Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis
2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090517
ISSN1525-2191
AutoresBenjamin D. Humphreys, Shuei‐Liong Lin, Akio Kobayashi, Thomas E. Hudson, Brian T. Nowlin, Joseph V. Bonventre, M. Todd Valerius, Andrew P. McMahon, Jeremy S. Duffield,
Tópico(s)Renal cell carcinoma treatment
ResumoUnderstanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor β1 treatment. However, using either red fluorescent protein or β-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive(+) mesenchymal cells give rise to adult CD73+, platelet derived growth factor receptor β+, smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin+ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease. Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor β1 treatment. However, using either red fluorescent protein or β-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive(+) mesenchymal cells give rise to adult CD73+, platelet derived growth factor receptor β+, smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin+ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease. Understanding the origin and differentiation pathways of myofibroblasts in vivo is critical for identifying new therapeutic strategies for fibrosing disease. Myofibroblasts, contractile cells that deposit pathological extracellular matrix, were first believed to derive from a specialized perivascular cell known as the hepatic stellate cell when studied in the liver. In health these cells store retinoic acid in intracellular vesicles and cultured stellate cells possess all of the hallmarks of myofibroblasts in vitro.1Friedman SL Roll FJ Boyles J Arenson DM Bissell DM Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix.J Biol Chem. 1989; 264: 10756-10762Abstract Full Text PDF PubMed Google Scholar In other organ systems, similar perivascular cells have been postulated to be the source of myofibroblasts, but have been hard to define.2Ivarsson M Sundberg C Farrokhnia N Pertoft H Rubin K Gerdin B Recruitment of type I collagen producing cells from the microvasculature in vitro.Exp Cell Res. 1996; 229: 336-349Crossref PubMed Scopus (28) Google Scholar, 3Schlondorff D The glomerular mesangial cell: an expanding role for a specialized pericyte.FASEB J. 1987; 1: 272-281Crossref PubMed Scopus (379) Google Scholar Mesoderm-derived cells, when cultured in vitro, differentiate into cells with hallmarks of myofibroblasts, including most notably mesenchymal stem cells from bone marrow, as well as mesangial cells of the kidney, and cultured monocyte-derived macrophages.4Campagnoli C Roberts IA Kumar S Bennett PR Bellantuono I Fisk NM Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow.Blood. 2001; 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13: 952-961Crossref PubMed Scopus (1621) Google Scholar It has been postulated that during kidney injury in vivo, epithelial cells undergo a phenotypic transition or can transdifferentiate into interstitial myofibroblasts by this same process of EMT.13Iwano 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 (1714) Google Scholar, 14Kalluri R Neilson EG Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2097) Google Scholar Subsequent studies both in vivo and in vitro support this hypothesis.15Li 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 (343) Google Scholar, 16Zavadil J Cermak L Soto-Nieves N Bottinger EP Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.EMBO J. 2004; 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Epithelial cells are known to respond to injury in several ways. They undergo morphological changes, lose polarity, acquire stress fibers, and migrate along the basement membrane.17Thadhani R Pascual M Bonventre JV Acute renal failure.N Engl J Med. 1996; 334: 1448-1460Crossref PubMed Scopus (1511) Google Scholar They up-regulate inflammatory genes and genes that enhance their ability to survive in a hostile environment.18Ouellette AJ Malt RA Sukhatme VP Bonventre JV Expression of two “immediate early” genes, Egr-1 and c-fos, in response to renal ischemia and during compensatory renal hypertrophy in mice.J Clin Invest. 1990; 85: 766-771Crossref PubMed Scopus (99) Google Scholar, 19Yoshida T Tang SS Hsiao LL Jensen RV Ingelfinger JR Gullans SR Global analysis of gene expression in renal ischemia-reperfusion in the mouse.Biochem Biophys Res Commun. 2002; 291: 787-794Crossref PubMed Scopus (45) Google Scholar In addition, they express some genes shared by embryonic mesenchymal cells transitioning to epithelium during nephrogenesis.20Dressler GR Woolf AS Pax2 in development and renal disease.Int J Dev Biol. 1999; 43: 463-468PubMed Google Scholar, 21Stark K Vainio S Vassileva G McMahon AP Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4.Nature. 1994; 372: 679-683Crossref PubMed Scopus (896) Google Scholar, 22Witzgall R Brown D Schwarz C Bonventre JV Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.J Clin Invest. 1994; 93: 2175-2188Crossref PubMed Scopus (531) Google Scholar Thus it has been suggested that in response to injury epithelial cells undergo EMT, recapitulating primitive mesenchymal cells of the intermediate mesoderm.9Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar This, however, is misleading since intermediate mesoderm cells do not express inflammatory and cell-survival genes that injured adult epithelial cells up-regulate, and expression of a limited number of genes shared by embryonic mesenchyme such as α smooth muscle actin (SMA), by itself, does not define injured epithelial cells as mesenchymal.23Ichimura T Asseldonk EJ Humphreys BD Gunaratnam L Duffield JS Bonventre JV Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells.J Clin Invest. 2008; 118: 1657-1668Crossref PubMed Scopus (555) Google Scholar, 24Mazzucchelli L Protein S100A4: too long overlooked by pathologists?.Am J Pathol. 2002; 160: 7-13Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 25Zavadil J Bottinger EP TGF-beta and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1383) Google Scholar Neoplastic epithelial cells have the capacity to metastasize, share some characteristics with myofibroblasts, and express or down-regulate key regulators of metastasis such as mts1 (S100A4 or FSP-1), Twist, Snail, and β-catenin, genes whose expression can also be activated in cultured epithelial cells.26Tulchinsky EM Grigorian MS Ebralidze AK Milshina NI Lukanidin EM Structure of gene mts1, transcribed in metastatic mouse tumor cells.Gene. 1990; 87: 219-223Crossref PubMed Scopus (22) Google Scholar, 27Vega S Morales AV Ocana OH Valdes F Fabregat I Nieto MA Snail blocks the cell cycle and confers resistance to cell death.Genes Dev. 2004; 18: 1131-1143Crossref PubMed Scopus (676) Google Scholar, 28Yang J Mani SA Donaher JL Ramaswamy S Itzykson RA Come C Savagner P Gitelman I Richardson A Weinberg RA Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3068) Google Scholar Proponents of the hypothesis that myofibroblasts in inflammation and scarring derive from epithelial cells have drawn on these observations to extend the term EMT to mean epithelial-to-myofibroblast transition. Interstitial myofibroblasts are the principle source of interstitial collagens, including fibrillar collagens I and III. They are widely held to be the primary cell in the injured kidney that lays down the interstitial matrix that becomes fibrotic (For review see29Kaissling B Le Hir M The renal cortical interstitium: morphological and functional aspects.Histochem Cell Biol. 2008; 130: 247-262Crossref PubMed Scopus (146) Google Scholar). Many myofibroblasts express the actin fiber, αSMA that correlates with contractile and activated morphology, and recent studies confirmed that in the fibrotic kidney more than 80% of these produce fibrillary collagen.30Lin SL Kisseleva T Brenner DA Duffield JS Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar Although this is not specific to interstitial myofibroblasts (αSMA is also expressed by vascular smooth muscle cells), αSMA has long been used as a marker of myofibroblasts. Although it is widely accepted that primary epithelial cells cultured in vitro up-regulate genes that result in a myofibroblast phenotype,9Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar, 25Zavadil J Bottinger EP TGF-beta and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1383) Google Scholar and generate fibrillar collagens, the evidence that this occurs in vivo is less well-established. There are some published examples of epithelial cells transgressing intact or disrupted basement membrane or cells co-expressing established epithelial and fibroblast markers in vivo,31Cheng S Lovett DH Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation.Am J Pathol. 2003; 162: 1937-1949Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 32Ng YY Huang TP Yang WC Chen ZP Yang AH Mu W Nikolic-Paterson DJ Atkins RC Lan HY Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.Kidney Int. 1998; 54: 864-876Crossref PubMed Scopus (346) Google Scholar, 33Tyler JR Robertson H Booth TA Burt AD Kirby JA Chronic allograft nephropathy: intraepithelial signals generated by transforming growth factor-beta and bone morphogenetic protein-7.Am J Transplant. 2006; 6: 1367-1376Crossref PubMed Scopus (38) Google Scholar but histological snapshots do not prove a lineage relationship, and cells may express a variety of antigens during injury. In our own extensive studies of injured epithelial cells in kidney repair, we concluded that non-epithelial cells do not migrate from interstitium into the tubule.34Humphreys BD Valerius MT Kobayashi A Mugford JW Soeung S Duffield JS McMahon AP Bonventre JV Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar Similarly, we have never observed a cell outside of the confines of the epithelial basement membrane that was positive for markers of epithelial injury. Explanations for a failure to make these observations in fixed tissues include the hypothesis that a cell exiting the confines of the basement membrane rapidly loses epithelial markers and only subsequently gains myofibroblast markers.35Liu 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 (960) Google Scholar However, in vitro, epithelial cells can express both fibroblast markers and epithelial markers simultaneously.36Fan JM Ng YY Hill PA Nikolic-Paterson DJ Mu W Atkins RC Lan HY Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro.Kidney Int. 1999; 56: 1455-1467Crossref PubMed Scopus (477) Google Scholar Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we have performed lineage analysis of both renal epithelial cells and interstitial stromal cells during fibrosis in vivo. Transgenic or knock-in mice with lineage-restricted expression of bacterial Cre recombinase were used for genetic tracking of three cell populations. The HoxB7-Cre driver is expressed exclusively in the mesonephric duct and its derivatives, resulting in labeling of collecting duct epithelium and ureteral epithelium of adult kidney.37Yu J Carroll TJ McMahon AP Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.Development. 2002; 129: 5301-5312PubMed Google Scholar In the Six2-Cre transgenic mouse, expression of Cre occurs in cap-mesenchyme and labels all non-ureteric, bud-derived, nephron epithelia, including podocytes, proximal tubule, loop of Henle, and connecting segment, but it does not label any interstitial cell population.34Humphreys BD Valerius MT Kobayashi A Mugford JW Soeung S Duffield JS McMahon AP Bonventre JV Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 38Kobayashi A Valerius MT Mugford JW Carroll TJ Self M Oliver G McMahon AP Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell. 2008; 3: 169-181Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar FoxD1 is a well characterized marker of renal stromal cells, but not epithelia, during development, and we used FoxD1-Cre knock-in mice to genetically label renal stroma.39Hatini V Huh SO Herzlinger D Soares VC Lai E Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.Genes Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (422) Google Scholar We crossed these three Cre drivers against two different reporter lines to permanently and heritably label all epithelial cells of the entire nephron in adult mouse kidney or all stromal cells.34Humphreys BD Valerius MT Kobayashi A Mugford JW Soeung S Duffield JS McMahon AP Bonventre JV Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 38Kobayashi A Valerius MT Mugford JW Carroll TJ Self M Oliver G McMahon AP Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell. 2008; 3: 169-181Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar We demonstrate that, contrary to the prevailing model, kidney epithelial cells do not become myofibroblasts in vivo during fibrotic disease. Rather, we show by genetic tracing that myofibroblasts derive from interstitial pericytes/perivascular fibroblasts. The Six2-GC and HoxB7-Cre mice are described.38Kobayashi A Valerius MT Mugford JW Carroll TJ Self M Oliver G McMahon AP Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell. 2008; 3: 169-181Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar FoxD1-GFP-Cre (FoxD1-GC) and FoxD1-GFP-Cre-ER(t2) (FoxD1-GCE) knock-in mice were generated using standard techniques. Full details of lines, their generation and use in identifying kidney lineage will be documented elsewhere (A.K. and A.P.M., unpublished data). Z/Red, NLSCre-Red, R26-LacZ mice and R26R mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The studies were performed on a mixed background consisting of 129/B6. FoxD1-GCE+; R26R or FoxD1-GCE−; R26R pregnant females were given 6 mg of tamoxifen, i.p. in corn oil at embryonic day (e)10.5 or corn oil alone. All surgeries were performed according to protocols overseen by Animal Resources and Comparative Medicine at Harvard University. The complete unilateral ureteric obstruction (UUO) procedure is described elsewhere.30Lin SL Kisseleva T Brenner DA Duffield JS Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar Unilateral ischemia-reperfusion injury was performed as described, except mice underwent 35 minutes of clamping instead of 25 minutes.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar In brief, ureteral obstruction was performed through a left flank incision under general anesthesia. The ureter was identified and tied at the level of the lower pole of the kidney with two separate silk ties. Ischemia reperfusion injury to the left kidney only was performed through a similar incision under general anesthesia, at core body temperature of 36.7–37.2°C. A surgical clamp was placed over the artery and vein for 35 minutes and then removed. Return of blood flow was confirmed. Mice were anesthetized, sacrificed, and immediately perfused via the left ventricle with ice-cold PBS for 2 minutes. Kidneys were hemi-sectioned and portions were snap frozen in liquid nitrogen. Other kidneys were fixed in 10% neutral buffered formalin at 4°C for 12 hours, processed, embedded in paraffin wax, sectioned, and stained with periodic acid-Schiff, picrosirius red or Jones’ Silver stain using standard procedures. Quantitative morphometry was performed as previously reported.41Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1277) Google Scholar Tubule injury was assessed by a semiquantitative scoring system as previously described.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar In brief injury was graded in periodic acid Schiff-stained sections on a scale from 0 to 3 (0, no changes; 1, changes affecting <25%; 2, changes affecting 25% to 50%; 3, changes affecting 50% to 100% of the section). To preserve native red fluorescent protein (RFP) epifluorescence which we found was quenched by prolonged fixation, some kidneys were fixed in PLP fixative (4% paraformaldehyde [PFA], 75 mmol/L l-lysine, 10 mmol/L sodium periodate) for 1 hour or 2 hours at 4°C, cryopreserved in 18% sucrose, and frozen, and 5-μm cryosections were prepared for direct visualization or immunofluorescence as described.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar Other kidneys were perfusion fixed with 4% PFA for 2 minutes, followed by sagittal hemisection and further fixation for 1 hour in 4% PFA, then cryopreserved in 18% sucrose before sectioning. Bromodeoxyuridine labeling was performed as previously described.42Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 167: 1207-1219Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar Primary antibodies against the following proteins were used: DsRed (Rabbit, 1:500, Cat. No. PM005; MBL, Nagoya, Japan), E-cadherin (Rabbit, 1:200, Cat. No. 610404, BD Biosciences, San Jose, CA), αSMA (mouse fluorescein isothiocyanate-conjugated, 1:400, #F3777, mouse Cy3-conjugated, 1:400 #C6198, Sigma, St. Louis MO), platelet-derived growth factor receptor (PDGFR)β (1:500, Gift William Stallcup, Burnham Inst.), S100A4 (1:400 DAKO A5114 and 1:400 Abcam Ab27957 [both antibodies were tested and gave comparable results]). Primary antibodies against β-galactosidase (β-gal) were tested (Sigma #B0271, #G6282; Cappel #55976, Promega #Z378A) to detect the LacZ gene product, β-gal, by immunofluorescence. All four antibodies against β-gal were tested in frozen and formalin-fixed, paraffin-embedded sections but none of these gave a reliable signal by immunofluorescence or immunostain in adult kidneys of the R26-LacZ positive control mouse or Six2GC; R26R mice. It may be that β-gal enzymatic activity is the most reliable readout of LacZ expression in the fibrotic adult kidney. Affinity purified secondary antibodies were obtained from DAKO, Carpinteria CA, or Jackson Immunoresearch. All labeling with directly conjugated anti-αSMA antibodies was performed in 10% mouse serum to block nonspecific binding. In examples involving double-labeling with anti-αSMA antibodies, labeling with anti-DsRed antibodies followed by donkey anti-rabbit-Cy3 secondary was performed initially in 10% donkey serum, then after washing, directly conjugated anti-αSMA antibodies were applied in 10% mouse serum as previously described.30Lin SL Kisseleva T Brenner DA Duffield JS Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar Sections were mounted in Vectashield or Gel Mount or antifade Gold containing 4′, 6-diamino-2-phenylindole (VectorLabs, Burlingame CA). Epifluorescent images were taken with a Nikon TE2000 microscope, CoolSnap camera (Roper Scientific, Germany) and processed using IP lab software (BD Biosciences, San Jose, CA). Antibody enhancement of RFP signal was performed as previously described and gave identical results in vivo to native RFP fluorescence (data not shown).34Humphreys BD Valerius MT Kobayashi A Mugford JW Soeung S Duffield JS McMahon AP Bonventre JV Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar Confocal images were generated using a Nikon C1 D-Eclipse confocal microscope as previously described.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar Projection images were generated from 10 Z-stack images that were acquired at 0.1-μm steps. To allow for comparison between sections, all confocal settings including exposure time were kept constant between sections. LacZ activity was measured by standard 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining protocol on PFA-fixed frozen kidney sections.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar Care was taken to perform X-gal staining at neutral pH, and was performed for 16 hours at 37°C. After washing, sections were either counterstained with nuclear fast red or were postfixed (4% PFA 2 minutes), then were immunolabeled as detailed above before aqueous mounting. Morphometry of LacZ stained area in tissues sections was assessed using Fovea Pro Software as previously described.40Duffield JS Park KM Hsiao LL Kelley VR Scadden DT Ichimura T Bonventre JV Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar Kidney proximal tubule cells were cultured exactly as described.43Sheridan AM Schwartz JH Kroshian VM Tercyak AM Laraia J Masino S Lieberthal W Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia.Am J Physiol. 1993; 265: F342-F350PubMed Google Scholar After 7 days of culture the epithelial cells were confluent, then treated with transforming growth factor (TGF)β1 (10 ng/ml) or no additional treatment. Four days later, cells were fixed with 4% PFA 5 minutes, washed ×3 with PBS, permeabilized with 0.1% TX100, 5 minutes, washed ×3 with PBS, then labeled with antibodies as described in above. To study the process of EMT in the kidney we used a well-established mouse model o
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