Epithelial-to-Mesenchymal Transition Is a Potential Pathway Leading to Podocyte Dysfunction and Proteinuria
2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.070057
ISSN1525-2191
AutoresYingjian Li, Young Sun Kang, Chunsun Dai, L Kiss, Xiao‐Yan Wen, Youhua Liu,
Tópico(s)Lymphatic System and Diseases
ResumoPodocyte dysfunction plays an essential role in the pathogenesis of proteinuria and glomerulosclerosis. However, the mechanism underlying podocyte dysfunction in many common forms of chronic kidney diseases remains poorly understood. Here we tested the hypothesis that podocytes may undergo epithelial-to-mesenchymal transition after injury. Conditionally immortalized mouse podocytes were incubated with transforming growth factor (TGF)-β1, a potent fibrogenic cytokine that is up-regulated in the diseased kidney. TGF-β1 suppressed the slit diaphragm-associated protein P-cadherin, zonula occludens-1, and nephrin, a change consistent with loss of the epithelial feature. Meanwhile, TGF-β1 induced the expression of the intermediate filament protein desmin and interstitial matrix components fibronectin and collagen I. Furthermore, TGF-β1 promoted the expression and secretion of matrix metalloproteinase-9 by podocytes. Functionally, TGF-β1 increased albumin permeability across podocyte monolayers, as demonstrated by a paracellular albumin influx assay. The expression of Snail, a key transcriptional factor that has been implicated in initiating epithelial-to-mesenchymal transition, was induced by TGF-β1, and ectopic expression of Snail suppressed P-cadherin and nephrin in podocytes. In vivo, in addition to loss of nephrin and zonula occludens-1, mesenchymal markers such as desmin, fibroblast-specific protein-1, and matrix metalloproteinase-9 could be observed in glomerular podocytes of diabetic nephropathy. These results suggest that podocyte dedifferentiation and mesenchymal transition could be a potential pathway leading to their dysfunction, thereby playing a role in the genesis of proteinuria. Podocyte dysfunction plays an essential role in the pathogenesis of proteinuria and glomerulosclerosis. However, the mechanism underlying podocyte dysfunction in many common forms of chronic kidney diseases remains poorly understood. Here we tested the hypothesis that podocytes may undergo epithelial-to-mesenchymal transition after injury. Conditionally immortalized mouse podocytes were incubated with transforming growth factor (TGF)-β1, a potent fibrogenic cytokine that is up-regulated in the diseased kidney. TGF-β1 suppressed the slit diaphragm-associated protein P-cadherin, zonula occludens-1, and nephrin, a change consistent with loss of the epithelial feature. Meanwhile, TGF-β1 induced the expression of the intermediate filament protein desmin and interstitial matrix components fibronectin and collagen I. Furthermore, TGF-β1 promoted the expression and secretion of matrix metalloproteinase-9 by podocytes. Functionally, TGF-β1 increased albumin permeability across podocyte monolayers, as demonstrated by a paracellular albumin influx assay. The expression of Snail, a key transcriptional factor that has been implicated in initiating epithelial-to-mesenchymal transition, was induced by TGF-β1, and ectopic expression of Snail suppressed P-cadherin and nephrin in podocytes. In vivo, in addition to loss of nephrin and zonula occludens-1, mesenchymal markers such as desmin, fibroblast-specific protein-1, and matrix metalloproteinase-9 could be observed in glomerular podocytes of diabetic nephropathy. These results suggest that podocyte dedifferentiation and mesenchymal transition could be a potential pathway leading to their dysfunction, thereby playing a role in the genesis of proteinuria. Proteinuria, the clinical manifestation of structural and functional defects in glomerular filtration barrier, occurs often in the early stage of many forms of primary glomerular diseases. A large body of evidence suggests that the podocyte foot processes and slit diaphragm are pivotal components of the glomerular filter, and disruption of their integrity is a critical event in the development of proteinuria and nephrotic syndrome in a variety of inherited and acquired glomerular disorders.1Pavenstädt H Kriz W Kretzler M Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307PubMed Google Scholar, 2Tryggvason K Wartiovaara J Molecular basis of glomerular permselectivity.Curr Opin Nephrol Hypertens. 2001; 10: 543-549Crossref PubMed Scopus (204) Google Scholar, 3Benzing T Signaling at the slit diaphragm.J Am Soc Nephrol. 2004; 15: 1382-1391Crossref PubMed Scopus (221) Google Scholar Many genetic studies have underscored that podocyte slit diaphragm-associated proteins, such as nephrin and podocin, play an essential role in establishing the size-selective filtration barrier of the kidney, and mutations or deletions of the genes encoding these proteins are consequently associated with the development of proteinuria in both animal models and patients.4Roselli S Heidet L Sich M Henger A Kretzler M Gubler MC Antignac C Early glomerular filtration defect and severe renal disease in podocin-deficient mice.Mol Cell Biol. 2004; 24: 550-560Crossref PubMed Scopus (226) Google Scholar, 5Kim JM Wu H Green G Winkler CA Kopp JB Miner JH Unanue ER Shaw AS CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility.Science. 2003; 300: 1298-1300Crossref PubMed Scopus (424) Google Scholar, 6Tryggvason K Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar, 7Schwarz K Simons M Reiser J Saleem MA Faul C Kriz W Shaw AS Holzman LB Mundel P Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.J Clin Invest. 2001; 108: 1621-1629Crossref PubMed Scopus (523) Google Scholar However, mutations in the slit diaphragm-associated proteins are rare in most common forms of chronic kidney diseases such as diabetic nephropathy.8Pettersson-Fernholm K Forsblom C Perola M Groop PH Polymorphisms in the nephrin gene and diabetic nephropathy in type 1 diabetic patients.Kidney Int. 2003; 63: 1205-1210Crossref PubMed Scopus (20) Google Scholar, 9Sako M Nakanishi K Obana M Yata N Hoshii S Takahashi S Wada N Takahashi Y Kaku Y Satomura K Ikeda M Honda M Iijima K Yoshikawa N Analysis of NPHS1. NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome.Kidney Int. 2005; 67: 1248-1255Crossref PubMed Scopus (76) Google Scholar, 10Hingorani SR Finn LS Kowalewska J McDonald RA Eddy AA Expression of nephrin in acquired forms of nephrotic syndrome in childhood.Pediatr Nephrol. 2004; 19: 300-305Crossref PubMed Scopus (32) Google Scholar In this regard, the pathogenesis of podocyte dysfunction in the vast majority of acquired proteinuric kidney diseases remains to be elucidated. Podocytes are specialized, terminally differentiated visceral epithelial cells that reside on the glomerular basement membrane (GBM) outside the glomerular capillaries.1Pavenstädt H Kriz W Kretzler M Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307PubMed Google Scholar, 3Benzing T Signaling at the slit diaphragm.J Am Soc Nephrol. 2004; 15: 1382-1391Crossref PubMed Scopus (221) Google Scholar In response to injurious stimuli, they often undergo a range of adaptive changes, including hypertrophy, dedifferentiation, detachment, and apoptosis, depending on the severity and duration of the injury.11Shankland SJ The podocyte's response to injury: role in proteinuria and glomerulosclerosis.Kidney Int. 2006; 69: 2131-2147Crossref PubMed Scopus (679) Google Scholar, 12Durvasula RV Shankland SJ Podocyte injury and targeting therapy: an update.Curr Opin Nephrol Hypertens. 2006; 15: 1-7Crossref PubMed Scopus (88) Google Scholar, 13Wiggins RC The spectrum of podocytopathies: a unifying view of glomerular diseases.Kidney Int. 2007; 71: 1205-1214Crossref PubMed Scopus (603) Google Scholar Because of their limited proliferative capacity, podocyte detachment from GBM and apoptosis will inevitably lead to cell depletion or drop out, which could reduce podocyte density, results in an impaired glomerular filtration, and causes proteinuria.14Schiffer M Bitzer M Roberts IS Kopp JB ten Dijke P Mundel P Bottinger EP Apoptosis in podocytes induced by TGF-beta and Smad7.J Clin Invest. 2001; 108: 807-816Crossref PubMed Scopus (559) Google Scholar, 15Susztak K Raff AC Schiffer M Bottinger EP Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes. 2006; 55: 225-233Crossref PubMed Scopus (924) Google Scholar, 16Macconi D Bonomelli M Benigni A Plati T Sangalli F Longaretti L Conti S Kawachi H Hill P Remuzzi G Remuzzi A Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury.Am J Pathol. 2006; 168: 42-54Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 17Wharram BL Goyal M Wiggins JE Sanden SK Hussain S Filipiak WE Saunders TL Dysko RC Kohno K Holzman LB Wiggins RC 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 (606) Google Scholar However, podocyte depletion often takes place in the advanced stage of chronic kidney disease in which proteinuria is already prominent. Recent experimental evidence also demonstrates that podocyte detachment and apoptosis significantly lag behind the onset of proteinuria, arguing against a causative role of podocyte loss in the genesis of proteinuria.18Dai C Stolz DB Bastacky SI St-Arnaud R Wu C Dedhar S Liu Y Essential role of integrin-linked kinase in podocyte biology: bridging the integrin and slit diaphragm signaling.J Am Soc Nephrol. 2006; 17: 2164-2175Crossref PubMed Scopus (121) Google Scholar, 19El-Aouni C Herbach N Blattner SM Henger A Rastaldi MP Jarad G Miner JH Moeller MJ St-Arnaud R Dedhar S Holzman LB Wanke R Kretzler M Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis.J Am Soc Nephrol. 2006; 17: 1334-1344Crossref PubMed Scopus (135) Google Scholar In this context, it is conceivable to speculate that the aberrant regulation of podocyte differentiation or function, rather than podocyte depletion, could be an initial cause of proteinuria in many clinical circumstances. Similar to the cells in most parts of the nephron, podocytes are developmentally derived from the metanephric mesenchyme through mesenchymal-to-epithelial transdifferentiation. This raises an interesting possibility that the podocyte may undergo epithelial-to-mesenchymal transition (EMT), a process of reverse embryogenesis that occurs in diseased kidney as well as in many other organs under pathological conditions.20Xia JL Dai C Michalopoulos GK Liu Y Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation.Am J Pathol. 2006; 168: 1500-1512Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 21Willis BC duBois RM Borok Z Epithelial origin of myofibroblasts during fibrosis in the lung.Proc Am Thorac Soc. 2006; 3: 377-382Crossref PubMed Scopus (412) Google Scholar It is widely recognized that renal tubular epithelial cells are able to undergo EMT after chronic injury22Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 23Iwano M Plieth D Danoff TM Xue C Okada H Neilson EG Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1773) Google Scholar, 24Strutz F Zeisberg M Renal fibroblasts and myofibroblasts in chronic kidney disease.J Am Soc Nephrol. 2006; 17: 2992-2998Crossref PubMed Scopus (273) Google Scholar; a process that is believed to play a critical role in generating matrix-producing fibrogenic cells and in causing tubular atrophy and dysfunction.25Liu 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 (988) Google Scholar, 26Kalluri R Neilson EG Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2172) Google Scholar In analogy to tubular EMT, we hypothesized that mesenchymal transition of podocytes after injury may play a vital role in causing podocyte dysfunction that ultimately leads to a defective glomerular filtration. In this study, we have investigated the possibility of podocyte EMT in a conditionally immortalized mouse podocyte cell line by incubating with transforming growth factor (TGF)-β1, a potent EMT inducer that is up-regulated in diseased kidneys.27Liu Y Renal fibrosis: new insights into the pathogenesis and therapeutics.Kidney Int. 2006; 69: 213-217Crossref PubMed Scopus (915) Google Scholar Our results suggest that EMT could be a potential pathway leading to podocyte dysfunction and proteinuria under pathological conditions. The conditionally immortalized mouse podocyte cell line was kindly provided by Dr. Peter Mundel (Mount Sinai School of Medicine, New York, NY).28Mundel P Reiser J Zuniga Mejia Borja A Pavenstadt H Davidson GR Kriz W Zeller R Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (787) Google Scholar To propagate podocytes, cells were cultured at 33°C in RPMI 1640 medium supplemented with 10% fetal bovine serum and 10 U/ml mouse recombinant interferon-γ (R&D Systems, Minneapolis, MN) to enhance the expression of a thermosensitive T antigen. To induce differentiation, podocytes were grown under nonpermissive conditions at 37°C in the absence of interferon-γ for 14 days. Podocytes were treated under differentiating condition with recombinant TGF-β1 at the concentration of 2 ng/ml, unless otherwise indicated. For some experiments, podocytes were kept in RPMI 1640 medium supplemented with 100 nmol/L 1,25-dihydroxyvitamin D3 and 1 μmol/L all-trans retinoic acid (Sigma, St. Louis, MO) to induce nephrin expression, as recently described elsewhere.29Takano Y Yamauchi K Hiramatsu N Kasai A Hayakawa K Yokouchi M Yao J Kitamura M Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin.Am J Physiol. 2007; 292: F1573-F1582Google Scholar Cells were then collected at different time points for subsequent analyses. For some studies, podocytes were transfected with Snail expression vector (pHA-Snail; provided by A. Garcia de Herreros, Universitat Pompeu Fabra, Barcelona, Spain) by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Mouse kidney tissues were prepared from a uninephrectomized diabetic model, as described previously.30Dai C Yang J Bastacky S Xia J Li Y Liu Y Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice.J Am Soc Nephrol. 2004; 15: 2637-2647Crossref PubMed Scopus (108) Google Scholar Briefly, male CD-1 mice underwent uninephrectomy 1 week before intravenous injection of streptozotocin (Sigma) at 150 mg/kg body weight. A group of mice that underwent sham operation and received no streptozotocin injection served as normal control. Three months after injection of streptozotocin, mice were sacrificed, and the kidney was analyzed as described previously.30Dai C Yang J Bastacky S Xia J Li Y Liu Y Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice.J Am Soc Nephrol. 2004; 15: 2637-2647Crossref PubMed Scopus (108) Google Scholar Human diabetic kidney specimens were obtained from diagnostic renal biopsy performed at the University of Pittsburgh Medical Center. As normal controls, nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used. All studies involving animal model and human tissues were approved by the Institutional Animal Care and Use Committee and the Institutional Review Board, respectively, at the University of Pittsburgh. Western blot analysis for specific protein expression was performed essentially according to an established procedure.31Li Y Yang J Dai C Wu C Liu Y Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis.J Clin Invest. 2003; 112: 503-516Crossref PubMed Scopus (349) Google Scholar, 32Yang J Shultz RW Mars WM Wegner RE Li Y Dai C Nejak K Liu Y Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy.J Clin Invest. 2002; 110: 1525-1538Crossref PubMed Scopus (236) Google Scholar The primary antibodies used were as follows: rat monoclonal anti-P-cadherin (R&D Systems), rabbit polyclonal anti-desmin (MP Biomedicals, Solon, OH), anti-α-tubulin, and anti-matrix metalloproteinase (MMP)-9 (Sigma), and anti-fibronectin (sc-9068; Santa Cruz Biotechnology, Santa Cruz, CA). Quantification was performed by measuring the intensity of the bands with the use of the National Institutes of Health Image analysis software. Indirect immunofluorescence staining was performed using an established procedure.31Li Y Yang J Dai C Wu C Liu Y Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis.J Clin Invest. 2003; 112: 503-516Crossref PubMed Scopus (349) Google Scholar, 33Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar Briefly, cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes at −20°C, followed by blocking with 20% normal donkey serum in phosphate-buffered saline (PBS) for 30 minutes at room temperature. Cells were incubated with the specific primary antibodies against P-cadherin, desmin, fibronectin, and MMP-9, as described above, and rabbit polyclonal anti-ZO-1 (61-7300, Invitrogen). To visualize the primary antibodies, cells were stained with cyanine Cy2-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred. Cells were double stained with 4′,6-diamidino-2-phenylindole HCl to visualize the nuclei. Kidney sections from paraffin-embedded tissues were prepared at 4-μm thickness and stained for WT-1 and TGF-β type I receptor (Santa Cruz Biotechnology), desmin, MMP-9, ZO-1, nephrin (Progen, Heidelberg, Germany) and fibroblast-specific protein-1 (FspI, also known as S100A4; DAKO, Carpinteria, CA), respectively, using a routine procedure.33Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar Stained slides were viewed under an Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Nikon, Melville, NY). Zymographic analysis of MMP proteolytic activity in the supernatant of cultured cells was performed according to the method described previously.22Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 32Yang J Shultz RW Mars WM Wegner RE Li Y Dai C Nejak K Liu Y Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy.J Clin Invest. 2002; 110: 1525-1538Crossref PubMed Scopus (236) Google Scholar Briefly, a constant amount of protein from the conditioned media (15 μg) was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel containing 1 mg/ml of gelatin (Bio-Rad Laboratories, Hercules, CA). After electrophoresis, the gel was incubated at 37°C for 16 to 36 hours in a developing buffer containing 50 mmol/L Tris-HCl, pH 7.6, 0.2 mol/L NaCl, 5 mmol/L CaCl2, and 0.02% Brij 35. The gel was then stained with a solution of 30% methanol, 10% glacial acetic acid, and 0.5% Coomassie blue G250, followed by destaining in the same solution without dye. Proteinase activity was detected as unstained bands on a blue background representing areas of gelatin digestion. Total RNA was prepared using a TRIzol RNA isolation system according to the instructions specified by the manufacturer (Invitrogen). The first strand of cDNA was synthesized using 2 μg of RNA in 20 μl of reaction buffer using AMV-RT (Promega, Madison, WI) and random primers at 42°C for 30 minutes. PCR was performed using a standard PCR kit on 1-μl aliquots of cDNA and HotStarTaq polymerase (Qiagen Inc., Valencia, CA) with specific primer pairs. The sequences of primer pairs are shown in Table 1. The PCR products were size fractionated on a 1.0% agarose gel and detected by ethidium bromide staining. No detectable signal was found in a parallel control tube without RT (data not shown).Table 1The Sequences of the PCR Primer Pairs Used in This StudyGeneGenBank accession numberSenseAntisenseProduct sizeZO-1NM_0093865′-TAGCACGGACAGTAGACACA-3′5′-ATGGAAGTTGGGGTTCATAG-3′635 (α+)FibronectinNM_0102335′-CGAGGTGACAGAGACCACAA-3′5′-CTGGAGTCAAGCCAGACACA-3′149Collagen INM_0077425′-ATCTCCTGGTGCTGATGGAC-3′5′-ACCTTGTTTGCCAGGTTCAC-3′154DesminNM_0100435′-TGCAGCCACTCTAGCTCGTA-3′5′-GACATGTCCATCTCCACCTG-3′150MMP-9NM_0135995′-CACCACCACAACTGAACCAC-3′5′-CTCAGAAGAGCCCGCAGTAG-3′199MMP-2NM_0086105′-AAGGGGATCCAGGAGCTCTA-3′5′-GCTTGTCACGTGGTGTCAC-3′199SnailNM_0114275′-AGCCCAACTATAGCGAGCTG-3′5′-CCAGGAGAGAGTCCCAGATG-3′150β-ActinNM_0073935′-CAGCTGAGAGGGAAATCGTG-3′5′-CGTTGCCAATAGTGATGACC-3′150P-cadherinX063405′-GATTTTGAGGCTCAGGACCA-3′5′-GACCTTGGAAGGTGGAACAA-3′150NephrinNM_0194595′-CCCAGGTACACAGAGCACAA-3′5′-CTCACGCTCACAACCTTCAG-3′200 Open table in a new tab A simple albumin influx assay was used to evaluate the filtration barrier function of podocyte monolayer, as described previously.34Rico M Mukherjee A Konieczkowski M Bruggeman LA Miller RT Khan S Schelling JR Sedor JR WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.Am J Physiol. 2005; 289: F431-F441Crossref Scopus (33) Google Scholar Briefly, podocytes (5 × 103) were seeded onto the collagen-coated transwell filters (3-μm pore; Corning, New York, NY) in the top chamber and cultured under differentiating conditions. After 10 days, podocytes were serum-starved overnight and treated without or with 2 ng/ml of TGF-β1 for 48 hours. Cells were washed twice with PBS supplemented with 1 mmol/L MgCl2 and 1 mmol/L CaCl2 to preserve the cadherin-based junctions. The top chamber was then refilled with 0.15 ml of RPMI 1640 and the bottom chamber with 1 ml of RPMI 1640 supplemented with 40 mg/ml of bovine serum albumin and incubated at 37°C. A small aliquot of media from the top chamber was collected at different time points and the albumin concentration was determined using a bicinchoninic acid protein assay kit (Sigma). All data examined were expressed as mean ± SEM. Statistical analysis of the data were performed using SigmaStat software (Jandel Scientific Software, San Rafael CA). Comparison between groups was made using one-way analysis of variance, followed by Student-Newman-Keuls test. A P value of less than 0.05 was considered significant. Podocyte slit diaphragm, a modified P-cadherin-containing adherens junction, is an essential component of the glomerular filtration barrier. We first examined the expression of P-cadherin in podocytes after TGF-β1 treatment. As shown in Figure 1A, treatment of podocytes with TGF-β1 at a concentration of 2 ng/ml markedly suppressed P-cadherin protein expression in a time-dependent manner. Western blot analysis revealed that P-cadherin started to decrease as early as 24 hours and almost completely disappeared at 72 hours after TGF-β1 treatment. The suppression of P-cadherin expression by TGF-β1 in podocytes was also dose-dependent (Figure 1B). Immunofluorescence staining exhibited that P-cadherin was primarily localized in the cell-cell junctional sites of the differentiated podocytes (Figure 1C), and its staining primarily disappeared after TGF-β1 treatment (Figure 1D). Zonula occludens-1 (ZO-1), a tight junction-associated protein, locates at the slit diaphragm and is linked through catenin intermediates to the transmembrane proteins Neph1 and P-cadherin.35Reiser J Kriz W Kretzler M Mundel P The glomerular slit diaphragm is a modified adherens junction.J Am Soc Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar RT-PCR showed that TGF-β1 inhibited ZO-1 mRNA expression as early as 12 hours (Figure 2A). This suppression of ZO-1 by TGF-β1 was also dose-dependent (Figure 2B). Immunofluorescence staining showed abundant ZO-1 at the sites of cell-cell contacts, with a characteristic zipper-like pattern between the interdigitating processes of podocytes (Figure 2C). After TGF-β1 treatment, the overall density of ZO-1 staining was markedly decreased, and its zipper-like staining pattern was changed to a continuous belt along the cell borders (Figure 2D). Nephrin is a slit diaphragm protein that plays an essential role in establishing an effective glomerular filtration. Mutations or down-regulation of nephrin is associated with a defective filtration barrier, causing proteinuria.6Tryggvason K Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar Cultured mouse podocytes expressed little nephrin under normal differentiating conditions; however, significant nephrin expression was observed when they were incubated in the medium containing active vitamin D and retinoic acid, as recently reported.29Takano Y Yamauchi K Hiramatsu N Kasai A Hayakawa K Yokouchi M Yao J Kitamura M Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin.Am J Physiol. 2007; 292: F1573-F1582Google Scholar Using this culture system, we examined the effect of TGF-β1 on nephrin expression in podocytes. As shown in Figure 3, A and C, TGF-β1 at a concentration of 2 ng/ml repressed nephrin mRNA expression in a time-dependent manner. Similarly, TGF-β1 could inhibit nephrin expression in different concentrations ranging from 0.5 to 5 ng/ml (Figure 3, B and D). Together with the data presented above, these results suggest that TGF-β1 is able to induce podocyte dedifferentiation by suppressing P-cadherin, ZO-1, and nephrin expression. We next investigated whether TGF-β1 induces a mesenchymal conversion of podocytes. To this end, the expression of desmin, an intermediate filament protein, was examined in podocytes after TGF-β1 treatment. As illustrated in Figure 4, incubation of podocytes with TGF-β1 induced desmin mRNA and protein expression in a time- and dose-dependent manner. Immunostaining also demonstrated clearly the induction of desmin-positive intermediate filaments in the cytoplasm of podocytes after TGF-β1 treatment (Figure 4C). We also examined the expression of interstitial matrix components in podocytes on TGF-β1 stimulation. As shown in Figure 5, A and B, incubation of podocytes with TGF-β1 induced de novo expression of interstitial matrix components. Podocytes at basal conditions barely expressed interstitial type I collagen and fibronectin. However, after TGF-β1 stimulation, podocytes began to express an impressive amount of type I collagen and fibronectin mRNA. Western blot analysis and immunofluorescence staining also revealed a remarkable induction of fibronectin protein, which was assembled and deposited in the extracellular compartment (Figure 5, C and D). We further investigated the MMP-9 and MMP-2 expression in podocytes after TGF-β1 treatment by a wide variety of approaches, including RT-PCR, zymographic analysis, Western blot, and immunostaining. As shown in Figure 6A, MMP-9 mRNA was dramatically induced by TGF-β1 in podocytes. Zymographic analysis of the conditioned media demonstrated that TGF-β1 induced a marked increase in MMP-9 protein expression and secretion, and such MMP-9 induction was both time- and dose-dependent (Figure 6B). The striking induction of MMP-9 by TGF-β1 in podocytes was independently confirmed by Western blot analysis of the conditioned media (Figure 6C). TGF-β1 also marginally induced MMP-2 mRNA and increased MMP-2 secretion (Figure 6, A and B). Of interest, MMP-9 protein was apparently aggregated on the surface of podocytes after TGF-β1 treatment, as shown by immunofluorescence staining (Figure 6D). This pattern of MMP-9 distribution suggests a high local concentration of its protein in certain areas of cell surface. To assess the functional consequence of podocyte EMT, we examined the filtration barrier function of podocyte by using a paracellular permeability influx assay.34Rico M Mukherjee A Konieczkowski M Bruggeman LA Miller RT Khan S Schelling JR Sedor JR WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.Am J Physiol. 2005; 289: F431-F441Crossref Scopus (33) Google Scholar As depicted in Figure 7A, this simple assay measured the albumin flux rate across the differentiated podocyte monolayer. Differentiated podocytes were incubated with TGF-β1 for 48 hours to induce podocyte EMT, and then subjected to albumin influx assay. As shown in Figure 7B, compared with the controls, TGF-β1 treatment resulted in a greater albumin influx across the podocyte monolayer. These results indicate that
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