Nitro-oleic Acid Inhibits The High Glucose-induced Epithelial-mesenchymal Transition In Human Peritoneal Mesothelial Cells

Background:In recent years,the prevalence of end-stage renal disease(ESRD)has been rapidly increasing,driven by the aging of population and the increased prevalence of hypertension and diabetes,placing a heavy economic burden on society.Renal replacement therapy(RRT),through peritoneal dialysis,hemodialysis or renal transplantation,is a life-sustaining treatment for patients with ESRD.According to a recent report,more than 272,000 people are undergoing peritoneal dialysis(PD)worldwide,representing 11%of the world dialysis population.Compared with hemodialysis(HD),PD has the advantages of being simple,economical,and hemodynamically stable,and it better preserves residual renal function.However,long-term peritoneal dialysis using glucose-based PD solutions can lead to morphological and functional changes of the peritoneum,such as denudation of the mesothelial cells from the peritoneum,extracellular matrix accumulation,epithelial-mesenchymal transition(EMT),neoangiogenesis and increased peritoneal thickness,which eventually result in peritoneal fibrosis and loss of ultrafiltration capacity.High glucose(HG),glucose-degradation products,hyperosmolality,acidic pH,recurrent peritonitis,etc.,have been implicated as important factors in the pathogenesis of peritoneal fibrosis.As the EMT is associated with pro-fibrotic signalling,it is important in the early pathogenesis of peritoneal fibrosis.Some studies have examined other osmotic agents,such as sorbitol,mannitol,xylitol and icodextrin,but none have shown superior efficacy or safety compared with that provided by glucose.Several studies have demonstrated that TGF-?1 is a key mediator that regulates the EMT and peritoneal fibrosis,and high glucose and glucose-degradation products can stimulate TGF-?1 production in mesothelial cells.TGF-?1 can exert biological functions through Smad-dependent and Smad-independent pathways,and most profibrotic activities are mediated by Smad-dependent pathways[3].By binding to T?RI,TGF-?1 phosphorylates receptor-regulated Smad2/3,and phospho-Smad2/3 are released from the receptor complex and translocate into the nucleus to regulate the expression of target genes,such as Snail,?-SMA and collagen I.As an electrophilic nitroalkene fatty acid,nitro-oleic acid(OA-NO2)is generated endogenously by NO-and NO2--mediated redox reactions.OA-NO2 can be detected in normal tissues at nanomolar concentrations,and increased production is demonstrated in response to metabolic stress and inflammation.By reversible Michael addition reactions with the nucleophilic residues of the target proteins,OA-NO2 can exhibit multiple biological effects.Animal and cell experiments have confirmed that OA-NO2 has properties that contribute to anti-inflammation,anti-oxidative stress,anti-apoptosis,and the regulation of lipid metabolism.In endothelial cells(ECs),OA-NO2 downregulated the development of the TGF-?1-induced EMT and the activation of both Smad2 and Smad3.Another study showed that OA-NO2 combined with losartan inhibited glomerulosclerosis and the expression of TGF-?1,?-SMA,collagen ?,PAI-1 and FN in the kidneys of mice with diabetic nephropathy.However,little is known about the role of OA-NO2 in peritoneal fibrosis.Here,we studied the effects of OA-NO2 on the high glucose-induced epithelial-mesenchymal transition(EMT)in human peritoneal mesothelial cells(HPMCs)and the relevant mechanisms.Part ? High glucose induces the occurrence of epithelial-mesenchymal transition in human peritoneal mesothelial cells.Objective:To elucidate whether high glucose could induce the occurrence of EMT in HPMCs.Methods:1.Cell culture:The human peritoneal mesothelial cells were cultured in DMEM/F12 supplemented with 10%fetal bovine serum,100 ?g/ml streptomycin and 100 U/ml penicillin at 37? with a 5%CO2 atmosphere.Subconfluent cells were cultured in serum-free DMEM/F12 medium for overnight starvation prior to each experiment.2.The HPMCs were exposed to four different concentrations of glucose(control,1.5%,2.5%,4.25%)for 24 hours.(1)Investigating whether different concentrations of glucose have cytotoxic effects on HPMCs by CCK-8 experiments.(2)Detecting the protein expression level of the epithelial marker,E-cadherin and the protein expression levels of the mesenchymal markers,N-cadherin,?-SMA and snail by Western blot analysis3.The HPMCs were exposed to 2.5%glucose for different time course(0 h,2 h,12 h,24 h,and 48 h).And the protein expression levels of TGF-?1/Smads signalling pathway in the HPMCs were detected by Western blot analysis.Results:1.Compared with control group,1.5%and 2.5%glucose did not induce significant toxicity in the HPMCs but 4.25%glucose induced significant toxicity in the HPMCs.2.2.5%and 4.25%glucose significantly decreased the protein expression level of the epithelial marker,E-cadherin,and increased the protein expression levels of the mesenchymal markers,N-cadherin,?-SMA and Snail,compared with levels in the control group.Additionally,1.5%glucose significantly increased ?-SMA and Snail protein expression levels compared with levels in the control group.However,its effect on the protein expression of E-cadherin and N-cadherin was not statistically significant.3.The HPMCs exposed to 2.5%glucose for 2 hours significantly increased the TGF-?1 protein expression level and increased the phosphorylation levels of Smad2 and Smad3.Conclusions:High glucose induces the occurrence of EMT and the activation of TGF-?1/Smads signalling pathway in HPMCs.Part ? Nitro-oleic acid inhibits the high glucose-induced epithelial-mesenchymal transition in human peritoneal mesothelial cellsObjective:To elucidate whether Nitro-oleic acid could inhibit the high glucose-induced EMT in HPMCs and the related mechanisms.Methods:1.The HPMCs were exposed to different concentrations of OA-NO2(0 ?M,1?M,2 ?M,4 ?M,or 8 ?M)for 24 hours.And we investigated whether different concentrations of OA-NO2 have cytotoxic effects on HPMCs by CCK-8 experiments.2.The HPMCs were preincubated with OA-NO2 for 2 hours(2 ?M,4 ?M),and then were exposed to four 2.5%glucose for 24 hours.(1)Detecting the protein expression level of the epithelial marker,E-cadherin and the protein expression levels of the mesenchymal markers,N-cadherin,?-SMA and snail by Western blot analysis.(2)Detecting the protein expression level of the epithelial marker,E-cadherin and the protein expression level of the mesenchymal markers,?-SMA by Immunofluorescence.(3)Detecting the protein expression levels of TGF-?1/Smads signalling pathway in the HPMCs by Western blot analysis.(4)Detecting the protein expression levels of JNK,ERK,PKC and PI3K/AKT signalling pathways in the HPMCs by Western blot analysis.Results:1.Compared with control group,exposure to 1 ?M,2?M and 4 ?M concentrations of OA-NO2 for 24 hours did not induce significant toxicity in the HPMCs but 8 ?M concentration of OA-NO2 induced significant toxicity in the HPMCs.2.Pre-treatment with OA-NO2 significantly increased the protein expression level of epithelial marker E-cadherin and decreased the protein expression levels of mesenchymal markers N-cadherin,?-SMA and Snail compared with their levels in the cells treated with 2.5%glucose alone.3.Pre-treatment with OA-NO2 significantly decreased the protein expression level of TGF-?1 and the phosphorylation levels of Smad2 and Smad3 compared with the expression and phosphorylation levels of these factors in the HG group.4.Pre-treatment with OA-NO2 significantly decreased the phosphorylation levels of ERK and JNK compared with the phosphorylation levels of these factors in the HG group.However,pre-treatment with OA-NO2 had no significant effect on the protein expression of AKT and PKC compared with the protein expression of these factors in the HG groupConclusions:Nitro-oleic acid could inhibit the high glucose-induced EMT and the activation of TGF-?1/Smads,ERK and JNK signalling pathways in HPMCs.Part ? Nitro-oleic acid inhibits the high glucose-induced the protein expression of NF-?B p65 and IL-6 secretion in human peritoneal mesothelial cells.Objective:To elucidate whether Nitro-oleic acid could inhibit the high glucose-induced the protein expression of NF-?B p65 and IL-6 secretio in HPMCsMethods:The HPMCs were preincubated with OA-NO2 for 2 hours(2 ?M,4 ?M),and then were exposed to four 2.5%glucose for 24 hours.(1)Detecting the protein expression level of NF-?B p65 by Western blot analysis.(2)Detecting the mRNA expression levels of TNF-?,IL-6 and IL-1? by Real-time PCR.(3)Detecting the protein levels of IL-6,IL-1? and TNF-? in the culture supernatants by enzyme-linked immunosorbent assays.Results:1.Pre-treatment with OA-NO2 significantly increased the protein expression level of NF-?B p65 compared with its level in the cells treated with 2.5%glucose alone.2.Pre-treatment with OA-NO2 significantly decreased the mRNA expression level of TNF-?,IL-6 and IL-1? compared with their levels in the cells treated with 2.5%glucose alone.3.High glucose could increase the secretion of IL-6 and that pre-treatment with ON-NO2 significantly decreased the secretion of IL-6 compared with the amount secreted from the cells in the HG group.However,IL-1? and TNF-? were undetectable in the culture supernatants of all the groups.Conclusions:Nitro-oleic acid inhibits the high glucose-induced the protein expression of NF-?B p65 and IL-6 secretion in human peritoneal mesothelial cells.