XBP1s Affect Ultrafiltration In Peritoneal Dialysis By Regulating AQPs Expression In Peritoneal Mesothelial Cells

BackgroundAs one of the important renal replacement therapies for end-stage renal disease(ESRD),peritoneal dialysis(PD)has been paid more and more attentions.However,the ultrafiltration failure(UFF)resulted in many patients unable to perform continues PD therapy due to the water-sodium retention and heart failure.A large number of the studies confirmed that the water clearance are closely related to aquaporins expression in peritoneum,and the water clearance in PD patients are attracting increasing attention,which was evidenced by some clinical research such as the ADEMEX research from Mexico and that from Hong Kong China.These studies confirmed that solute removal achieved to a certain degree will not increase the long-term survival of patients with PD,while the water clearance seems to be more important in reducing complications and increasing patients survival.The endoplasmic reticulum(ER)is a vital site for protein synthesis and modification,When ER is stimulated by specific factors,such as hypoxia,high glucose,calciumimbalance,infection,etc,the ER homeostasis is destroyed,leading to ER stress/ unfolded protein response.(UPR),characterized with accumulation of unfolded protein and misfolded protein.Then the UPR accelerates protein folding and removes out the misfolded protein,in order to make the cells into a new homeostasis.The UPR includes IRE1α、ATF6 and PERK pathway.Among them,the x-box-binding protein 1(XBP1)is a key molecule of IRE1α pathway.In UPR process,IRE1α splices unsliced XBP1(XBP1u)into spliced XBP1(XBP1s)by non-canonical splicing in cytoplasm.XBP1 s is a important transcription factor which controls cell differentiation,protein synthesis and secretion,inflammation,lipid metabolism and cancer.Because XBP1 s regulates protein expression,especially the membrane surface protein expression,it is reasonable to believe that XBP1 s may regulate the ultrafiltration of PD by regulating the expression of AQPs molecules.Objective1)To investigate the predominant expression of AQPs in peritoneum;to explore the relationship between AQPs and peritoneal dialysis ultrafiltration.2)To investigate the correlation between XBP1 s and AQPs expression.3)To verify the relationship between XBP1 s and AQPs expression.Methods:1)Peritoneal specimens were taken from patients undergoing peritoneal dialysis catheter insertion,immunohistochemical detection of human peritoneal tissue distribution and expression AQP1,2,3,4,5,7 position in the peritoneal tissue,and then by using Real-time PCR and Western Blotting to detect the m RNA and protein expression levels.2)The RNA and protein of human peritoneal mesothelial cell line(HPMCs)were extracted and the expression of AQP1,2,3,4,5,7 in peritoneal mesothelial cell line was detected by Real-time PCR and Western Blotting.3)The peritoneal dialysis effluent cells of ESRD patients were collected and divided into four groups according to the different amount of ultrafiltration.Western blotting was used to detect the expression of AQP1 in different patients,and to observe the expression of AQP1 and the relationship between patients with ultrafiltration.4)Using lentivirus infection strategies and puromycin screening to get XBP1 s overexpression and kock down HPMCs.The infection efficiency is detected by immunofluorescence,and the XBP1 s m RNA and protein expression is detected by real-time PCR and Western blotting respectively.5)The expression of AQP1 after upregulation or downregulation of XBP1 s was detected by Real-time PCR and Western blotting in XBP1 s overexpression and kock down-stabilized HPMCs cell lines.6)To construct the conditional overexpression and conditional knockout mouse model of XBP1 s,and finally obtain homozygous mice of XBP1flox/flox by breeding.7)We used Cre-AAV intraperitoneal injection to obtain peritoneum XBP1 s knockin and conditional knockout mice.After 28 days of injection of AAV,we performed peritoneal balance test(PET)on mice model to get ultrafiltration related data,and mice peritoneal specimens were taken for subsequent immunohistochemistry,Real-time PCR and Western blotting and other tests.8)The expression of AQP1 and the effect on the ultrafiltration were analyzed by experimental data after knockout and overexpression of XBP1 s in mice model.Results1)The dominant AQPs subtypes of peritoneal tissue is AQP1.We also detected a small amount of AQP3 expression,but does not detect other AQPs subtypes;However,we only detected AQP1 expression in HPMCs.2)Using Western Blot to detect the peritoneal dialysis effluent cells of different ultrafiltration volume,we found that the expression of AQP1 was positively correlated with the amount of ultrafiltration.3)XBP1s overexpression and knockdown stable HPMCs cell line were successfully constructed.4)The expression of AQP1 in XBP1 s knockdown cell line was inhibited.5)The expression of AQP1 in XBP1 s overexpressing cell line was up-regulated.6)The XBP1 s conditional overexpression and conditional knockout mice model were successfully constructed and the homozygous mice were obtained by cultivating and identification.7)Mice PET test results,immunohistochemistry and Western Blot and other results shown that compared with the control,XBP1 s knockout mice peritoneal tissue XBP1 s protein expression was down-regulated,accompanied by inhibition of AQP1 expression,ultrafiltration also decreased,while the expression of XBP1 s protein in XBP1 s overexpression mice was up-regulated and AQP1 was also up-regulated,and the amount of ultrafiltration was increased in the same time.Conclusion1)The predominant expression of AQPs subtype in peritoneal mesothelial cells is AQP1,and AQP1 is closely related to ultrafiltration in PD patient.2)XBP1s controls AQP1 expression in peritoneal mesothelial cells,therefore affects ultrafiltration in PD patient.Thus,XBP1 s is potential molecule target of UFF treatment in the future.