glomerulosclerosis.N Engl J Med. 2011; 365: 2398-2411Crossref PubMed Scopus (553) Google Scholar Progressive decrease in podocytes is considered a factor underlying FSGS.2.Kriz W. LeHir M. Pathways to nephron loss starting from glomerular diseases-insights from animal models.Kidney Int. 2005; 67: 404-419Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 3.Wharram B.L. Goyal M. Wiggins J.E. et al.Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (584) Google Scholar The sequence of events in podocyte loss–induced FSGS is known for the remnant kidney.4.Nagata M. Kriz W. Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis.Kidney Int. 1992; 42: 148-160Abstract Full Text PDF PubMed Scopus (226) Google Scholar In this model, slow podocyte loss results in presclerotic synechia formation, as observed in postadaptive secondary FSGS.4.Nagata M. Kriz W. Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis.Kidney Int. 1992; 42: 148-160Abstract Full Text PDF PubMed Scopus (226) Google Scholar Collapsing FSGS (cFSGS), a distinct form of FSGS, is characterized by more pronounced proteinuria and more rapid renal dysfunction as compared with other variants in the Columbia FSGS classification.1.D'Agati V.D. Kaskel F.J. Falk R.J. Focal segmental glomerulosclerosis.N Engl J Med. 2011; 365: 2398-2411Crossref PubMed Scopus (553) Google Scholar, 5.D'Agati V.D. Fogo A.B. 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Focal segmental glomerulosclerosis.N Engl J Med. 2011; 365: 2398-2411Crossref PubMed Scopus (553) Google Scholar the molecular mechanism underlying cFSGS remains unclear. cFSGS has a distinguishing morphological feature known as tuft collapse that is accompanied by marked epithelial cell hyperplasia, which is supposedly related to its progression.5.D'Agati V.D. Fogo A.B. Bruijn J.A. et al.Pathologic classification of focal segmental glomerulosclerosis: a working proposal.Am J Kidney Dis. 2004; 43: 368-382Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 7.Stokes M.B. Valeri A.M. Markowitz G.S. et al.Cellular focal segmental glomerulosclerosis: clinical and pathologic features.Kidney Int. 2006; 70: 1783-1792Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 8.Barisoni L. Kriz W. 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Smeets B. et al.Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells.Kidney Int. 2006; 70: 338-344Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14.Asano T. Niimura F. Pastan I. et al.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 (71) Google Scholar, 15.Smeets B. Uhlig S. Fuss A. et al.Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 2604-2615Crossref PubMed Scopus (203) Google Scholar showed that hyperplastic cells expressed parietal epithelial cell (PEC) markers but not podocyte markers, although others have interpreted lack of podocyte markers as an indicator of podocyte dedifferentiation.8.Barisoni L. Kriz W. Mundel P. et al.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 (6) Google Scholar Probably, heterogeneous origins are responsible for these lesions.16.Bariéty J. Bruneval P. Activated parietal epithelial cells or dedifferentiated podocytes in FSGS: can we make the difference?.Kidney Int. 2006; 69: 194Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Genetically tagged podocytes in murine cFSGS models showed that hyperplastic lesions were essentially negative for podocyte-tagged cells but positive for PEC markers.9.Suzuki T. Matsusaka T. Nakayama M. et al.Genetic podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis.Am J Pathol. 2009; 174: 1675-1682Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14.Asano T. Niimura F. Pastan I. et al.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 (71) Google Scholar, 15.Smeets B. Uhlig S. Fuss A. et al.Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 2604-2615Crossref PubMed Scopus (203) Google Scholar More importantly, in a transgenic mouse expressing human CD25 on podocyte (NEP25 mouse), specific antibody to human CD25 (an immunotoxin called LMB2) treatment induced extensive podocyte loss and histology similar to human cFSGS.14.Asano T. Niimura F. Pastan I. et al.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 (71) Google Scholar, 17.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (213) Google Scholar These findings indicate that acute decrease in podocytes followed by PEC hyperplasia may be a pathogenic route leading to cFSGS. In this study, we focused on the Notch signaling pathway as a possible mechanism underlying these events. Notch signaling is a highly conserved determinant of cell-fate decisions during organogenesis via the asymmetrical differentiation of developing cells.18.Bray S.J. Notch signalling: a simple pathway becomes complex.Nat Rev Mol Cell Biol. 2006; 7: 678-689Crossref PubMed Scopus (1979) Google Scholar, 19.Cheng 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 (286) Google Scholar It is also involved in cell migration, proliferation, phenotypic changes, and apoptosis in many diseases.20.Sonoshita M. Aoki M. Fuwa H. et al.Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling.Cancer Cell. 2011; 19: 125-137Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21.Bao B. Wang Z. Ali S. et al.Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells.Cancer Lett. 2011; 307: 26-36Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 22.Bielesz B. Sirin Y. Si H. et al.Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans.J Clin Invest. 2010; 120: 4040-4054Crossref PubMed Scopus (278) Google Scholar, 23.Niranjan 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 (326) Google Scholar During kidney development, Notch signaling expression is detected transiently in candidate podocytes and PECs, and is important for glomerulogenesis.24.Chen L. Al-Awqati Q. Segmental expression of Notch and Hairy genes in nephrogenesis.Am J Physiol Renal Physiol. 2005; 288: F939-F952PubMed Google Scholar, 25.Piscione 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 Moreover, Notch signaling is involved in several kidney diseases and was detected in podocytes in which it induces apoptosis.23.Niranjan 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 (326) Google Scholar On the basis of these observations, we hypothesized that the Notch signaling pathway might induce significant epithelial changes that could provide new insights into cFSGS pathogenesis. For this aim, we used NEP25 mouse as an experimental cFSGS model and a recently established immortalized PEC line to determine the role of Notch signaling in PEC responses to podocyte loss.14.Asano T. Niimura F. Pastan I. et al.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 (71) Google Scholar, 17.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (213) Google Scholar, 26.Ohse T. Pippin J.W. Vaughan M.R. et al.Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.J Am Soc Nephrol. 2008; 19: 1879-1890Crossref PubMed Scopus (68) Google Scholar The therapeutic potential of Notch inhibition was also tested in our mouse cFSGS model. Our results suggest that acute podocyte loss followed by glomerular collapse and PEC migration via Notch1-dependent phenotypic changes is a novel pathogenic mechanism underlying cFSGS. Furthermore, inhibition of Notch signaling in vivo potentially worsens cFSGS, suggesting an underlying wound healing mechanism for cFSGS pathology. NEP25 mice administered the immunotoxin (LMB2) developed progressive proteinuria beginning on day 8 (Figure 1a). Histological analysis showed normal kidney morphology in vehicle-treated NEP25 mice (controls; Figure 1b). Conversely, in LMB2-treated mice (LMB2 mice), 9.44±0.62% glomeruli exhibited segmental or global collapse and 27.67±2.12% glomeruli exhibited extracapillary hypercellularity that formed adhesions with the glomerular tuft. These glomeruli closely resembled those in human cFSGS (Figure 1c). LMB2 mice, but not controls (Figure 1d), had marked decrease in podocytes on β-galactosidase (β-gal) staining (Figure 1e). In the sections examined, >95% of the cells in the extracapillary lesions expressed claudin-1 (a PEC marker) and were negative for β-gal, a podocyte label. In LMB2 mice, the numbers of WT1-positive cells (podocytes) progressively decreased and cleaved caspase 3–positive cells were rarely observed throughout the observation period (Figure 1f). The PEC score was significantly higher for LMB2 mice than for controls on day 12 (Figure 1g). Cleaved caspase 3– and Ki-67–positive cells were considered PECs but not podocytes according to results of double labeling for β-gal and claudin-1 (Figure 1h–k). Electron micrographs revealed severe podocyte injury and initial PEC migration toward the bared glomerular capillary during the early events in this model (Figure 1ll′). These results showed that PEC hyperplasia occurred concurrently with acute podocyte depletion and that proliferating/apoptotic cells were PECs but not podocytes. Immunofluorescence staining for cleaved Notch1, which reflects Notch signaling activation, was barely detectable in control glomeruli (Figure 2aa′′). Conversely, cleaved Notch1 staining intensity was markedly increased in podocytes and PECs during the early disease phase (Figure 2bb′′) and was rarely colocalized with podocalyxin (a podocyte marker) during the advanced phase in LMB2 mice (Figure 2cc′′′). Immunohistochemical staining of serial sections revealed that cleaved Notch1 exclusively colocalized with claudin-1 (Figure 2dd′). Similarly, Jagged1 (a Notch ligand) and Hes1 (a Notch target gene) were specifically increased in PEC lesions in LMB2 mice (Figure 2ee′). These results demonstrated that active Notch signaling proteins were present in hyperplastic lesions comprising PECs in diseased LMB2 mice. To determine any changes in mRNA levels of components of the Notch signaling pathway, quantitative reverse transcriptase–PCR (qRT–PCR) was performed for isolated glomeruli. Statistically significant increases in mRNA expression of Notch-related genes (Notch1, Notch4, Jagged1, Delta1, and Hes1) were observed in LMB2 mice on day 12 compared with controls (Figure 2f). Among Notch receptors, Notch1 was most significantly increased. Among the Notch ligands, Jagged1 and Delta1 exhibited significant increases. Hes1 was the only significantly enhanced downstream gene. Consistent with its protein expression in immunohistochemistry, claudin-1 mRNA levels were markedly increased in LMB2 mice. These results demonstrated that the mRNA and protein levels were increased for several Notch receptors and ligands in hyperplastic lesions comprising PECs in this mouse model. Human cFSGS biopsy samples with typical glomerular features were also examined (Figure 3a). Immunostaining revealed that cleaved Notch1 was increased in extracapillary cells and colocalized with claudin-1 but not with the podocyte marker Nestin (Figure 3aa′ and b). Jagged1 and Hes1 also colocalized in this lesion (Figure 3cc′). These results showed that Notch signaling expression in PECs were similar in our mouse model and human cFSGS. To investigate a functional role for Notch signaling in PECs at the cellular level, we used a recently established immortalized PEC cell line.26.Ohse T. Pippin J.W. Vaughan M.R. et al.Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.J Am Soc Nephrol. 2008; 19: 1879-1890Crossref PubMed Scopus (68) Google Scholar We also examined a possible role for TGF-β1 because it could induce Notch signaling in numerous cell types,27.Fuxe J. Vincent T. Garcia de Herreros A. Transcriptional crosstalk between TGF-β and stem cell pathways in tumor cell invasion: role of EMT promoting Smad complexes.Cell Cycle. 2010; 9: 2363-2374Crossref PubMed Scopus (273) Google Scholar, 28.Zavadil J. Cermak L. Soto-Nieves N. et al.Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.EMBO J. 2004; 23: 1155-1165Crossref PubMed Scopus (631) Google Scholar, 29.Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis.J Am Soc Nephrol. 2010; 21: 212-222Crossref PubMed Scopus (711) Google Scholar and is increased in FSGS.30.Kim J.H. Kim B.K. Moon K.C. et al.Activation of the TGF-beta/Smad signaling pathway in focal segmental glomerulosclerosis.Kidney Int. 2003; 64: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Treating cultured PECs with TGF-β1 resulted in increased Jagged1, Notch1, and Hes1 mRNA levels after 6–12h, as per qRT–PCR (Figure 4a). Hes1 mRNA exhibited a significantly higher expression (17.99±5.85-fold increase), whereas other downstream genes were not significantly affected. This specificity was confirmed using pharmacological inhibition of Notch signaling by preincubation with dibenzazepine (DBZ), a γ-secretase inhibitor that significantly reduced Hes1 mRNA expression (Figure 4b). In association with Notch induction by TGF-β1 in cultured PECs, the mRNA levels of the epithelial cell marker E-cadherin decreased, whereas those of the mesenchymal markers α-smooth muscle actin, vimentin, and Snail1 increased (Figure 4c). Preincubation with DBZ clearly preserved E-cadherin and restricted the expression of mesenchymal markers (Figure 4d–g), indicating that Notch-dependent phenotypic changes were mediated by TGF-β1 in PECs. Notch signaling is involved in cell migration, proliferation, phenotypic changes, and apoptosis.20.Sonoshita M. Aoki M. Fuwa H. et al.Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling.Cancer Cell. 2011; 19: 125-137Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21.Bao B. Wang Z. Ali S. et al.Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells.Cancer Lett. 2011; 307: 26-36Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 22.Bielesz B. Sirin Y. Si H. et al.Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans.J Clin Invest. 2010; 120: 4040-4054Crossref PubMed Scopus (278) Google Scholar, 23.Niranjan 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 (326) Google Scholar Cultured PEC migration was assessed using a Boyden chamber motility assay for three cell groups: control, TGF-β1-treated, and TGF-β1+DBZ-treated cells. The number of migrating PECs was significantly increased after TGF-β1 treatment compared with control treatment (Figure 5a, b). Notably, DBZ pretreatment significantly reduced cell migration (Figure 5c and d). Conversely, a cell proliferation assay showed no significant differences among these three groups within 48h (Figure 5e). The proportion of apoptotic cells, as determined by cleaved caspase 3 expression, was also not significantly different among these three groups (Figure 5f). These results indicated that TGF-β1-induced PEC migration was dependent on the Notch signaling pathway and that TGF-β1-mediated Notch signaling had no effect on cell proliferation or apoptosis. To determine the consequences of hyperplastic PECs on disease outcomes and a role for Notch in our cFSGS model, we administered DBZ (5μg/g body weight (BW)) to LMB2 mice. DBZ-treated LMB2 mice had significantly increased proteinuria (Figure 6a) that was associated with reduced PEC score compared with untreated LMB2 mice (Figure 6b). The numbers of WT1-positive cells were not significantly different (Figure 6c). However, urinary casts reflecting histological damage were far more frequent in DBZ-treated LMB2 mice than in untreated mice (Figure 6d and e). The proportions of urinary casts in the cortex, which reflected tubulointerstitial damage, were significantly higher in DBZ-treated LMB2 mice than in untreated mice (Figure 6f). Electron micrographs showed PEC migration and that these cells covered a glomerular tuft with or without underlying podocytes in LMB2 mice (Figure 7a and b). Conversely, DBZ-treated LMB2 mice had bare PEC lesions and severe podocyte damage, including villous transformation, diffuse foot process effacement, and vacuolation (Figure 7c and d).Figure 7Transmission electron micrographs of glomeruli in LMB2 and dibenzazepine (DBZ)-treated LBM2 mice. (a) A low-magnification view of a glomerulus in an LMB2 mouse showed a collapsed glomerular tuft with segmental hyperplastic parietal epithelial cells (PECs; original magnification × 200; bar=38μm). PECs migrated onto the glomerular tuft from the presumed entry site (arrowhead), and clusters or single PECs had attached to the collapsed tuft surface. (b) Higher magnification view of the inset in a showing the collapsed tuft covered by migrated PECs (original magnification × 1000; bar=12μm). (c) A low-magnification view of a glomerulus in a DBZ-treated LMB2 mouse showed a segmentally collapsed glomerular tuft and no PEC lesion (original magnification × 200; bar=38μm). (d) Higher magnification view of the inset in c (original magnification × 600; bar=12μm). Podocytes on the collapsed glomerular tuft exhibited severe foot process effacement, a pseudocyst, and villous transformation.View Large Image Figure ViewerDownload (PPT) cFSGS in our murine model was initiated by podocyte-specific injury. Genetically tagging podocytes with β-gal and WT1 immunostaining indicated notable podocyte depletion. Hyperplastic epithelium essentially expressed claudin-1 and was negative for β-gal, indicating that PECs are major component of cFSGS. It is noteworthy that glomerular and tubulointerstitial histology was similar to that observed in human cFSGS. Because epithelial hyperplasia is the major characteristic of presclerotic lesions in cFSGS, understanding the molecular mechanism that induces PEC hyperplasia could provide important insights into cFSGS pathogenesis. Our in vivo and in vitro findings strongly suggest that the Notch signaling pathway plays a novel role in cFSGS pathogenesis. First, hyperplastic PECs in LMB2-treated NEP25 mice distinctly expressed cleaved Notch1 (an activated Notch signaling molecule), Jagged1, and Hes1, whereas cleaved Notch1 expression was minimal in mice without LMB2 treatment. This aberrant Notch signaling expression was confirmed by increased mRNA levels in isolated glomeruli. Claudin-1-positive hyperplastic PECs in human cFSGS also expressed these molecules. These results suggest that Notch signaling molecules are aberrantly expressed in activated PECs in cFSGS in mice and humans. Among the pleiotropic functions regulated by Notch signaling,20.Sonoshita M. Aoki M. Fuwa H. et al.Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling.Cancer Cell. 2011; 19: 125-137Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21.Bao B. Wang Z. Ali S. et al.Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells.Cancer Lett. 2011; 307: 26-36Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 22.Bielesz B. Sirin Y. Si H. et al.Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans.J Clin Invest. 2010; 120: 4040-4054Crossref PubMed Scopus (278) Google Scholar, 23.Niranjan 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 (326) Google Scholar proliferation, migration, and transdifferentiation were likely responsible for the PEC lesions in our model. Although PEC lesion formation depends on cell proliferation and migration, our previous work with a cFSGS model of anti-glomerular basement membrane serum–treated p21-deficient mice found only a single PEC attached to naked glomerular basement membrane without hyperplasia.9.Suzuki T. Matsusaka T. Nakayama M. et al.Genetic podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis.Am J Pathol. 2009; 174: 1675-1682Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar This suggests that proliferation and migration of PECs do not always occur together or that they may be mediated by different mechanisms. To investigate the cellular mechanisms of Notch signaling in PEC lesions, we used the recently established immortalized PEC line with TGF-β1 as a Notch inducer.26.Ohse T. Pippin J.W. Vaughan M.R. et al.Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.J Am Soc Nephrol. 2008; 19: 1879-1890Crossref PubMed Scopus (68) Google Scholar The Boyden chamber migration assay demonstrated TGF-β1-mediated PEC migration, which was significantly reduced by Notch inhibition. Conversely, TGF-β1-induced Notch activation did not lead to cell proliferation. TGF-β1 also induces cell cycle inhibitors, such as p21, in vitro.31.Wada T. Pippin J.W. Terada Y. et al.The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis.Kidney Int. 2005; 68: 1618-1629Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Thus, lack of cell proliferation with TGF-β1-induced Notch activation does not exclude the possibility that Notch signals have no effect on PEC proliferation in vivo. We propose that no single factor mediates PEC lesion formation in cFSGS. Further studies are required to establish this hypothesis. Cell migration is occasionally associated with transdifferentiation into different cell types (for example, the epithelial–mesenchymal transition) and is considered a cell motility phenotype.32.Acloque H. Adams M.S. Fishwick K. et al.Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.J Clin Invest. 2009; 119: 1438-1449Crossref PubMed Scopus (1046) Google Scholar, 33.Zeisberg M. Neilson E.G. Biomarkers for epithelial-mesenchymal transitions.J Clin Invest. 2009; 119: 1429-1437Crossref PubMed Scopus (1733) Google Scholar, 34.Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (966) Google Scholar The immortalized PEC line exhibited significant increases in mesenchymal phenotype mRNAs, including those for α-smooth muscle actin, vimenti
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