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• W2127890209 abstract "Peritoneal fibrosis, a major complication of peritoneal dialysis, limits the effectiveness of peritoneal dialysis as a treatment of end-stage renal disease. Preventing this complication by identifying targets for therapy has recently received much attention. In the present study, we showed that Notch signaling was highly activated in rats in peritoneal dialysis fluid-induced fibrotic peritoneum, as indicated by increased expression of Jagged-1, Notch-1, and HES-1. Blocking Notch signaling activation by intraperitoneal injection of a γ-secretase inhibitor, DAPT, significantly attenuated peritoneal fibrosis as indicated by the decreased expression of α-smooth muscle actin, collagen I, and vascular endothelial growth factor as well as increased expression of E-cadherin. Moreover, compared with control rats, DAPT-treated rats had a thinner peritoneum with less extracellular matrix accumulation, a lower mass transfer of glucose, and a higher ultrafiltration rate. In addition, transforming growth factor (TGF)-β1 induced Notch signaling activation in primary rat peritoneal mesothelial cells. DAPT blocked this TGF-β1–induced Notch signaling activation and therefore significantly inhibited TGF-β1–induced expression of α-smooth muscle actin, collagen I, and vascular endothelial growth factor. Thus, a γ-secretase inhibitor that interferes with Notch signaling prevents biochemical, histological, and functional consequences of peritoneal fibrosis through inhibiting epithelial to mesenchymal transition of rat peritoneal mesothelial cells. These results support the use of γ-secretase inhibitors as a novel therapeutic approach for peritoneal fibrosis. Peritoneal fibrosis, a major complication of peritoneal dialysis, limits the effectiveness of peritoneal dialysis as a treatment of end-stage renal disease. Preventing this complication by identifying targets for therapy has recently received much attention. In the present study, we showed that Notch signaling was highly activated in rats in peritoneal dialysis fluid-induced fibrotic peritoneum, as indicated by increased expression of Jagged-1, Notch-1, and HES-1. Blocking Notch signaling activation by intraperitoneal injection of a γ-secretase inhibitor, DAPT, significantly attenuated peritoneal fibrosis as indicated by the decreased expression of α-smooth muscle actin, collagen I, and vascular endothelial growth factor as well as increased expression of E-cadherin. Moreover, compared with control rats, DAPT-treated rats had a thinner peritoneum with less extracellular matrix accumulation, a lower mass transfer of glucose, and a higher ultrafiltration rate. In addition, transforming growth factor (TGF)-β1 induced Notch signaling activation in primary rat peritoneal mesothelial cells. DAPT blocked this TGF-β1–induced Notch signaling activation and therefore significantly inhibited TGF-β1–induced expression of α-smooth muscle actin, collagen I, and vascular endothelial growth factor. Thus, a γ-secretase inhibitor that interferes with Notch signaling prevents biochemical, histological, and functional consequences of peritoneal fibrosis through inhibiting epithelial to mesenchymal transition of rat peritoneal mesothelial cells. These results support the use of γ-secretase inhibitors as a novel therapeutic approach for peritoneal fibrosis. Peritoneal dialysis (PD) is a convenient and inexpensive therapy for patients with end-stage renal disease. In long-term PD, the effectiveness is markedly limited mainly by the fibrotic changes in the peritoneal membrane.1Williams JD Craig KJ Topley N Williams GT Peritoneal dialysis: changes to the structure of the peritoneal membrane and potential for biocompatible solutions.Kidney Int Suppl. 2003; : S158-S161Crossref PubMed Scopus (71) Google Scholar, 2Krediet RT Lindholm B Rippe B Pathophysiology of peritoneal membrane failure.Perit Dial Int. 2000; 20 Suppl 4: S22-S42PubMed Google Scholar Thus, there is a pressing need for the understanding of the molecular pathogenesis of peritoneal fibrosis and the development of effective therapy for preventing peritoneal fibrosis. The monolayer of peritoneal mesothelial cells is the key structure of the biological and physical barrier that are involved in regulating permeability and ultrafiltration in PD.3Tamura M Osajima A Nakayamada S Anai H Kabashima N Kanegae K Ota T Tanaka Y Nakashima Y High glucose levels inhibit focal adhesion kinase-mediated wound healing of rat peritoneal mesothelial cells.Kidney Int. 2003; 63: 722-731Crossref PubMed Scopus (41) Google Scholar In patients chronically exposed to the peritoneal dialysis fluid (PDF), there is a loss of mesothelial cells and the replacement of the peritoneal membrane by fibrous tissue.4Williams JD Craig KJ Topley N Von Ruhland C Fallon M Newman GR Mackenzie RK Williams GT Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479PubMed Google Scholar, 5Mortier S Faict D Schalkwijk CG Lameire NH De Vriese AS Long-term exposure to new peritoneal dialysis solutions: effects on the peritoneal membrane.Kidney Int. 2004; 66: 1257-1265Crossref PubMed Scopus (156) Google Scholar Recent studies revealed an important role of mesothelial cells in peritoneal injury through the epithelial-to-mesenchymal transition (EMT) induced by PDF. Submesothelial myofibroblasts, which participate in extracellular matrix accumulation (ECM) and angiogenesis, can originate from mesothelial cells through EMT.6Aroeira LS Aguilera A Selgas R Ramirez-Huesca M Perez-Lozano ML Cirugeda A Bajo MA del Peso G Sanchez-Tomero JA Jimenez-Heffernan JA Lopez-Cabrera M Mesenchymal conversion of mesothelial cells as a mechanism responsible for high solute transport rate in peritoneal dialysis: role of vascular endothelial growth factor.Am J Kidney Dis. 2005; 46: 938-948Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 7Kalluri R Neilson EG Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2103) Google Scholar Therefore, EMT is an early event in peritoneal membrane fibrogenesis and is likely mediated by transforming growth factor (TGF)-β both in mesothelial cell culture and in vivo.8Aroeira LS Aguilera A Sanchez-Tomero JA Bajo MA del Peso G Jimenez-Heffernan JA Selgas R Lopez-Cabrera M 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, 9Del Peso G Jimenez-Heffernan JA Bajo MA Aroeira LS Aguilera A Fernandez-Perpen A Cirugeda A Castro MJ de Gracia R Sanchez-Villanueva R Sanchez-Tomero JA Lopez-Cabrera M Selgas R Epithelial-to-mesenchymal transition of mesothelial cells is an early event during peritoneal dialysis and is associated with high peritoneal transport.Kidney Int Suppl. 2008; : S26-S33Crossref PubMed Scopus (102) Google Scholar Notch signaling is an ancient cell signaling system that regulates cell fate specification, stem cell maintenance, and initiation of differentiation in embryonic and postnatal tissues.10Calvi LM Adams GB Weibrecht KW Weber JM Olson DP Knight MC Martin RP Schipani E Divieti P Bringhurst FR Milner LA Kronenberg HM Scadden DT Osteoblastic cells regulate the haematopoietic stem cell niche.Nature. 2003; 425: 841-846Crossref PubMed Scopus (2803) Google Scholar, 11Huppert SS Le A Schroeter EH Mumm JS Saxena MT Milner LA Kopan R Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1.Nature. 2000; 405: 966-970Crossref PubMed Scopus (285) Google Scholar, 12Artavanis-Tsakonas S Rand MD Lake RJ Notch signaling: cell fate control and signal integration in development.Science. 1999; 284: 770-776Crossref PubMed Scopus (4907) Google Scholar Four Notch receptors isoforms, namely Notch-1,13Ellisen LW Bird J West DC Soreng AL Reynolds TC Smith SD Sklar J TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.Cell. 1991; 66: 649-661Abstract Full Text PDF PubMed Scopus (1447) Google Scholar Notch-2,14Weinmaster G Roberts VJ Lemke G Notch2: a second mammalian Notch gene.Development. 1992; 116: 931-941Crossref PubMed Google Scholar Notch-3,15Lardelli M Dahlstrand J Lendahl U The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium.Mech Dev. 1994; 46: 123-136Crossref PubMed Scopus (261) Google Scholar and Notch-4,16Uyttendaele H Marazzi G Wu G Yan Q Sassoon D Kitajewski J Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene.Development. 1996; 122: 2251-2259Crossref PubMed Google Scholar and five ligands, Jagged-117Lindsell CE Shawber CJ Boulter J Weinmaster G Jagged: a mammalian ligand that activates Notch1.Cell. 1995; 80: 909-917Abstract Full Text PDF PubMed Scopus (540) Google Scholar and Jagged-218Shawber C Nofziger D Hsieh JJ Lindsell C Bogler O Hayward D Weinmaster G Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway.Development. 1996; 122: 3765-3773Crossref PubMed Google Scholar belonging to the Serrate family, and Delta-1,19Bettenhausen B Gossler A Efficient isolation of novel mouse genes differentially expressed in early postimplantation embryos.Genomics. 1995; 28: 436-441Crossref PubMed Scopus (22) Google Scholar Delta-3,20Dunwoodie SL Henrique D Harrison SM Beddington RS Mouse Dll3: a novel divergent Delta gene, which may complement the function of other Delta homologues during early pattern formation in the mouse embryo.Development. 1997; 124: 3065-3076PubMed Google Scholar and Delta-like 421Rao PK Dorsch M Chickering T Zheng G Jiang C Goodearl A Kadesch T McCarthy S Isolation and characterization of the notch ligand delta4.Exp Cell Res. 2000; 260: 379-386Crossref PubMed Scopus (53) Google Scholar belonging to the Delta family, have been identified in mammals. The pathway is activated through an interaction of a Notch receptor with a Jagged or Delta-like ligand leading to proteolytic cleavages of Notch receptor at two distinct sites. The cleavage releases the Notch intracellular domain (NICD) such that it can enter the nucleus and function as a transcription activator. Importantly, the second cleavage is mediated by the γ-secretase complex, and effective inhibition of Notch activation can be achieved by pharmacological inhibition of this proteolytic activity. Once within the nucleus, NICD interacts with CSL (RBP-Jk/CBF1) and Mastermind to generate a large transcriptional activator complex and activates transcription of downstream target genes.22Mumm JS Kopan R Notch signaling: from the outside in.Dev Biol. 2000; 228: 151-165Crossref PubMed Scopus (846) Google Scholar In mammals, primary target genes of the Notch-intracellular domain/RBP-J complex include the HES (Hairy/Enhancer of Split)23Jarriault S Brou C Logeat F Schroeter EH Kopan R Israel A Signalling downstream of activated mammalian Notch.Nature. 1995; 377: 355-358Crossref PubMed Scopus (1215) Google Scholar, 24Bailey AM Posakony JW Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.Genes Dev. 1995; 9: 2609-2622Crossref PubMed Scopus (505) Google Scholar and HEY (HES-related with YRPW motif, also named HERP, HES-related repressor protein)25Iso T Sartorelli V Poizat C Iezzi S Wu HY Chung G Kedes L Hamamori Y HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling.Mol Cell Biol. 2001; 21: 6080-6089Crossref PubMed Scopus (184) Google Scholar, 26Kokubo H Lun Y Johnson RL Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.Biochem Biophys Res Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar, 27Leimeister C Schumacher N Steidl C Gessler M Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos.Mech Dev. 2000; 98: 175-178Crossref PubMed Scopus (87) Google Scholar family of genes, which act as transcription factors. Notch has recently been shown to promote EMT during cardiac valve formation.28Timmerman LA Grego-Bessa J Raya A Bertran E Perez-Pomares JM Diez J Aranda S Palomo S McCormick F Izpisua-Belmonte JC de la Pompa JL Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.Genes Dev. 2004; 18: 99-115Crossref PubMed Scopus (749) Google Scholar Moreover, an upregulation of Notch ligand Jagged-1 expression was detected in the kidney of a model of progressive interstitial fibrosis induced by ureteral obstruction.29Morrissey J Guo G Moridaira K Fitzgerald M McCracken R Tolley T Klahr S Transforming growth factor-beta induces renal epithelial jagged-1 expression in fibrotic disease.J Am Soc Nephrol. 2002; 13: 1499-1508Crossref PubMed Scopus (86) Google Scholar In epithelial cells from mammary gland, kidney tubules, and epidermis, TGF-β induces the Notch target gene Hey1 at the onset of EMT in a Smad3-dependent process.30Zavadil 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; 23: 1155-1165Crossref PubMed Scopus (630) Google Scholar However, despite a most recent report showing expression of Jagged-1 in peritoneal mesothelial cells,31Choi JH Park JT Davidson B Morin PJ Shih Ie M Wang TL Jagged-1 and Notch3 juxtacrine loop regulates ovarian tumor growth and adhesion.Cancer Res. 2008; 68: 5716-5723Crossref PubMed Scopus (99) Google Scholar little is known about the expression pattern and functional role of the Notch signaling pathway in normal and injured peritoneum induced by long term PD. In the present study, we investigated the role of Notch signaling in the progression of peritoneal fibrosis induced by PDF. Our results demonstrated that the components of Notch signaling are expressed and activated in fibrotic peritoneum induced by PDF. Moreover, TGF-β induced the expression of Notch signaling components during the process of EMT of primary rat mesothelial cells (RPMCs). Because γ-secretase inhibitor (GSI) has been extensively used for inhibiting Notch signaling both in vitro32Lewis HD Perez Revuelta BI Nadin A Neduvelil JG Harrison T Pollack SJ Shearman MS Catalytic site-directed gamma-secretase complex inhibitors do not discriminate pharmacologically between Notch S3 and beta-APP cleavages.Biochemistry. 2003; 42: 7580-7586Crossref PubMed Scopus (70) Google Scholar and in vivo33Shih Ie M Wang TL Notch signaling, gamma-secretase inhibitors, and cancer therapy.Cancer Res. 2007; 67: 1879-1882Crossref PubMed Scopus (362) Google Scholar and also been clinically tested for T cell acute lymphoblastic leukemia,34Lewis HD Leveridge M Strack PR Haldon CD O'Neil J Kim H Madin A Hannam JC Look AT Kohl N Draetta G Harrison T Kerby JA Shearman MS Beher D Apoptosis in T cell acute lymphoblastic leukemia cells after cell cycle arrest induced by pharmacological inhibition of notch signaling.Chem Biol. 2007; 14: 209-219Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 35Kogoshi H Sato T Koyama T Nara N Tohda S Gamma-secretase inhibitors suppress the growth of leukemia and lymphoma cells.Oncol Rep. 2007; 18: 77-80PubMed Google Scholar we used GSI to treat RPMCs and found that GSI dramatically inhibited TGF-β–induced EMT of RPMCs. Most importantly, we demonstrated that GSI could significantly attenuate peritoneum fibrosis and prevent a loss of peritoneal function in vivo. These results support the use of GSI as a novel therapeutic approach for PD-related peritoneal fibrosis. Antibodies against α–smooth muscle actin (α-SMA) and GAPDH were from Sigma-Aldrich (St. Louis, MO). Notch-1(sc-6015) and HES-1 antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Antibody specific for NICD (ab52301) was from Abcam (Cambridge, MA). Vascular endothelial growth factor (VEGF) antibody was purchased from Thermo Fisher Scientific Inc (Fremont, CA). Goat anti-type collagen antibody was obtained from Southern Biotech (Birmingham, AL). Jagged-1 antibody and secondary HRP-conjugated antibodies were from Cell Signaling (Danvers, MA). FITC-conjugated donkey anti-goat IgG antibody was purchased from Jackson Immuno Research Laboratories Inc (West Grove, PA). Fluorescent phallotoxin-labeled secondary antibodies were from Invitrogen (Carlsbad, CA). γ-secretase inhibitor DAPT was purchased from Calbiochem (San Diego, CA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Male Sprague-Dawley (SD) rats weighing 150 to 180 g were obtained from the Experiment Animal Centre at the Northern Campus of Sun Yat-sen University. All animal experiments were approved by the Committee on Animal Experimentation of Sun Yat-sen University and performed in compliance with the university’s Guidelines for the Care and Use of Laboratory Animals. Thirty male SD rats were randomly allocated into five groups: rats in group A (n = 6) served as normal controls; rats in group B (n = 6) and group C (n = 6) received daily intraperitoneal injections of PDF named Dianeal® PD-2 Peritoneal Dialysis Solution with 4.25% Dextrose (4.25% Dianeal; Baxter HealthCare, Deerfield, IL) at 100 ml/kg of body weight36Nakav S Kachko L Vorobiov M Rogachev B Chaimovitz C Zlotnik M Douvdevani A Blocking adenosine A2A receptor reduces peritoneal fibrosis in two independent experimental models.Nephrol Dial Transplant. 2009; 24: 2392-2399Crossref PubMed Scopus (15) Google Scholar; rats in group D (n = 6) were intraperitoneally injected with 10 μmol/L DAPT together with 4.25% Dianeal; rats in group E (n = 6) received the same amount of DMSO (the vehicle for DAPT) as group D together with 4.25% Dianeal. Rats of group B were sacrificed at 14 days and the rest of rats were sacrificed at 28 days after initial treatment. Peritoneal function tests were performed as previously described.37Nie J Dou X Hao W Wang X Peng W Jia Z Chen W Li X Luo N Lan HY Yu XQ Smad7 gene transfer inhibits peritoneal fibrosis.Kidney Int. 2007; 72: 1336-1344Crossref PubMed Scopus (57) Google Scholar Briefly, for the peritoneal ultrafiltration rate, 4.25% Dianeal was administered intraperitoneally to the rats at 90 ml/kg body weight before being euthanized. Four hours later, the peritoneal fluid was removed for ultrafiltration measurement. Net ultrafiltration was the volume of fluid removed after four hours minus the volume of fluid administered. For glucose transportation assay, glucose was measured by a standard enzymatic test on a Hitachi automated chemistry analyzer (Hitachi 7170, Japan). Mass transfer of glucose from the peritoneum was calculated using the formula: (initial dialysate glucose × initial volume) − (final dialysate glucose × final volume). These values were corrected for animal weight at the time of euthanasia. Four-μm paraffin sections from the anterior abdominal wall were stained with hematoxylin and eosin and Masson trichrome. The thickness (μm) of the peritoneum was measured in each animal using a micrometer fitted into the eyepiece of the microscope and expressed as the means ± SD. Each section was measured at 10 random sites. For immunofluoresence analysis, 10-μm paraffin-embedded sections from the visceral peritoneum were cut and dehydrated in xylene, followed by microwave antigen retrieval. Sections were then blocked with PBS containing 5% BSA for 30 minutes at 37°C. Sections were incubated overnight at 4°C with indicated primary antibodies diluted in 3% BSA in PBS and secondary antibodies for 50 minutes at 37°C. Samples were mounted in fixation medium (Biomeda, Foster City, CA). DAPI (Sigma-Aldrich, St. Louis, MO) was used to stain the nucleus. Images were analyzed and collected with Zeiss LSM 510 Confocal Imaging System (Zeiss, Germany). The isolation and culture of RPMCs was performed according to our previously reported method.37Nie J Dou X Hao W Wang X Peng W Jia Z Chen W Li X Luo N Lan HY Yu XQ Smad7 gene transfer inhibits peritoneal fibrosis.Kidney Int. 2007; 72: 1336-1344Crossref PubMed Scopus (57) Google Scholar Briefly, RPMCs were prepared by infusing 30 ml of 0.25% trypsinase-0.2% EDTA-Na2 into the rat abdominal cavity. The fluid was removed from the peritoneal cavity one hour later under sterile conditions. To harvest RPMCs, cellular components were isolated by centrifugation and then washed with PBS and suspended in DMEM/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 12% (v/v) FCS (Invitrogen, Carlsbad, CA). Cells were placed into 25-cm2 culture flasks and incubated overnight at 37°C. Nonadherent cells were removed the next day, and the adherent population was incubated at 37°C in 5% CO2 in fresh culture medium. RPMCs in this study were derived from two to four passages grown as a monolayer to subconfluency. To induce EMT, RPMCs were seeded into 35-mm diameter tissue culture plates. When 60% confluent, cells were cultured in serum-free medium for 24 hours and then indicated amount of TGF-β1 was added for various time period. To examine the effect of γ-secretase inhibitor DAPT on TGF-β1–induced EMT, RPMCs were pre-incubated for 15 minutes with 10 μmol/L DAPT before TGF-β1 treatment. Total peritoneal RNA was isolated from visceral peritoneum using Trizol Reagent (Invitrogen, Carlsbad, CA) and was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) as previously described.37Nie J Dou X Hao W Wang X Peng W Jia Z Chen W Li X Luo N Lan HY Yu XQ Smad7 gene transfer inhibits peritoneal fibrosis.Kidney Int. 2007; 72: 1336-1344Crossref PubMed Scopus (57) Google Scholar Amplified cDNA was used as the template DNA, and PCR was performed with TaqDNA polymerase and specific primers. The steps were as follows: denaturation at 95°C for 5 minutes followed by denaturation at 95°C for 30 seconds, hybridization at 60°C for 30 seconds, and elongation at 72°C for 45 seconds, for 25–32 cycles. The following PCR primers were used: Jagged-1: forward, 5′-GCCAAGTGGGATGACGACT-3′, reverse, 5′-GCAACAGCAGCGATAAGTGA-3′; Notch-1: forward, 5′-GCAAGAAGAAGCGGAGAG-3′, reverse, 5′-AGCTGGCACCCTGATAGATG-3′; HES-1: forward, 5′-CAGATGACCGCCGCTCTCA-3′, reverse, 5′-GCGACACTGCGTTAGGACCC-3′; GAPDH: forward, 5′-AGATCCACAACGGATACATT-3′, reverse, 5′-TCCCTCAAGATTGTCAGCAA-3′. Tissue and cells were lysed in lysis buffer (50 mmol/L Hepes pH 7.5, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L NaF, 10 mmol/L Na4P2O7, 1 mmol/L Na3VO5, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and 20 μg/ml aprotinin). Protein was quantified by the Bradford assay (Bio-Rad, Hercules, CA), equal amount of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). After blocking in 5% skim milk for 1 hour at room temperature, membranes were incubated with indicated primary antibody at 4°C overnight followed by horseradish peroxidase-conjugated second antibody for 1 hour at room temperature and detected by chemiluminescence (Amersham Biosciences, Piscataway, NJ). Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals using NIH Image analysis program. The results were expressed as mean ± SD. Statistical analysis was performed using SPSS13.0. Data were analyzed using one-way analysis of variance followed by post hoc test. A value of P < 0.05 was considered as statistically significant. To investigate the expression of Notch signaling components in fibrotic peritoneum, we generated a rat model of peritoneal fibrosis induced by daily intraperitoneal injection of PDF containing 4.25% glucose at 100 ml/kg of body weight, and rats were sacrificed either 14 days or 28 days after the initial treatment. As shown in Figure 1, A and B, the mRNA levels of Jagged-1, Notch-1, and the Notch downstream target HES-1 was hardly detected in normal peritoneum by RT-PCR, but their expression was gradually increased in rats after 14 days of PDF treatment. The mRNA expression of Jagged-1, Notch-1, and HES-1 in 28 days group was readily detected and was significantly higher than that in control group. Similarly, the protein level of Jagged-1 and HES-1 was increased gradually after PDF treatment, and a significant increase was detected in 28 days group compared with that of control group (Figure 2, A and B). Because the cleavage of Notch-1 is an indicator of Notch signaling activation, we examined the protein level of NICD by Western blot and detected a significant increase of NICD in the peritoneum after 28 days of PDF treatment (Figure 2). The expression of NICD, Jagged-1, and HES-1 at the protein level was further documented by immunofluorescence. As shown in Figure 3, A–C, positive staining for NICD, Jagged-1, and HES-1 was readily detected in the mesothelial cells on the surface of the peritoneum and in cells in the submesothelial areas after 28 days of PDF treatment. In contrast, few cells with positive staining for NICD, Jagged-1, and HES-1 expression were detected in normal peritoneum. We next examined the localization of the upregulated HES-1 as a marker of activated Notch signaling by immunofluorescence. In the fibrotic peritoneum, the HES-1–positive staining was colocalized with α-SMA, a phenotypic marker of myofibroblast and a hallmark of EMT of mesothelial cells, in the cells on the surface of the peritoneum and in the submesothelial area (Figure 3D). These data indicated that the upregulation of Notch signaling occurs in myofibroblasts and transdifferentiated mesothelial cells. These data demonstrated that Notch signaling was highly activated in fibrotic peritoneum induced by PDF.Figure 2The protein level of Notch signaling components was increased in fibrotic peritoneum induced by PDF. A: Normal rats (A1 to A6) or rats that received daily intraperitoneal injections of 4.25% Dianeal (100 ml/kg) for 14 days (B1 to B6) or 28 days (C1 to C6) were sacrificed. The protein levels of NICD, Jagged-1, and HES-1 were analyzed by Western blot. GAPDH was used to verify equivalent loading. Each panel represents an independent experiment with two animals per group. B: Graphic representation of relative abundance of NICD, Jagged-1, and HES-1 normalized to GAPDH. Data are given as mean ± SD (n = 6). *P < 0.05 versus normal rats. **P < 0.01 versus normal rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunofluorescence evidence for the increased Notch signaling activation in fibrotic peritoneum. Peritoneal sections of normal rats or rats on 28-day PDF treatment (100 ml/kg) were paraffin-fixed and stained with antibodies against NICD (A), Jagged-1 (B), and HES-1 (C). D: Peritoneal sections of rats on 28-day PDF treatment (100 ml/kg) were paraffin-fixed and costained with antibodies against HES-1(red) and α-SMA (green). Nuclei were stained with DAPI (blue). Images (magnification ×400) were taken by confocal microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the potential clinical implication of GSI, we further tested the possibility that blocking Notch signaling activity by GSI could attenuate peritoneal fibrosis in vivo. Rats were administrated with 10 μmol/L N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S- phenylglycine t-butyl ester (DAPT), a highly active GSI, by intraperitoneal injection together with 4.25% Dianeal (100 ml/kg) for 28 days. Control rats received the same amount of DMSO (the vehicle for DAPT) together with 4.25% Dianeal (100 ml/kg). The efficiency of DAPT on inhibiting Notch signaling was demonstrated by decreased protein level of NICD and HES-1 (Figure 4, A and B). As expected, peritoneal fibrosis developed on day 28 after PDF treatment that was characterized by a marked peritoneal thickening, significant ECM accumulation, and a loss of linear mesothelial cells in rats that received PDF alone and in control rats receiving both DMSO and PDF. However, these features of peritoneal fibrosis remarkably attenuated in DAPT-treated rats. As shown in Figure 5, A–C, the thickness of peritoneum was significantly reduced in the DAPT-treated rats accompanied by less ECM accumulation. Moreover, the rates of mass transfer of glucose (MTG) and ultrafiltration rate, representative indexes of peritoneal function, were preserved in the DAPT-treated rats in comparison with those treated with PDF (Figure 6, A and B). Moreover, the preservation of peritoneal function by DAPT was accompanied by the attenuation in expression of several proteins characteristic of EMT and peritoneal fibrosis. By both Western blot analysis and immunofluorescence staining, the expression of α-SMA, collagen I, and VEGF was markedly attenuated in DAPT-treated rats compared with PDF treated rats, whereas the E-cadherin expression was increased significantly in DAPT-treated rats (Figure 7, A–C).Figure 5DAPT treatment attenuated peritoneal fibrosis induced by PDF. A: Hematoxylin and eosin staining of paraffin-fixed parietal peritoneal sections. B: Masson Trichrome staining of paraffin-fixed parietal peritoneal sections. C: Semiquantification of the thickness of peritoneal membrane. Data are mean ± SD (n = 6). **P < 0.01 versus normal rats. ##P < 0.01 versus PDF-treated and vehicle control rats. N indicates normal rats; P, PDF-treated rats; V, vehicle control rats treated with DMSO together with PDF. D, Rats treated with DAPT toge" @default.
• W2127890209 created "2016-06-24" @default.
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• W2127890209 date "2010-02-01" @default.
• W2127890209 modified "2023-10-17" @default.
• W2127890209 title "Preventive Effect of Notch Signaling Inhibition by a γ-Secretase Inhibitor on Peritoneal Dialysis Fluid-Induced Peritoneal Fibrosis in Rats" @default.
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