The Role Of Galectin-1 On Epithelial To Mesenchymal Transition Induced By High Glucose Peritoneal Dialysate In Human Peritoneal Mesothelial Cells

Background:Peritoneal Dialysis (PD) is one of three methods of renal replacement therapy.Ultrafiltration failure is one of the most important reasons for discontinuation of peritoneal dialysis.Numerous studies have confirmed that high concentrations of glucose dialysate caused peritoneal structure and function changes,and peritoneal cell injury and repair and even Epithelial-to-mesenchymal Transition (EMT), leading to peritoneal fibrosis (PF) and ultrafiltration failure (UFF).But the molecular mechanism of peritoneal fibrosis has not fully understood.Galectin-1 was the first reported mammalian galectin, involved in cell adhesion, proliferation, differentiation, apoptosis, inflammation and tumor metastasis, invasion, growth. Our former research use proteomics have discovered that high glucose stimulate peritoneum mesothelial cell result in upgrade the expresses of galectin-1,suggesting that galectin-1 may play a role in peritoneal fibrosis.It is reported that galectin-1 suppress the expression of collagen I in kidney epithelial cell through block up TGF-β/Smad signal pathway.However,the role of galectin-1 on epithelial to mesenchymal transition induced by peritoneal dialysate in human peritoneal mesothelial cells has not been reported.vimentin and zo-1 is commonly used EMT-related indicators. Vimentin is the major middle fiber in the stromal cells,which exists in mesodermal origin cells,such as fibroblasts,endothelial cells and leukocytes,together with microtubules and microfilaments form a cell scaffold network to maintain cell integrity. Tight junction associated protein 1 (zonula occluden-1,ZO-1)is composed of tight junctions of stents, to maintain vascular and peritoneal dialysis fluid osmotic pressure gradient.Upgrade expression of Vimentin and loss expression of zo-1 suggest cell may occur EMT.RNA interference induced by double-stranded RNA with homologous mRNA degradation, causing post-transcriptional gene silencing has been successfully used in gene function and the relationship between signaling systems downstream and upstream molecules,it may become another new means after the antisense and ribozyme. Objective:To investigate the expression of galectin-1 and EMT-related marker vimentin,zo-1 induce by different glucose concentrations of dialysate on HPMCs and the relationship between galectin-1 and EMT.Methods:HPMCs were randomly divided into four groups:normal control group (5.5mmol/L glucose),1.5% PDS group,2.5% PDS group and 4.25% PDS group.Real-time PCR and Western blot were used to analyze the mRNA and protein expression of galectin-1,Vimentin and zo-1,analysis the relationship between galectin-1 and Vimentin,zo-1.Results:(1)Under normal culture conditions, HPMC express galectin-1, only express few vimentin and basis of zo-1.(2)Treatment of HPMCs with 4.25% PDS different time resulted in a time-dependent increased in the protein expression of galectin-1, 12h,24h,48h galectin-1 protein expression compared with control group were 1.09 times,1.22 times and 1.42 times has statistically significant (P <0.05).72h galectin-1 protein expression was slightly decreased, compared with the control group were 1.37 times, has significantly different(P<0.05).(3)Treatment of HPMCs with different glucose concentrations of peritoneal dialysis solution resulted in a glucose concentrations depen-dent.Galectin-1mRNA expression were 1.22 times,1.535 times and 2.34 times(P<0.05)compare to control group.In 1.5%PDS group,2.5%PDS group,4.25%PDS group galectin-1 protein expression were 1.48 times, 2.01 times and 2.38 times respectively as compare with control group, has statistically significant (P<0.05).(4)HPMCs treated with different concentrations high glucose of peritoneal dialysis solution resulted in a dose-dependent increase in the expression of vimentin mRNA and protein level.Compared with control group, the expression of vimentin mRNA level were 1.16 times,1.27 times and 1.69 times (P<0.05),respectively. The expression of vimentin protein level were 1.28 times,1.51 times and 1.92.times (P<0.05) respectively as compared to control group.(5)HPMCs treated with different concentrations high glucose peritoneal dialysis solution resulted in a dose-dependent decrease in the expression of zo-1 mRNA and protein level.Compared with control group, the expression of zo-1 mRNA level were 77%,43%(P<0.05)and 20%(P<0.05),respectively. The expression of zo-1 protein level were 88%,52%(P<0.05) and 40%(P<0.05) respectively as compared to control group. (6)There was a positive correlation between the expressions of galectin-1 and vimentin mRNA of HPMCs (r=0.864,P<0.05).There was a negative correlation between the expressions of galectin-1 and zo-1 mRNA of HPMCs(r=-0.604,P<0.05).There was a positive correlation between the expressions of galectin-1 and vimentin protein of HPMCs (r=0.917,P<0.05).There was a negative correlation between the expressions of galectin-1 and zo-1 protein of HPMCs (r=-0.869,P<0.05).Conclusion:(1)galectin-1 was expressed in HPMCs,and high glucose peritoneal dialysis fluid can upregulate the expression of galectin-1 in a time and dose dependent manner in HPMCs.(2) high glucose peritoneal dialysis fluid can induced HPMCs EMT.(3)galectin-1 related to HPMCs EMT induced by high glucose peritoneal dialysis fluid. Objective:Transfect galectin-1 siRNA in peritoneal mesothelial cells to inhibit galectin-1 expression, detect vimentin, zo-1 expression, investigate whether galectin-1 can inhibit high glucose peritoneal dialysis-induced EMT.Methods:Use liposome transfected siRNA to HPMCs,HPMCs were randomly divided into three groups:normal control group (untransfected HPMC+5.5mmol/L glucose), high glucose group (untran-sfected HPMC +4.25% PDS)and transfected group (transfected galectin-1 siRNA in HPMC +4.25% PDS),Real-time PCR and Western blot were used to analyze the mRNA and protein expression of galectin-1,Vimentin and zo-1.Analysis the relationship between galectin-1 and Vimentin, zo-1.Results:(1)Treatment of HPMCs with 4.25% peritoneal dialysate for 48h, galectin-1 mRNA and protein expression were upgraded compared with the control group (P<0.05).vimentin mRNA and protein expression compared with control group increased, zo-1 mRNA and protein expression is lower than control group,has statistically significant(P <0.05). (2)After transfect galectin-1 siRNA to HPMCs,compared with high glucose group, galectin-1 mRNA and protein expression were inhibited (P<0.05), while vimentin expression has significantly reduced, zo-1 expression was markly up-regulated, has significant differences(P <0.05).Conclusion:galectin-1 siRNA may suppress high glucose peritoneal dialysate induced HPMCs EMT.