XBP1 Modulates Endoplasmic Reticulum And Mitochondria Crosstalk Via Affecting NLRP3 Inflammasome In Renal Ischemia Reperfusion Injury

BackgroundFunctional status of mitochondria and endoplasmic reticulum(ER)lie at the center of matters in renal ischemia reperfusion injury(IRI).X-box binding protein 1(XBP1)is one of important transcription factors in endoplasmic reticulum stress(ERS).Studies have showed that NLR family pyrin domain-containing 3(NLRP3)inflammasome is strongly correlated to renal IRI.It is established that XBP1 may function as the upstream regulator of NLRP3.However,it still remains unknown how regulates and whether influence ER or mitochondria by the potent XBP1 protein in the context of renal IRI.This study aims to investigate the molecular mechanism and function of XBP1-NLRP3 signaling that influenced ER-mitochondria crosstalk in renal ischemia reperfusion injury in vivo and in vitro.MethodsWide type(WT)mice and XBP1+/-mice were subjected to 45 min of unilateral renal warm ischemia,followed by removing the other kidney and 24 h of reperfusion.XBP1 expression was downregulated by transfecting mouse renal tubular epithelial(TCMK-1)cells with small-interfering RNA.TCMK-1 cells were exposed to 24 h hypoxia(5%CO2,1%O2,and 94%N2),and 2 h reoxygenation.Tissue or cell injury was assessed by blood urea nitrogen(BUN)and creatinine(CR),histological staining,flow Cytometry,staining of terminal deoxynucleotidyl transferase-mediated nick end labeling and dihydroethidium,and transmission electron microscope(TEM).Protein expression or location was analyzed using Western blot,immunohistochemistry,immunofluorescence staining,immune electron microscopy and enzyme-linked immuno sorbent assay.Bioinformatics analyses were applied to predict the possibility of XBP1 binding directly to the NLRP3 promoter,which were then confirmed by a luciferase reporter assay.ResultsThe kidney damage of XBP1+/-mice alleviated as the levels of BUN,CR,interleukin-1β and interleukin-18 decreased after IRI compared to WT mice.Meanwhile,protected mitochondria in the kidney of XBP1+/-mice were confirmed by reduction of total ROS,cytochrome c,Caspase-9 and change of mitochondrial morphology under TEM.Disruption of XBP1 markedly raised the survival rate.In the parallel in vitro study,inhibition of XBP1 led to reduction of NLRP3 expression,which was accompanied by reduced Caspase-1,GSDMD and other apoptotic molecules activation.Besides,XBP1 interference suppressed NLRP3-mediated mitochondrial damage and decreased mitochondrial ROS production in TCMK-1 cells,in addition to weakening of the co-localization of ER,mitochondria and NLRP3.The luciferase assays revealed that the spliced XBP1 isoform was likely to bound directly to the NLRP3 promoter.ConclusionDownregulation of XBP1,as one of ERS-specific transcription factors,suppresses expression of NLRP3 colocalizing with mitochondria,which in turn relieves mitochondrial damage,improves renal function and raises the survival rate of mice in renal IRI.This might be one of essential signaling pathways on ER-mitochondria crosstalk in acute kidney injury.Besides,XBP1,binding directly to the NLRP3 promoter,might be a novel regulator and potential therapeutic target for the sterile renal injury.