High Glucose-induced TGF-β₁ Secretion In Human Peritoneal Mesothelial Cells Via Reactive Oxygen Species Signaling Pathway

Background:Continuous ambulatory peritoneal dialysis(CAPD) is one of the main replacement therapy of end stage renal disease(ESRD). Peritoneal fibrosis(PF) and ultrafiltration failure(UFF) induced by long-term peritoneal dialysis is the main cause which forces patients to drop out the therapy of peritoneal dialysis.Recent study has shown that non-physiological dialysate,especially dialysate containing high glucose could be an important cause of peritoneal dialysis-related peritoneal fibrosis,but the mechanisms by which high glucose induces peritoneal fibrosis remain unclear.Reactive oxygen species(ROS) are molecules or ions formed by the incomplete one-electron reduction of oxygen.These reactive oxygen intermediates include superoxides,peroxides,hydroxyl radical,singlet oxygen and lipid peroxidation.They contribute to the microbicidal activity of phagocytes,regulation of signal transduction and gene expression,the oxidative damage to nucleic acids,proteins and lipids.In diabetic nephropathy,ROS could mediate renal fibrosis through activing signaling pathways such as PKC,MAPK and transcription factors such as NF-κB,AP-1,upregulating expression of genes and proteins such as TGF-β₁,FN,Angâ…¡.Transforming growth factor-β₁ (TGF-β₁) is the most potent fibrogenic cytokine and is considered to play a principal role in high glucose-induced peritoneal fibrosis.So we hypothesize that ROS may be intracellular signal transduction molecules which play a role in high glucose-induced TGF-β₁ secretion in human peritoneal mesothelial cells(HPMCs),however there is no direct evidence of this investigation.Objective:This study was aimed to investigate the effect of high glucose on TGF-β₁ secretion and ROS generation in HPMCs.Meanwhile, we applied different concentrations of antioxidant N-acetylcysteine(NAC) to pretreat HPMCs to explore whether high glucose induces TGF-β₁ secretion in HPMCs via ROS signaling pathway.Methods:Mesothelial cells were isolated from human omental specimens by trypsin disaggregation,cells were cultured in DMEM medium supplemented with 15%FBS under an atmosphere of 5%CO₂ in a humidified incubator at 37℃.The cultured cells were identified by inverted phase contrast microscopy and immunocytochemistry staining with cytokeratin and vimentin antibodies respectively.HPMCs were randomly divided into two groups:1.High glucose groups(containing 1.5%,2.5%,4.25%D-Glucose),serum-free DMEM containing 0.1% glucose was used as the control group,each group was cultured for 24h; control group and 1.5%high glucose group were cultured for 12h,24h and 48h respectively;2.NAC pretreatment groups(containing 0.1mM, 1.0mM,10mM NAC),serum-free DMEM containing 0mM NAC was used as the control group,each group was pretreated with NAC for 1h, then was cultured with 1.5%high glucose for 24h.TGF-β₁ in supernatant fluid was measured by enzyme linked immunosorbent assay(ELISA);the generation of intracellular ROS were assessed indirectly by measuring the fluorescent product from the oxidation of a fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA),and using fluorescence microscope and multi-functional microplate reader to detect the relative fluorescence intensity(RFI) of dichlorofluorescin(DCF).Results:1.More than 96%of the cultured cells were proved to be HPMCs by identification.2.High glucose significantly induced the TGF-β₁ secretion in HPMCs with a dose-dependent and time-dependent manner(P＜0.05),the group of 4.25%high glucose and cultured for 48h showed the highest level of TGF-β₁ secretion in HPMCs.3.High glucose significantly stimulated the ROS generation in HPMCs with a dose-dependent and time-dependent manner(P＜0.05),the group of 4.25%high glucose and cultured for 48h showed the highest level of ROS generation in HPMCs.4.Antioxidant NAC significantly inhibited high glucose-induced ROS generation and TGF-β₁ secretion in HPMCs with a dose-dependent manner(P＜0.05),the 10mM NAC group showed the most obvious effect of inhibition.Conclusions:1.High glucose significantly induces the TGF-β₁ secretion in HPMCs,antioxidant inhibits the high glucose-induced TGF-β₁ secretion.2.High glucose significantly stimulates the ROS generation in HPMCs,antioxidant inhibits the high glucose-induced ROS generation.3.As a consequence of the above,high glucose induces TGF-β₁ secretion in HPMCs via ROS signaling pathway.