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Matrix-Stiffness–Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis

2015; Elsevier BV; Volume: 185; Issue: 9 Linguagem: Inglês

10.1016/j.ajpath.2015.05.019

ISSN

1525-2191

Autores

Wan-Chun Chen, Hsi‐Hui Lin, Ming‐Jer Tang,

Tópico(s)

Cancer-related gene regulation

Resumo

The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression. The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression. Renal fibrosis, a common pathologic condition in progressive chronic kidney disease, is characterized by excessive crosslinking or deposition of the extracellular matrix, particularly of collagenous fibers. With the use of the unilateral ureteral obstruction (UUO) model, we found that the fibrotic kidney was stiffer than the normal kidney (Y.C. Yeh, unpublished data). Accumulated data indicate that matrix stiffness, one of the mechanical forces acting on cells, has a large impact as chemical stimuli on the regulation of cell proliferation, apoptosis, and differentiation.1Engler A.J. Sen S. Sweeney H.L. Discher D.E. 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Considering the importance of matrix stiffness in regulating cellular behavior and tissue function, we speculate whether increasing stiffness in the microenvironment is a prerequisite for the fibrotic response of PTs. With the use of the ex vivo primary culture system and matrices with tunable stiffness, we found that soft matrix retained primary mice PT epithelial cells (mPTECs) at a tubular-like structural characteristics with differentiated phenotypes and growth arrest. EMT induced by transformation growth factor-β1, a key mediator in renal fibrosis,11Meng X.M. Chung A.C. Lan H.Y. Role of the TGF-beta/BMP-7/Smad pathways in renal diseases.Clin Sci (Lond). 2013; 124: 243-254Crossref PubMed Scopus (282) Google Scholar is also inhibited by soft matrix.12Chen W.C. Lin H.H. Tang M.J. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.Am J Physiol Renal Physiol. 2014; 307: F695-F707Crossref PubMed Scopus (40) Google Scholar In the present work, we studied the detailed mechanisms underlying the matrix stiffness-modulated behavior of mPTECs, particularly in relation to proliferation. On the basis of the microarray profiling of mPTECs on culture dishes, we found that a subfamily of Krüppel-like factors (Klfs) was markedly altered during culture (Table 1). Klfs, highly conserved zinc finger-containing transcription factors (TFs), are key regulators of cellular functions. To date, 17 Klfs have been identified.13Suske G. Bruford E. Philipsen S. Mammalian SP/KLF transcription factors: bring in the family.Genomics. 2005; 85: 551-556Crossref PubMed Scopus (301) Google Scholar Among these, the expressions of Klf4 and Klf5 are highly restricted in the epithelium of several organs. Klf4 and Klf5 antagonize each other by physical competition in controlling expression of target genes, which are related to proliferation, differentiation, apoptosis, development, and disease.14Dang D.T. Zhao W. Mahatan C.S. Geiman D.E. Yang V.W. Opposing effects of Kruppel-like factor 4 (gut-enriched Kruppel-like factor) and Kruppel-like factor 5 (intestinal-enriched Kruppel-like factor) on the promoter of the Kruppel-like factor 4 gene.Nucleic Acids Res. 2002; 30: 2736-2741Crossref PubMed Google Scholar, 15McConnell B.B. Ghaleb A.M. Nandan M.O. Yang V.W. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology.BioEssays. 2007; 29: 549-557Crossref PubMed Scopus (209) Google Scholar, 16Ghaleb A.M. Nandan M.O. Chanchevalap S. Dalton W.B. Hisamuddin I.M. Yang V.W. 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In this study, we clarify the role of the inverse expression pattern of Klf5 and Klf4 in mPTECs.Table 1Lists of the Top Six Up-Regulated and Down-Regulated Transcription Factors in Mouse Proximal Tubule Epithelial Cells During ex Vivo CultureRankGeneFull name∗Acquired from the NCBI Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore).D1/D0 (fold)D3/D0 (fold)Up-regulated 1Klf5Kruppel-like factor 5 (NM_009769)25.228.3 2Runx1Runt related transcription factor 1 (NM_009821)16.727.8 3Atf5Activating transcription factor 5 (NM_030693)15.25.5 4Ybx3 (Csda)Cold shock domain protein A (NM_011733)7.710.5 5E2f3E2F transcription factor 3 (NM_010093)6.34.6 6Atf1Activating transcription factor 1 (NM_007497)5.81.9Down-regulated 1Klf2Kruppel-like factor 2 (NM_008452)−8.6−4.2 2Atf3Activating transcription factor 3 (NM_007498)−8.4−4.6 3Sp5Trans-acting transcription factor 5 (NM_022435)−6.9−4.5 4Pitx2Paired-like homeodomain transcription factor 2 (NM_001042502)−6.5−8.4 5Klf15Kruppel-like factor 15 (NM_023184)−5.4−16.9 6Klf4Kruppel-like factor 4 (NM_010637)−5.0−1.7D, day.∗ Acquired from the NCBI Nucleotide Database (http://www.ncbi.nlm.nih.gov/nuccore). Open table in a new tab D, day. Klf5 is highly expressed in proliferating epithelial cells during development and in adult tissues.15McConnell B.B. Ghaleb A.M. Nandan M.O. Yang V.W. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology.BioEssays. 2007; 29: 549-557Crossref PubMed Scopus (209) Google Scholar, 17Ema M. Mori D. Niwa H. Hasegawa Y. Yamanaka Y. Hitoshi S. Mimura J. Kawabe Y. Hosoya T. Morita M. Shimosato D. Uchida K. Suzuki N. Yanagisawa J. Sogawa K. Rossant J. Yamamoto M. Takahashi S. Fujii-Kuriyama Y. 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Kruppel-like factor 4 inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin gene expression.J Biol Chem. 2010; 285: 16854-16863Crossref PubMed Scopus (130) Google Scholar Inducible expression of Klf4 blocks cell cycle by inhibiting cyclin D1 in both normal and cancer cells, suggesting that Klf4 functions as a tumor suppressor.15McConnell B.B. Ghaleb A.M. Nandan M.O. Yang V.W. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology.BioEssays. 2007; 29: 549-557Crossref PubMed Scopus (209) Google Scholar, 27Shimizu Y. Takeuchi T. Mita S. Notsu T. Mizuguchi K. Kyo S. Kruppel-like factor 4 mediates anti-proliferative effects of progesterone with G0/G1 arrest in human endometrial epithelial cells.J Endocrinol Invest. 2010; 33: 745-750Crossref PubMed Google Scholar, 28Chen X. Johns D.C. Geiman D.E. Marban E. Dang D.T. Hamlin G. Sun R. Yang V.W. Kruppel-like factor 4 (gut-enriched Kruppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle.J Biol Chem. 2001; 276: 30423-30428Crossref PubMed Scopus (212) Google Scholar, 29Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Gut-enriched Kruppel-like factor represses cyclin D1 promoter activity through Sp1 motif.Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar In addition, Klf4 is essential for maintaining cancer stem cells or stem cells.30Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar, 31Yu F. Li J. Chen H. Fu J. Ray S. Huang S. Zheng H. Ai W. 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Transcriptional control of terminal nephron differentiation.Am J Physiol Renal Physiol. 2008; 294: F1273-F1278Crossref PubMed Scopus (24) Google Scholar, 34Saifudeen Z. Dipp S. Fan H. El-Dahr S.S. Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation.Am J Physiol Renal Physiol. 2005; 288: F899-F909Crossref PubMed Scopus (36) Google Scholar Klf4 in glomerular podocytes facilitates cell function and a reduction in proteinuria.35Hayashi K. Sasamura H. Nakamura M. Azegami T. Oguchi H. Sakamaki Y. Itoh H. KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria.J Clin Invest. 2014; 124: 2523-2537Crossref PubMed Scopus (88) Google Scholar Here, we report that inverse expression of Klf5 and Klf4 was switched between soft and stiff matrices. Yes-associated protein 1 (YAP1)-transduced mechanical cues from matrix stiffness may regulate the inverse expression of Klf5 and Klf4, subsequently deciding the cellular fate. Furthermore, the inverse expression of Klf5 and Klf4 were also evaluated in kidneys from normal and UUO mice. Primary mPTECs from mice kidneys were harvested and cultured as previously described.12Chen W.C. Lin H.H. Tang M.J. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.Am J Physiol Renal Physiol. 2014; 307: F695-F707Crossref PubMed Scopus (40) Google Scholar All procedures were reviewed and approved through the Institute of Animal Care and Use Committee at the Medical College of National Cheng Kung University (Tainan, Taiwan). RNA obtained from freshly isolated mPTECs and mPTECs cultured on culture dishes for 1 and 3 days were purified and quantified by OD260 nm by a ND-1000 spectrophotometer (Nanodrop Technology, Wilmington, DE) then qualitated by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with RNA 6000 nano labchip kit. One microgram of total RNA was amplified by a low RNA input fluor linear amp kit (Agilent Technologies) and labeled with cyanin 3 (CyDye; PerkinElmer, Boston, MA) during the in vitro transcription process. Cyanin 3–labled complementary RNA (1.65 μg) was fragmented to an average size of approximately 50 to 100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled complementary RNA was then pooled and hybridized to oligo microarray (Agilent Technologies) at 60°C for 17 hours. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner at 535 nm for cyanin 3. Scanned images are analyzed by Feature extraction 10.5 software (Agilent Technologies), an image analysis and normalization software used to quantify signal and background intensity for each feature. The data discussed in this publication was deposited in National Center for Biotechnology Information's Gene Expression Omnibus36Edgar R. Domrachev M. Lash A.E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.Nucleic Acids Res. 2002; 30: 207-210Crossref PubMed Scopus (8467) Google Scholar (http://www.ncbi.nlm.nih.gov/geo; accession number GSE69217). Ingenuity pathway analysis version 8.7 (Ingenuity Systems, Inc., Redwood City, CA) software was used for functional network analysis of the microarray result; Table 1 summarizes the results. All procedures were reviewed and approved by the Institute of Animal Care and Use Committee at the Medical College of National Cheng Kung University, Taiwan. UUO was performed in 1-month-old male C57BL/6 mice with an established procedure, as previously described.37Yeh Y.C. Wei W.C. Wang Y.K. Lin S.C. Sung J.M. Tang M.J. Transforming growth factor-{beta}1 induces Smad3-dependent {beta}1 integrin gene expression in epithelial-to-mesenchymal transition during chronic tubulointerstitial fibrosis.Am J Pathol. 2010; 177: 1743-1754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 38Wu M.J. Wen M.C. Chiu Y.T. Chiou Y.Y. Shu K.H. Tang M.J. Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis.Kidney Int. 2006; 69: 2029-2036Crossref PubMed Scopus (144) Google Scholar In mice subjected to UUO, the contralateral unligated kidney was used as a control organ. After UUO surgery, mice were sacrificed at various time points, and their kidneys were removed. Paraffin-fixed tissues were used for immunohistochemistry (IHC). Lysates from the whole kidney (including cortex and medulla) or cortex only were used for Western blot analysis. For in vivo lysyl oxidase inhibition, β-aminopropionitrile (BAPN; 200 mg/kg body weight; Sigma-Aldrich, St. Louis, MO) was injected via the i.p. route daily. In the control group, normal saline was used instead of BAPN. The injection was started 1 day before UUO surgery and persisted until the end of the experiment. In mice subjected to 5/6 nephrectomy (Nx), the left kidney was exposed, and the upper and lower poles were tied with a polyglycolic acid suture line, followed by right nephrectomy. Then, the peritoneum and skin were sutured. Seventeen weeks after 5/6 Nx surgery, mice were sacrificed, and their kidneys were removed for the IHC experiments. IHC was performed, as previously described.39Lee P.T. Lin H.H. Jiang S.T. Lu P.J. Chou K.J. Fang H.C. Chiou Y.Y. Tang M.J. Mouse kidney progenitor cells accelerate renal regeneration and prolong survival after ischemic injury.Stem Cells. 2010; 28: 573-584PubMed Google Scholar Primary antibodies against Klf4, cyclin D1, proliferating cell nuclear antigen (Santa Cruz Biotechnology, Santa Cruz, CA), Klf5, YAP1 (Novus), phospho-extracellular signal-regulated kinase (p-ERK), and ERK (Cell Signaling, Boston, MA) were used for IHC detection. Part of the IHC experiments were performed by double staining polymer detection systems (BioTnA, Taiwan). 293T (human embryonic kidney cell line), LLC-PK1 (porcine proximal tubule cell line), TCMK-1 (mice proximal tubule cell line), MDCK (dog distal tubule cell line), M1 (mouse collecting duct cell line), and NRK49F (rat renal fibroblasts) cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 5% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin under 5% CO2 at 37°C. Matrices composed of Matrigel (MG; BD Biosciences PharMingen, San Jose, CA) were prepared as previously described.12Chen W.C. Lin H.H. Tang M.J. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.Am J Physiol Renal Physiol. 2014; 307: F695-F707Crossref PubMed Scopus (40) Google Scholar The Young's moduli of these matrices were measured with atomic force microscopy (AFM). Briefly, the Young's modulus of the MG is approximately 66.0 ± 0.3 Pa, and both the dish and MG-coated dish are approximately giga Pa. Western blot analysis was performed as previously described.40Wei W.C. Lin H.H. Shen M.R. Tang M.J. Mechanosensing machinery for cells under low substratum rigidity.Am J Physiol Cell Physiol. 2008; 295: C1579-C1589Crossref PubMed Scopus (58) Google Scholar The cell lysates were harvested, resolved on SDS-PAGE, and then electrophoretically blotted onto nitrocellular paper. The primary antibodies used in this study are listed as follows: Klf4, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology), Klf5 (Millipore, Temecula, CA), α-smooth muscle actin (Sigma-Aldrich), β1 integrin (BD Biosciences PharMingen), p-ERK, and ERK (Cell Signaling). Total RNA was extracted with TRIzol reagent (Invitrogen-Molecular Probes, Carlsbad, CA) according to the manufacturer's instructions. RNA quality was verified and reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). PCR was performed with specific primer sets at 94°C for 5 minutes, followed by 27 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and a final step at 72°C for 7 minutes. The cDNA was then used as a template for PCR with the use of primers specific for mouse cyclin D1 (forward, 5′-CACACGGACTACAGGGGAGT-3′; reverse, 5′-CAAGGGAATGGTCTCCTTCA-3′); mouse Klf5 (forward, 5′-AGACGGCAGTAATGGACACC-3′; reverse, 5′-GATGTTGGCCTTCACGTACT-3′); mouse Klf4 (forward, 5′-TAGCCTAAATGATGGTGCTTGGTG-3′; reverse, 5′-TGTTCTGCTTAAGGCATACTTGGG-3′), and mouse glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-ACGGCACAGTCAAGGCTGAG-3′; reverse, 5′-GGAGGCCATGTAGACCATGAGG-3′). The PCR products were separated on a 1.2% agarose gel that contained ethidium bromide and was visualized under a UV transilluminator. Immunofluorescence staining was performed as previously described.41Yeh Y.C. Wu C.C. Wang Y.K. Tang M.J. DDR1 triggers epithelial cell differentiation by promoting cell adhesion through stabilization of E-cadherin.Mol Biol Cell. 2011; 22: 940-953Crossref PubMed Scopus (58) Google Scholar The primary antibodies used in this study are listed as follows: cyclin D1, Klf4 (Santa Cruz Biotechnology), Klf5, and YAP1 (Novus, Littleton, CO). After washing with phosphate-buffered saline, the cells were incubated with the secondary antibody for anti-mouse or rabbit IgG conjugated with Alexa 488 (Invitrogen-Molecular Probes) and/or phalloidin-tetramethylrhodamine isothiocyanate (Sigma-Aldrich) and 10 μg/mL Hoechst 33258 for 1 hour. The imaging was performed from sequential z-series scans with a confocal microscope (FV-1000; Olympus, Tokyo, Japan). cyclin D1, Klf5, and YAP1 in the apical, middle, and basal regions of cells were recolored green, red, and blue, respectively. The Max XY projection images were reconstructed from a stack of recolored confocal images by ImageJ software version 1.410 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Nuclear and cytoplasmic fractions were obtained with the REAP (Rapid, Efficient and Practical) method.42Suzuki K. Bose P. Leong-Quong R.Y. Fujita D.J. Riabowol K. REAP: a two minute cell fractionation method.BMC Res Notes. 2010; 3: 294Crossref PubMed Scopus (282) Google Scholar Briefly, cells grown on dishes were washed with phosphate-buffered saline and then scraped from dishes. After quickly spinning, the pellets were resuspended in 900 μL ice-cold 0.1% NP-40 (Calbiochem, La Jolla, CA) in phosphate-buffered saline and titrated to mechanically disrupt the cytoplasmic membranes. Lysate (300 μL) was divided into aliquots as the whole cell lysate. After the second centrifugation, 300 μL of the supernatant fluid was divided into aliquots as the cytoplasmic fraction. The resulting pellet was washed with 1 mL ice-cold 0.1% NP-40 and centrifuged again. The pellet was then resuspended with 180 μL 1× Laemmli sample buffer and designated the nuclear fraction. One hundred microliter of 4× Laemmli sample buffer was added to the whole cell and cytoplasmic fractions. Each fraction was sonicated with microprobes and then boiled for 1 minute. Finally, the whole cell, cytoplasmic, and nuclear fractions were examined by Western blot analysis. For measurements of mechanical properties of tissue/cell, JPK NanoWizard II AFM with BioCell (JPK Instruments, Berlin, Germany) was equipped and manipulated as previously described.43Chiou Y.W. Lin H.K. Tang M.J. Lin H.H. Yeh M.L. The influence of physical and physiological cues on atomic force microscopy-based cell stiffness assessment.PLoS One. 2013; 8: e77384Crossref PubMed Scopus (56) Google Scholar Fresh kidney tissue samples were sliced at a thickness of 100 μm with a microtome. Tissue slices were glued to a glass coverslip with a small drop of nail polish, and only the intact side of the cortex was immediately subjected to AFM measurements. Tipless cantilevers (Arrow-TL1-50; Nanoworld, Neuchâtel, Switzerland) modified with 5-μm diameter polystyrene bead were used to measure tissue and cells. The spring constants of all cantilevers were calibrated via the thermal noise method in liquid before each measurement and valued 0.03 N/m. The indenting force was set at 1 nN. Force-distance curves were collected and calculated with JPK package software version 4.6.62 (JPK Instruments), which was based on the Hertz model. For transient transfection, 293T cells, LLC-PK1 cells, and mPTECs were plated on culture dishes for 24 hours before transfection with Lipofectamine 3000 plus reagent according to the manufacturer's instructions (Life Technologies, Inc., Carlsbad, CA). The plasmids of p-mCherry and pLM-mCherry-Klf4 were purchased from Addgene Inc. (Cambridge, MA). After transfection, images of the transfected cells were taken to measure the cell spreading area, and then lyzed for RT-PCR. To further enrich the mCherry-positive cells in 293T cells, cells were sorted by flow cytometry. For immunostaining in transfected mPTECs, the medium was replaced for another 48 hours of culture after transfection. The cells were then fixed and costained with cyclin D1 and Hoechst 33258. To knockdown Klf5 in mPTECs cells, 21-mer shRNA against mouse Klf5 expressed in pLKO.1 vector was purchased from National RNAi Core Facility (Taipei, Taiwan). The sequence for shKlf5 is 5′-TCCGATAATTTCAGAGCATAA-3′. Cell proliferation was evaluated by Click-iT EdU (5-ethynyl-2′-deoxyuridine) Alexa Fluor 488 Imaging Kit (Invitrogen-Molecular Probes) as previously described.12Chen W.C. Lin H.H. Tang M.J. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.Am J Physiol Renal Physiol. 2014; 307: F695-F707Crossref PubMed Scopus (40) Google Scholar Briefly, mPTECs were cultured

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