Title: Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis
Abstract: Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis. Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis. Pericytes are mesenchyme-derived perivascular cells attached to the abluminal surface of capillaries.1Allt G. Lawrenson J.G. Pericytes: cell biology and pathology.Cells Tissues Organs. 2001; 169: 1-11Crossref PubMed Scopus (375) Google Scholar They share developmental origins with fibroblasts, and there may be plasticity between pericytes attached to capillaries and fibroblasts embedded in adjacent collagenous matrix; however, unlike fibroblasts, pericytes have vital functions in regulating microvascular stability, angiogenesis, capillary permeability, capillary flow, and capillary basement membrane synthesis.1Allt G. Lawrenson J.G. Pericytes: cell biology and pathology.Cells Tissues Organs. 2001; 169: 1-11Crossref PubMed Scopus (375) Google Scholar We have previously shown that pericytes are the major sources of scar-producing myofibroblasts during kidney injury, and we have identified adult kidney pericytes and perivascular fibroblasts are derived from Foxd1-expressing progenitors, positive for collagen I(α1)-GFP (Coll-GFP+), platelet-derived growth factor receptor β (PDGFR-β+), and CD73 (CD73+) and negative for α smooth muscle actin (α-SMA−) and CD45 (CD45−).2Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. 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 (647) Google Scholar, 3Humphreys B.D. Lin S.L. Kobayashi A. Hudson T.E. Nowlin B.T. Bonventre J.V. Valerius M.T. McMahon A.P. Duffield J.S. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 4Chang F.C. Chou Y.H. Chen Y.T. Lin S.L. Novel insights into pericyte-myofibroblast transition and therapeutic targets in renal fibrosis.J Formos Med Assoc. 2012; (DOI:)http://dx.doi.org/10.1016/j.jfma.2012.09.008Google Scholar Recently, spinal cord pericytes were identified as major progenitors of scar tissue in the central nervous system, intestinal pericytes as a source of myofibroblasts in models of colitis, and hepatic stellate cells, the major precursor of myofibroblasts in liver disease, have been determined to be specialized pericytes of the hepatic sinusoid,5Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. Iwaisako K. Moore-Morris T. Scott B. Tsukamoto H. Evans S.M. Dillmann W. Glass C.K. Brenner D.A. 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Many independent studies support the notion of perivascular resident mesenchymal cells, not injured tubular epithelial cells, as the major source of myofibroblasts in kidneys.9Picard N. Baum O. Vogetseder A. Kaissling B. Le Hir M. Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat.Histochem Cell Biol. 2008; 130: 141-155Crossref PubMed Scopus (127) Google Scholar, 10Asada N. Takase M. Nakamura J. Oguchi A. Asada M. Suzuki N. Yamamura K. Nagoshi N. Shibata S. Rao T.N. Fehling H.J. Fukatsu A. Minegishi N. Kita T. Kimura T. Okano H. Yamamoto M. Yanagita M. Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Crossref PubMed Scopus (260) Google Scholar, 11Faulkner J.L. Szcykalski L.M. Springer F. Barnes J.L. 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Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.J Am Soc Nephrol. 2012; 23: 868-883Crossref PubMed Scopus (147) Google Scholar Our investigations so far have focused on pericyte-endothelial crosstalk, because pericytes form direct communications with endothelial cells of peritubular capillaries at peg and socket junctions, where direct cell-cell signaling has been thought to occur.13Chen Y.T. Chang F.C. Wu C.F. Chou Y.H. Hsu H.L. Chiang W.C. Shen J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. Lin S.L. Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis.Kidney Int. 2011; 80: 1170-1181Crossref PubMed Scopus (238) Google Scholar, 14Lin S.L. Chang F.C. Schrimpf C. Chen Y.T. Wu C.F. Wu V.C. Chiang W.C. Kuhnert F. Kuo C.J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. 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Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival.Dev Biol. 2003; 264: 275-288Crossref PubMed Scopus (314) Google Scholar We have recently shown that Coll-GFP+ kidney pericytes function identically to brain pericytes in migrating to and stabilizing capillary networks, functions that require expression of tissue inhibitor of metalloproteinase 3 (TIMP-3).15Schrimpf C. Xin C. Campanholle G. Gill S.E. Stallcup W. Lin S.L. Davis G.E. Gharib S.A. Humphreys B.D. Duffield J.S. Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.J Am Soc Nephrol. 2012; 23: 868-883Crossref PubMed Scopus (147) Google Scholar These pericyte functions are lost when Coll-GFP+ pericytes transition to myofibroblasts.15Schrimpf C. Xin C. Campanholle G. Gill S.E. Stallcup W. Lin S.L. Davis G.E. Gharib S.A. Humphreys B.D. Duffield J.S. Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.J Am Soc Nephrol. 2012; 23: 868-883Crossref PubMed Scopus (147) Google Scholar Furthermore, we reported that endothelial activation at vascular endothelial cell growth factor (VEGF) receptor 2 and PDGFR-β signaling by pericytes are two critical signaling pathways that link endothelial activation with pericyte transition to myofibroblasts.14Lin S.L. Chang F.C. Schrimpf C. Chen Y.T. Wu C.F. Wu V.C. Chiang W.C. Kuhnert F. Kuo C.J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis.Am J Pathol. 2011; 178: 911-923Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Our studies showed that these signaling events alone are sufficient to drive microvascular rarefaction, inflammation, and fibrosis in models of kidney disease.14Lin S.L. Chang F.C. Schrimpf C. Chen Y.T. Wu C.F. 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Sijmonsma T.P. Jugold M. Akeson A.L. Traykova-Brauch M. Hosser H. Hähnel B. Gröne H.J. Koesters R. Kriz W. Effects of increased renal tubular vascular endothelial growth factor (VEGF) on fibrosis, cyst formation, and glomerular disease.Am J Pathol. 2009; 175: 1883-1895Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 22Yang L. Besschetnova T.Y. Brooks C.R. Shah J.V. Bonventre J.V. Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16: 535-543Crossref PubMed Scopus (878) Google Scholar Therefore, epithelial signaling events must somehow be transmitted across the tubular basement membrane to pericytes to drive interstitial fibrosis. These obscure molecular signaling events are the focus of the studies we report here.In previous investigations of embryonic microvascular development, endothelial cells have been shown to be a source of both PDGF and transforming growth factor β-1 (TGF-β1), cytokines that regulate pericyte attachment, differentiation, and angiogenesis.17Dickson M.C. Martin J.S. Cousins F.M. Kulkarni A.B. Karlsson S. Akhurst R.J. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice.Development. 1995; 121: 1845-1854Crossref PubMed Google Scholar, 23Zhang J. Cao R. Zhang Y. Jia T. Cao Y. Wahlberg E. Differential roles of PDGFR-alpha and PDGFR-beta in angiogenesis and vessel stability.FASEB J. 2009; 23: 153-163Crossref PubMed Scopus (129) Google Scholar, 24Lindahl P. Johansson B.R. Levéen P. Betsholtz C. 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Schönig K. Greiner O. Miloud T. Jauch A. Bode M. Felsher D.W. Glick A.B. Kwiatkowski D.J. Bujard H. Horst J. von Knebel Doeberitz M. Niggli F.K. Kriz W. Grone H.J. Koesters R. An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.Nat Med. 2008; 14: 979-984Crossref PubMed Scopus (192) Google Scholar Therefore, epithelial transgenic overexpression of TGF-β1, which stimulates epithelial cell dedifferentiation and autophagy, must stimulate pericyte to myofibroblast transition by epithelial cell to pericyte crosstalk.34Koesters R. Kaissling B. Lehir M. Picard N. Theilig F. Gebhardt R. Glick A.B. Hähnel B. Hosser H. Gröne H.J. Kriz W. Tubular overexpression of transforming growth factor-beta1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells.Am J Pathol. 2010; 177: 632-643Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Our aim in the present study was to identify the mechanism by which TGF-β1 signaling from injured tubular epithelial cells can activate pericytes to drive progressive kidney fibrosis.Materials and MethodsColl-GFP MiceColl-GFP transgenic mice were generated on the C57BL6 background as described previously.2Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. 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 (647) Google Scholar In brief, 3.2 kb of the collagen I(α1) (Col1a1) promoter and enhancer with the open reading frame of enhanced GFP yielded the highest levels of GFP expression when COL1A1 gene transcripts were generated.Mouse Models of Kidney FibrosisUnilateral ureteral obstruction (UUO) was performed in adult (8 to 12 weeks) C57BL6 wild-type or Coll-GFP mice as described previously.2Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. 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 (647) Google Scholar Briefly, the left ureter was ligated twice using 4-0 nylon surgical sutures at the level of the lower pole of kidney. All animal studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine.Culture of Kidney PericytesPurification of kidney pericytes from normal kidney was performed as described previously.13Chen Y.T. Chang F.C. Wu C.F. Chou Y.H. Hsu H.L. Chiang W.C. Shen J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. Lin S.L. Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis.Kidney Int. 2011; 80: 1170-1181Crossref PubMed Scopus (238) Google Scholar Kidney was diced, incubated at 37°C for 1 hour with Liberase (0.5 mg/mL; Roche Applied Science, Indianapolis, IN) and DNase (100 U/mL; Roche Applied Science) in Hank’s balanced salt solution. After centrifugation, cells were resuspended in 5 mL of PBS/1% bovine serum albumin, and filtered (40-μm mesh). Pericytes were purified by isolating GFP+PDGFR-α+ cells using a fluorescence-activated cell sorting (FACS) system (FACSAria; BD Biosciences, San Jose, CA), and then total RNA was isolated or purified cells were cultured in Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum. The primary cultured cells used in the present study were between passages 4 and 8 and have been characterized previously.13Chen Y.T. Chang F.C. Wu C.F. Chou Y.H. Hsu H.L. Chiang W.C. Shen J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. Lin S.L. Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis.Kidney Int. 2011; 80: 1170-1181Crossref PubMed Scopus (238) Google ScholarPurification and Culture of PTECsPurification of proximal tubular epithelial cells (PTECs) from normal and day-7 UUO kidneys was performed as described previously.36Lin S.L. Li B. Rao S. Yeo E.J. Hudson T.E. Nowlin B.T. Pei H. Chen L. Zheng J.J. Carroll T.J. Pollard J.W. McMahon A.P. Lang R.A. Duffield J.S. Macrophage Wnt7b is critical for kidney repair and regeneration.Proc Natl Acad Sci USA. 2010; 107: 4194-4199Crossref PubMed Scopus (326) Google Scholar Kidney was diced, incubated at 37°C for 1 hour with collagenase (0.5 mg/mL; Worthington Biochemical, Lakewood, NJ) and soybean trypsin inhibitor (0.5 mg/mL; Gibco; Life Technologies, Carlsbad, CA) in Dulbecco’s modified Eagle’s medium/F12 basal medium. After centrifugation, cells were resuspended in 5 mL of PBS/1% bovine serum albumin, and filtered (40-μm mesh). Cells were labeled with Lotus tetragonolobus lectin (LTL)-fluorescein isothiocyanate (Vector Laboratories, Burlingame, CA), anti–CD45-PE, and anti–Kim1-biotin antibodies (RMT1-4) (1:200; eBioscience, San Diego, CA), followed by streptavidin-allophycocyanin (Jackson ImmunoResearch Laboratories, West Grove, PA). Normal and injured PTECs were sorted by FACSAria cell sorting (BD Biosciences) for LTL+CD45− cells and Kim1+CD45− cells, and then total RNA was purified using an RNeasy system (Qiagen, Valencia, CA). Day-7 UUO kidney proximal tubular cells were cultured in Dulbecco’s modified Eagle’s medium/F12 with 1× insulin-transferrin-selenium and hydrocortisone (0.5 μmol/L; Sigma-Aldrich, St. Louis, MO) using established methods that maintained tubular epithelial characteristics.37Sheridan A.M. Schwartz J.H. Kroshian V.M. Tercyak A.M. 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 The primary cultured tubular epithelial cells used in the present study were between passages 4 and 8. In some experiments, after 48-hour treatment of PTECs with TGF-β1 (5 ng/mL), the drug was washed out and the cells continued in culture for 24 hours. The conditioned medium was then collected and added to serum-starved kidney pericytes. Control antibody 13C4, anti–TGF-β antibody (1D11; Genzyme, Framingham, MA), and anti–PDGFR-β antibody (100 μg/mL) were added in the pericyte culture with conditioned medium. Cell cycle, cell number, and gene expression of kidney pericytes were analyzed after 24 hours.Blocking TGF-β1 Signaling in VivoMice were injected intraperitoneally with 13C4, 1D11 (5 mg/kg/every other day), or the transforming growth factor β receptor I (TGF-βRI) inhibitor SB431542 (5 mg/kg per day; Tocris Bioscience, Bristol, UK) 2 hours before surgery, and then as scheduled until sacrifice on day 4 or day 10 (n = 6 per group).Blocking TGF-β1 Signaling in VitroNormal kidney pericytes were incubated with TGF-β1 (10 ng/mL; R&D Systems, Minneapolis, MN) in the presence of antibody 13C4 (100 μg/mL), 1D11 (100 μg/mL), or SB431542 (5 μg/mL). The extent of Smad2 phosphorylation was determined by Western blot analysis. In some experiments, SP600125 (10 μmol/L; Sigma-Aldrich) and SB203580 (10 μmol/L; Sigma-Aldrich) were used to block c-jun NH2-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38, respectively.Tissue Preparation and HistologyMouse tissues were prepared and stained as described previously.2Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. 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 (647) Google Scholar Primary antibodies against the following proteins were used for immunolabeling: α-SMA-Cy3 (clone 1A4; Sigma-Aldrich), laminin α4 (R&D Systems), Ki-67, PDGFB, TGF-βRII (Abcam, Cambridge, UK), p-Smad2, p-histone H3 (Ser10) (Cell Signaling Technology, Danvers, MA), proliferating cell nuclear antigen (PCNA) (Thermo Scientific, Fremont, CA), TGF-β1 and Nidogen (Santa Cruz Biotechnology, Santa Cruz, CA), and NG2 (gift from W. Stallcup). Fluorescent conjugated secondary antibody labeling (Jackson ImmunoResearch Laboratories), colabeled with DAPI, mounting with Vectashield medium (Vector Laboratories), and image capture and processing were performed as described previously. Quantification of specific cells in tissue sections was performed as described previously.14Lin S.L. Chang F.C. Schrimpf C. Chen Y.T. Wu C.F. Wu V.C. Chiang W.C. Kuhnert F. Kuo C.J. Chen Y.M. Wu K.D. Tsai T.J. Duffield J.S. Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis.Am J Pathol. 2011;