Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-β1 signaling
Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein–positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-β1(TGF-β1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-β1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-β1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner. Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein–positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-β1(TGF-β1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-β1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-β1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner. Peritoneal dialysis (PD) has been used as a beneficial treatment for end-stage kidney disease. However, peritoneal fibrosis remains a serious complication of long-term PD that leads to the failure of peritoneal function.1.Dobbie J. Morphology of the peritoneum in CAPD.Blood Purif. 1989; 7: 74-85Crossref PubMed Scopus (115) Google Scholar,2.Nakamoto H. Kawaguchi Y. Suzuki H. Encapsulating peritoneal sclerosis in patients undergoing continuous ambulatory peritoneal dialysis in Japan.Adv Perit Dial. 2002; 18: 119-123PubMed Google Scholar Peritoneal fibrosis arises in response to a variety of injurious factors, including bioincompatible dialysate components, uremic toxins, refractory or recurrent infectious peritonitis, and chronic inflammation.3.Margetts P. Bonniaud P. Basic mechanisms and clinical implications of peritoneal fibrosis.Perit Dial Int. 2003; 23: 530-541PubMed Google Scholar, 4.Mortier S. De Vriese A. Van de Voorde J. et al.Hemodynamic effects of peritoneal dialysis solutions on the rat peritoneal membrane: role of acidity, buffer choice, glucose concentration, and glucose degradation products.J Am Soc Nephrol. 2002; 13: 480-489PubMed Google Scholar, 5.Williams J. Craig K. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar The characteristic histological features of the fibrotic sclerosing peritoneum are loss of peritoneal mesothelial cells, abnormal proliferation of α-smooth muscle actin (α-SMA)-positive myofibroblasts, marked accumulation of collagen, and a progressive increase in the thickness of the submesothelial compact zone.5.Williams J. Craig K. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar,6.Mateijsen M. van der Wal A. Hendriks P. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525PubMed Google Scholar As the essential mechanism of peritoneal fibrosis, the epithelial-to-mesenchymal transition (EMT) has been identified in cultured human peritoneal mesothelial cells (HPMCs)7.Yang A. Chen J. Lin J. Myofibroblastic conversion of mesothelial cells.Kidney Int. 2003; 63: 1530-1539Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar and in PD patients.8.Yáñez-Mó M. Lara-Pezzi E. Selgas R. et al.Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells.N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (620) Google Scholar Many studies have reported that transforming growth factor-β1 (TGF-β1) has a pivotal role in the induction of EMT,7.Yang A. Chen J. Lin J. Myofibroblastic conversion of mesothelial cells.Kidney Int. 2003; 63: 1530-1539Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar,9.Margetts P. Bonniaud P. Liu L. et al.Transient overexpression of TGF-{beta}1 induces epithelial mesenchymal transition in the rodent peritoneum.J Am Soc Nephrol. 2005; 16: 425-436Crossref PubMed Scopus (240) Google Scholar typically characterized by reduced cell–cell adhesion, loss of epithelial polarity, and de novo expression of α-SMA and other fibrogenic mediators.10.Aroeira L. Aguilera A. Sánchez-Tomero J. et al.Epithelial to mesenchymal transition and peritoneal membrane failure in peritoneal dialysis patients: pathologic significance and potential therapeutic interventions.J Am Soc Nephrol. 2007; 18: 2004-2013Crossref PubMed Scopus (273) Google Scholar,11.Zavadil J. Böttinger E.P. TGF-beta and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1386) Google Scholar On binding to its receptors (types I and II serine/threonine kinases), TGF-β1 signaling leads to phosphorylation of receptor-regulated Smads (R-Smads) such as Smad2 and Smad3. Subsequently, R-Smads form a heteromultimer with Smad4, translocate to the nucleus, and regulate transcription of target genes.12.Shi Y. Massagué J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4817) Google Scholar The Smad proteins are thought to have an important role in the process of fibrosis. Various studies performed in vitro and in animal models have suggested that drugs, peptides, and gene therapy targeting fibrosis and/or angiogenesis could be useful for treating peritoneal fibrosis,13.Aroeira L. Lara-Pezzi E. Loureiro J. et al.Cyclooxygenase-2 mediates dialysate-induced alterations of the peritoneal membrane.J Am Soc Nephrol. 2009; 20: 582-592Crossref PubMed Scopus (51) Google Scholar, 14.Tanabe K. Maeshima Y. Ichinose K. et al.Endostatin peptide, an inhibitor of angiogenesis, prevents the progression of peritoneal sclerosis in a mouse experimental model.Kidney Int. 2007; 71: 227-238Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15.Nishino T. Miyazaki M. Abe K. et al.Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.Kidney Int. 2003; 64: 887-896Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 16.Guo H. Leung J. Lam M. et al.Smad7 transgene attenuates peritoneal fibrosis in uremic rats treated with peritoneal dialysis.J Am Soc Nephrol. 2007; 18: 2689-2703Crossref PubMed Scopus (45) Google Scholar whereas clinically available treatment for peritoneal fibrosis in PD patients remains limited at present. Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can be obtained from the bone marrow and many other tissues.17.Prockop D. Marrow stromal cells as stem cells for nonhematopoietic tissues.Science. 1997; 276: 71-74Crossref PubMed Scopus (4138) Google Scholar, 18.Lee R. Kim B. Choi I. et al.Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.Cell Physiol Biochem. 2004; 14: 311-324Crossref PubMed Scopus (820) Google Scholar, 19.Wang H. Hung S. Peng S. et al.Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord.Stem Cells. 2004; 22: 1330-1337Crossref PubMed Scopus (1130) Google Scholar MSCs have been used for the regeneration of various tissues because these cells have the capability to differentiate into a variety of lineages, including bone, cartilage, cardiac myocytes, and neurons.20.Horwitz E. Prockop D. Fitzpatrick L. et al.Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.Nat Med. 1999; 5: 309-313Crossref PubMed Scopus (1614) Google Scholar, 21.Yoo J. Barthel T. Nishimura K. et al.The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells.J Bone Joint Surg Am. 1998; 80: 1745-1757Crossref PubMed Scopus (781) Google Scholar, 22.Toma C. Pittenger M. Cahill K. et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation. 2002; 105: 93-98Crossref PubMed Scopus (2004) Google Scholar, 23.Kopen G. Prockop D. Phinney D. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.Proc Natl Acad Sci USA. 1999; 96: 10711-10716Crossref PubMed Scopus (1552) Google Scholar In addition to their regenerative capability, MSCs display immunomodulatory and antifibrotic activity that could be important in the response to injury.24.Caplan A. Dennis J. Mesenchymal stem cells as trophic mediators.J Cell Biochem. 2006; 98: 1076-1084Crossref PubMed Scopus (2319) Google Scholar The antifibrotic effects of cultured MSCs have been demonstrated in different animal models.25.Suga H. Eto H. Shigeura T. et al.IFATS collection: fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism.Stem Cells. 2009; 27: 238-249Crossref PubMed Scopus (116) Google Scholar,26.Li L. Zhang S. Zhang Y. et al.Paracrine action mediate the antifibrotic effect of transplanted mesenchymal stem cells in a rat model of global heart failure.Mol Biol Rep. 2009; 36: 725-731Crossref PubMed Scopus (130) Google Scholar Although ex vivo-expanded MSCs have been used in many basic studies and clinical trials, few authors have assessed the potential of MSCs to inhibit peritoneal fibrosis. Mesothelial cell transplantation has been studied in animal models and PD patients,27.Hekking L. Zweers M. Keuning E. et al.Apparent successful mesothelial cell transplantation hampered by peritoneal activation.Kidney Int. 2005; 68: 2362-2367Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,28.Di Paolo N. Sacchi G. Vanni L. et al.Autologous peritoneal mesothelial cell implant in rabbits and peritoneal dialysis patients.Nephron. 1991; 57: 323-331Crossref PubMed Scopus (38) Google Scholar and this approach may be one of the options for alleviating peritoneal membrane dysfunction. Because infusion of MSCs is to be and has few to be for clinical In this we whether infusion of MSCs could and ameliorate chlorhexidine gluconate peritoneal fibrosis in a rat the paracrine effect of we with human MSCs using a Transwell system and the expression of TGF-β1 and we the effects of MSC-conditioned medium on TGF-β1-induced Smad2 and the response of findings demonstrated that MSCs were in the inhibited and peritoneal fibrosis, the functional of the peritoneal membrane. Moreover, MSCs inhibited glucose-induced TGF-β1 and the TGF-β1-induced by of TGF-β1 signaling in by paracrine including secretion of hepatocyte growth factor staining was used to changes in cell and staining was used to peritoneal that injection of of of the submesothelial compact and that the thickness and of this On the thickness of the submesothelial compact not the rats treated with the and the rats that been treated with MSCs after In the cell of the compact was suppressed in rats treated with MSCs with rats the On peritoneal cell and thickness were suppressed in rats treated with MSCs with rats the and the peritoneal expression of α-SMA and as mesenchymal injection of α-SMA expression was in that in the of the submesothelial compact on days 7 and as as in muscle cells with MSCs suppressed the of the on days 7 and 14 with rats the that cells in the of the submesothelial compact on days 7 and with MSCs suppressed the of cells on days 7 and 14 with that in rats the I and are extracellular matrix proteins, and we showed that I and were in the submesothelial compact on 7 and showed an increase by 14 and with MSCs reduced the in I and on days 7 and 14 with the effect of the and stem cells (MSCs) suppressed I and in rats with peritoneal fibrosis. of I and expression in peritoneal tissues on days 7 and 14 in chlorhexidine gluconate rats treated with the and rats treated with The of and were on days 7 and 14 in rats treated with the whereas the of and were on days 7 and 14 in rats treated with infiltration into the we an accumulation of cells in the submesothelial on 7 and a increase on 14 with MSCs inhibited the infiltration of cells on days 7 and 14 with that in rats the The of cells showed for TGF-β1 of and The of cells for and TGF-β1 in the submesothelial 14 of MSCs inhibited the infiltration of cells for and TGF-β1 on days 7 and 14 with treatment with the Moreover, of Smad2 was in cells in the submesothelial on 7 and showed a increase on 14 with MSCs inhibited the of cells on days 7 and 14 with in rats the stem cells (MSCs) suppressed Smad2 expression in rats with peritoneal fibrosis. analysis of expression in peritoneal tissues on days 7 and 14 in chlorhexidine gluconate rats treated with and rats treated with The of cells 14 in rats treated with whereas was on days 7 and 14 in rats treated with peritoneal was performed on 14 to the functional of the peritoneal membrane. The of glucose from the dialysate and the of from the were in rats treated with the in whereas was in rats treated with MSCs and the of we green fluorescent protein MSCs after As a we MSCs injection injection As a MSCs were in the and of rats 3 these cells were not detected on 7 or The of MSCs in the peritoneum was on In MSCs were at in the peritoneum of rats injection of could not MSCs in other tissues such as the or in rats or rats at of that MSCs were in the tissues for only a few days. whether the effect of human MSCs on glucose-induced TGF-β1 and fibronectin gene expression in cell–cell were treated with for and were with the or with human MSCs using a Transwell system for treatment the expression of TGF-β1 and fibronectin with that in cells, whereas of with human MSCs in a of TGF-β1 and fibronectin mRNA expression with the in cells and that MSCs to suppress the response of in a paracrine manner. TGF-β1 is the growth factor that can in various of mechanisms and implications for fibrotic J Physiol Cell 2007; PubMed Scopus Google Scholar whether MSCs suppressed fibrotic through paracrine we from of MSCs and its effects on TGF-β1-induced of were with for and with TGF-β1 at changes of cultured after the are in were changes in the characteristic of and with TGF-β1 was following TGF-β1 whereas the was in with that in with analysis that inhibited TGF-β1-induced phosphorylation of Smad2 in TGF-β1 treatment for α-SMA protein expression and zonula occludens-1 protein these TGF-β1-induced and is that and bone morphogenic protein 7 have effects on TGF-β1 signaling and suppress R. et growth factor inhibits epithelial to transition in cells J Cell 2009; PubMed Scopus (115) Google Scholar, J. C. Liu Y. mechanism by hepatocyte growth factor epithelial to mesenchymal Am Soc Nephrol. 2005; 16: PubMed Scopus Google Scholar, M. J. H. et epithelial-to-mesenchymal transition and chronic renal Med. 2003; PubMed Scopus Google Scholar cultured and human MSCs in medium for and the of and in medium were by in the in a whereas was the of in On the other the of in and were the of not the effect of on and were with or antibody for its addition to analysis that inhibited phosphorylation of Smad2 in after of TGF-β1 and antibody the effect of the that secreted by MSCs inhibits TGF-β1-induced has the that MSCs have a therapeutic potential for the treatment of peritoneal fibrosis by TGF-β1 signaling in a paracrine manner. of MSCs peritoneal fibrosis in as by a of peritoneal and as as a of and In MSCs reduced the functional of the peritoneal membrane. that the beneficial effects of MSCs on peritoneal in as as in The MSCs were detected on the peritoneal 3 in rats following peritoneal fibrosis, were not in rats peritoneal fibrosis. findings that MSCs to the of peritoneal and immunomodulatory and antifibrotic effects in the of long-term of the cells. MSCs are to migrate to the of inflammation following into M. Abe R. Y. et al.Mesenchymal stem cells are into and to by into cell PubMed Scopus Google Scholar Although the mechanism of and by these cells is not expression of growth factors, and extracellular matrix receptors on the of MSCs may their adhesion, and infiltration into of A. E. N. et al.The in vitro of human bone marrow mesenchymal stem of and growth factor Cells. 2007; PubMed Scopus Google H. C. Mesenchymal stromal and clinical Cells. Google Scholar In this of MSCs into the peritoneal tissues was demonstrated in rats with peritoneal fibrosis, whereas MSCs were not detected in rats fibrosis. findings that the MSCs to the of peritoneal et Y. C. Y. et of bone marrow-derived cells into mesothelial cells in peritoneal using a peritoneal fibrosis mouse PubMed Scopus Google Scholar reported that bone cells and were in the for 3 after of in a mouse model of peritoneal fibrosis. that bone cells have an important role in the process of peritoneal and mesothelial In this of bone MSCs ex was only However, the and antifibrotic effects in the peritoneum were effects may be of of MSCs into functional peritoneal mesothelial cells and are likely to be due to paracrine of in and in vitro studies have suggested that paracrine mechanisms the of growth and by MSCs are with these Y. H. G. et effects of human mesenchymal stem cells on through 2009; 57: PubMed Scopus Google B. M. S. et growth stem cell renal Am Soc Nephrol. 2007; 18: PubMed Scopus Google Scholar an we performed to in the of was the of not In we could not effects of MSCs on signaling such as phosphorylation of and of transcription 3 not Although the mechanism of the effect of MSCs in our rat model of peritoneal fibrosis remains has the that the paracrine action may macrophages and not peritoneal mesothelial cells. In et K. A. et marrow stromal cells of macrophages to increase their Med. 2009; PubMed Scopus Google Scholar reported that MSCs through of was not by MSCs by As of the response to peritoneal or in PD various and growth from macrophages or mesothelial cells were suggested to to the progression of peritoneal fibrosis. TGF-β1 has been identified as the growth factor in peritoneal mesothelial A. Chen J. Lin J. Myofibroblastic conversion of mesothelial cells.Kidney Int. 2003; 63: 1530-1539Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, M. Lara-Pezzi E. Selgas R. et al.Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells.N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (620) Google Scholar, P. Bonniaud P. Liu L. et al.Transient overexpression of TGF-{beta}1 induces epithelial mesenchymal transition in the rodent peritoneum.J Am Soc Nephrol. 2005; 16: 425-436Crossref PubMed Scopus (240) Google Scholar, J. Aguilera A. Selgas R. et TGF-β1 the peritoneal membrane from dialysate-induced Am Soc Nephrol. 22: PubMed Scopus Google Scholar et Y. D. et al.Human umbilical mesenchymal stem cells fibrosis of J 2009; Full Text Full Text PDF PubMed Scopus Google Scholar reported that human umbilical MSCs suppressed fibrosis of and inhibited TGF-β1 expression that to in Smad2 In this we demonstrated that injection of MSCs suppressed TGF-β1 expression by macrophages in rat Moreover, with MSCs suppressed glucose-induced gene expression of TGF-β1 by in we showed that inhibited TGF-β1-induced phosphorylation of Smad2 and TGF-β1-induced changes in the protein of α-SMA and zonula that MSCs to suppress TGF-β1 signaling and TGF-β1-induced in a paracrine manner. Many have that MSCs essential growth M. Zhang A. et al.Paracrine mechanisms in adult stem cell signaling and PubMed Scopus Google Scholar and are of antifibrotic that display therapeutic potential in fibrosis and TGF-β1 R. et growth factor inhibits epithelial to transition in cells J Cell 2009; PubMed Scopus (115) Google Scholar, J. C. Liu Y. mechanism by hepatocyte growth factor epithelial to mesenchymal Am Soc Nephrol. 2005; 16: PubMed Scopus Google Scholar, M. J. H. et epithelial-to-mesenchymal transition and chronic renal Med. 2003; PubMed Scopus Google Scholar of studies that and have a role in peritoneal S. T. and hepatocyte growth factor dialysate-induced peritoneal Am Soc Nephrol. 2005; 16: PubMed Scopus Google Kim et and ameliorate glucose-induced epithelial-to-mesenchymal transition of peritoneal Am Soc Nephrol. 2009; 20: PubMed Scopus Google Scholar showed that the of in in a whereas was not In we demonstrated that secreted by MSCs is in the of the TGF-β1 signaling. In this of the MSCs were because of of However, immunomodulatory and antifibrotic effects of MSCs on peritoneal fibrosis were the of cells, and effects were In to the studies are to the effect of MSCs in other models of peritoneal fibrosis and and of In our that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner. Bone marrow was from rats by the and cultured as S. A. Miyazaki K. et of potential of mesenchymal cells proliferation in response to PubMed Scopus Google Scholar cells were used for was that these cells were MSCs by their into and with S. A. Miyazaki K. et of potential of mesenchymal cells proliferation in response to PubMed Scopus Google Scholar used rats at of injection of in in of was bone marrow MSCs in of were or was in at 7 and 14 days after injection, the rats were and the peritoneum was for peritoneal was performed on were with PD at the peritoneal was and were obtained by cardiac The peritoneal of glucose and were as the peritoneal of glucose from the dialysate and the of we used rats at of injection of in in of was or in of was as a in at MSCs obtained from the bone marrow of rats and in of were and 14 days after injection, rats were and the and were to the of of the animal were by the animal and of and were performed in with the of on the of and immunohistochemical staining of were performed as S. Y. A. et the progression of experimental peritoneal fibrosis in a rat Nephrol. 2009; PubMed Scopus Google Scholar The following were mouse antibody antibody I antibody antibody mouse antibody antibody antibody and antibody The of and were assessed in of the submesothelial compact by a and the was using a in The of cells for and of the submesothelial compact were in of human were obtained from patients undergoing or peritoneal The were isolated from human as E. L. M. et and of human peritoneal mesothelial cells.Kidney Int. Full Text PDF PubMed Scopus Google Scholar and were as in our T. N. M. et of glucose on of cultured human peritoneal mesothelial cells.J Am Soc Nephrol. Google Scholar and cells from the to were of the was by the of of and was obtained from performed using from different at bone marrow MSCs were from were into and to The medium was with medium or for the effect of of with human MSCs cell human MSCs were on the of with cultured MSCs or medium were into the of cultured for of TGF-β1 and fibronectin by was assessed by was by human to medium for days. were into and The medium was with medium or for and were in the or of human TGF-β1 of was performed with antibody at for its addition to were and to and were performed as Y. T. K. et inhibits fibronectin expression hepatocyte growth factor induction in human peritoneal mesothelial Dial. 14: PubMed Scopus Google Scholar and for TGF-β1 fibronectin and were obtained as The mRNA were for the of and were performed as S. Y. et inhibits transforming growth signaling and renal fibrosis and in Biol PubMed Scopus Google Scholar used in this were Smad2 mouse antibody occludens-1 and mouse antibody used in this were antibody or antibody were detected using the system The of was using of The for and were performed following the The were to the protein are as the analysis was performed using analysis of by and was to be was by for from the of and of by a from and by for kidney failure and from the of