Expression Of XBP1s In Human Peritoneal Mesothelial Cells Is Critical For High Glucose Induced Fibrosis

Background:In peritoneal dialysis,the peritoneum is a biological semipermeable membrane.The high-sugar peritoneal fluid and the blood in the peritoneal capillaries carry the transmembrane transport of substances,so as to achieve the purpose of blood purification.With the improvement of surgical methods and peritoneal dialysis devices,catheter-related infections and non-infective complications have been significantly reduced.In this case,peritoneal fibrosis has become the most serious problem for patients with long-term peritoneal dialysis.Peritoneal fibrosis can cause declined of dialysis efficacy and ultrafiltration failure.Eventually it leads to failure of peritoneal dialysis and the patient needs to switch to other renal replacement therapy.In recent years,there have been numerous studies on peritoneal dialysis-associated peritoneal fibrosis,and many new molecules and regulatory mechanisms have been discovered.It is suggested that there may be potential intervention sites in the early stage of fibrosis,but it is not enough to achieve clinical target therapy.X-box binding protein 1(XBP1)is a key transfactor,which is downstream of inositol reruiring enzyme 1(IRE1),it binds to the X box in the promoter sequence of the target genes.Then activate the transcription of them.IRE1/XBP1 is the most important and conserved signal pathway to deal with unfolded protein response(URP/endoplasmic reticulum stress,ERS).XBP1 s plays an important role in cell growth and differentiation,protein secretion,and apoptosis.XBP1 s participates in the secretion of the islet cells and salivary glands,the development of the embryonic liver and the growth and differentiation of the lymphocytes.Many literatures report that XBP1 s are closely related to the occurrence and development of various fibrotic diseases,such as idiopathic pulmonary fibrosis,scleroderma,hepatic fibrosis,cystic fibrosis,etc.But XBP1 s take a role as fibrosis has not been reported in human peritoneal mesothelial cells.Our previous studies found that XBP1 s mediates inflammatory response induced HPMCs inflammatory response by activating JNKsignaling pathway,and the use of XBP1 s inhibitors partially relieves inflammation-induced peritoneal fibrosis.However,the role and the mechanism of XBP1 s in high glucose-stimulated peritoneal mesothelial cells and peritoneal fibrosis remains unclear,and further studies are needed to prove this.Objective:1.To detect the expression of XBP1 s and fibrosis related molecules in peritoneal dialysis efflux cells in peritoneal dialysis effluent cells,and to investigate the relationship between XBP1 s and peritoneal fibrosis was preliminarily explored.2.To investigate the correlation between the expression of XBP1 s and fibrosis-related molecules in human peritoneal mesothelial cells stimulated by high glucose.3.To verify the effect of XBP1 s on peritoneal fibrosis at the cellular level by overexpressing and knocking down XBP1 s,and to observe its effect on the expression of fibrosis-related molecules.Methods:1.Effluent cells were isolated and cultured from Continuous Ambulatory Peritoneal Dialysis patients with nocturnal dialysate.XBP1 s and fibrosis-related markers such as E-cadherin,Collagen-I,Fibronectin,α-SMA,CTGF,TGF-β,Vimentin mRNA and protein expression were detected by qRT-PCR and Western blot,respectively.2.Human peritoneal mesothelial cells were stimulated with 120 mM Glu dlucose.The mRNA and protein expression of XBP1 s and fibrosis-related markers E-cadherin,Collagen-I,Fibronectin,α-SMA,CTGF,TGF-β,Vimentin were detected by qRT-PCR,Western blot and immunofluorescence,respectively.3.XBP1 s overexpression and shRNA lentivirus were used to infect human HPMCs to establish overexpressing and interfering cell lines.High glucose stimulation was also performed to observe the expression of fibrosis related molecules.4.XBP1 s overexpression and shRNA lentivirus were used to infect the HPMCs in the peritoneal effluent of CAPD patients,and the expression of fibrosis-related molecules was observed.Results:1.With the prolonged peritoneal dialysis time,the morphology of the peritoneal effluent cells changed from a paving stone to a fusiform shape.The results ofqRT-PCR and Western blot showed that with the prolonged peritoneal dialysis time,the mRNA and protein expression levels of XBP1 s increased,and the expression of E-cadherin in epithelial cells decreased.while fibroblast-related markers α-SMA,TGF-β,Vimentin,Fibronectin,CTGF,Collagen I expression increased.2.In high glucose-stimulated HPMCs,qRT-PCR and Western blot results showed that XBP1 s expression increased,and the expression of E-cadherin in epithelial cells decreased.while fibroblast-related markers α-SMA,TGF-β,Vimentin,Fibronectin,CTGF,Collagen I expression increased.3.Overexpression and interfering with human HPMCs cell lines were established using the lentivirus infection method,and the infection efficiency reached or exceeded80%.Overexpression of XBP1 s significantly enhanced the down-regulation of E-cadherin expression in epithelial cells under high glucose stress,and increased the expression of fibroblast phenotype markers such as α-SMA,TGF-β,Vimentin,Fibronectin,CTGF and Collagen I.Interfering of XBP1 s could significantly inhibit the downregulation of E-cadherin expression in epithelial cells under high glucose stress,and increase the expression of fibroblast phenotype markers such asα-SMA,TGF-β,Vimentin,Fibronectin,CTGF,Collagen I.4.Overexpression and interfering with human primary peritoneal mesothelial cells(derived from the culture of peritoneal effluent cells from patients with CAPD)were established using the lentivirus infection method,and infection efficiencies can reach or exceed 80%.The experimental results are the same as those in the third part.Conclusion:With the prolonged peritoneal dialysis time,the peritoneal mesothelial cells of CAPD patients changed from epithelial cells to fibroblasts,and the expression of XBP1 s was significantly positively correlated with this phenomenon.Overexpression and knockdown of XBP1 s by cell lines and primary human HPMCs were found.XBP1 s mediate high glucose-stimulated human HPMCs from epithelial cells to fibroblasts.These findings demonstrate that XBP1 s are one of the important molecules of peritoneal dialysis-related peritoneal fibrosis and provide potential therapeutic targets for the future treatment of peritoneal dialysis-related peritoneal fibrosis.