The Roles Of XBP1s In Inflammation-induced Peritoneal Fibrosis

Peritoneal dialysis is one of major therapeutic option for the treatment of ESRD andis based on the use of the peritoneum as a semipermeable membrane across whichultrafiltration and diffusion take place. Continuous exposure to nonphysiologic PDsolutions and episodes of peritonitis or hemoperitoneum cause inflammation and injury tothe peritoneal membrane, which undergoes fibrosis, angiogenesis, and hyalinizingvasculopathy. These morphologic alterations seem to be associated with increasedsmall-solute transport rate and with ultrafiltration dysfunction of the PM.Extracellular matrix and pro-inflammatory cytokines have been considered the mainentities responsible of the structural and functional alterations of the peritoneum. Morerecently, however, it has been shown that mesothelial cells may also play an active role inPM alteration. It has been demonstrated that during PD-induced inflammatory and repairresponses, The inflammatory response of HPMCs is a complex process that ischaracterized by the increased expression of the IL-1β，IL-6, which they contribute toinflammatory responses, fibrosis, and angiogenesis that ultimately lead to PM failure.A variety of mechanisms and linkages between transcription factors XBP1s and inflammation pathways, including the NF-κB, MAPK, ROS, etc. During Endoplasmicreticulum stress and inflammatory response, XBP1s，a key molecules, can promoteinflammatory response. XBP1s mediated by TLR-related proinflammatory cytokines IL-6,IL-8and TNF-α secretion in macrophages.To explore the role of XBP1s in the formation of inflammation-induced peritonealfibrosis in vitro and in vivo, which lays a foundation of further study on prevention andtreatment of peritoneal fibrosis induced by peritoneal dialysis.Objective:1) To explore the contacts role of XBP1s and inflammatory response of HPMC；2) To investigate the effect of XBP1s inhibitor on the formation of peritoneal fibrosis byCG-induced in rats.Methods:1) Human mesothelial cells were obtained from dialysis fluid taken randomly fromclinically stable patients who had a variety of treatment duration undergoing CAPD,the expression of XBP1s in MCs was detected with Western blotting assay, andanalysis of XBP1s and IL-1β by Real-time PCR in mesothelial cells from effluentwere obtained from53patients;2) HPMC were exposed to different IL-1β concentration, XBP1s and XBP1t weredetected by Real-time PCR, the ratio of XBP1s vs. total XBP1mRNA levels was aslodetermined in order to make sure of the appropriate concentration of IL-1β;3) Western blotting or real-time PCR demonstrating the effects of IL-1β for differentduration on XBP1s protein levels in HPMC; the expression of IL-1β mRNA wasdetermined by Real-time PCR, IL-6was detected by ELISA; Fibronectin wereanalysised by Western blotting;4) HPMC were treated with XBP1s inhibitor under IL-1β cultured, the level of XBP1s orFibronectin were determined by Western blotting, and the expression of IL-1β mRNA was detected by Real-time PCR, IL-6was analysised by ELISA;5) HPMC were treated with XBP1s inhibitor under IL-1β cultured, the expression ofJNK or P-JNK were analysised by Western blotting;6) The rats were randomly divided into three groups, the model group were infused CGdissolved in10%ethanol and saline every other day by intraperitoneal injection, thetreatment group were injected with the XBP1s inhibitor2hours before injection of CG,the rats in the control group were given a injection of saline and DMSO in a similarmanner. All rats were euthanized at the end of2weeks. The parietal peritoneumtissues of rats were harvested;7) The parietal peritoneum tissues of rats were stained with H-E or masson forobservation of the peritoneal structure; the expression of XBP1s was detected byWestern blotting; and the expression of XBP1, F4/80, α-SMA or CD31in parietalperitoneum was detected with immunohistochemistry assay.Results:1) Western blotting demonstrating XBP1s protein levels in MCs from patientsundergoing CAPD; the relative levels of XBP1s presented a correlation with PDduration, and the expression of XBP1s mRNA aslo presented a correlation with IL-1β;these results demonstrate that XBP1s signaling is activated particularly in PD patientswho had a long treatment duration undergoing CAPD;2) The levels of XBP1s, total XBP1mRNA, and mRNA splicing of total XBP1wereincreased in the HPMC exposed to different IL-1β concentration;3) The expression of XBP1s, IL-1β, IL-6and Fibronectin were upregulated in the HPMCexposed to different treatment duration induced by IL-1β;4) XBP1s was suppressed, and the expression of IL-1β, IL-6, Fibronectin were decreasedin HPMC treated with XBP1s inhibitor under IL-1β exposed;5) The phosphorylation activation of JNK were also downregulated in HPMC treatedXBP1s inhibitor under IL-1β cultured, the expression of JNK have no significant changes;6) The expression of XBP1s protein was increased in the parietal peritoneum tissues ofmodel group comparing with the control group; XBP1s inhibitor suppressed the highexpression of XBP1s in the treatment group;7) Compared with the control group, parietal peritoneum tissues were more thicker,matrix accumulation increased Significantly in the rats of model group; the results ofImmunohistochemistry showed that the level of XBP1, F4/80, α-SMA or CD31wereupregulated. Compared with the model group, administration of XBP1s inhibitorimproved the function and structure changes of the peritoneum in the treatment group;Conclusion:1) XBP1s mediated inflammatory response in IL-1β-induced HPMC, and possibly byactiving JNK signaling；2) XBP1s inhibitor plays a partial prevention role on peritoneal fibrosis in CG-inducedrats.