The Role And Mechanism Of XBP1 Inhibitor In Renal Ischemia Reperfusion Injury

Backgroud:Ischemia reperfusion injury(IRI)is one of the common causes for delayed function of renal allograft and is associated with poor long-term renal function.Complete and prolonged interruption of renal arterial blood flow occurs during renal transplantation or surgical procedures such as nephrolithotomy,parenchymal sparing surgery for renal tumours,and renal arterial surgeries.Prolonged renal ischemia can lead to Acute Kidney Injury(AKI).A variety of biochemical changes occur during ischemia and reperfusion,accompanied by a series of cellular events,including reactive oxygen species(ROS)release,apoptosis,necrosis,inflammatory cells infiltration and the release of the active medium,which can lead to tissue damage.Since the pathophysiological process of renal IRI is complex and IRI has high morbidity and mortality,Novel treatment strategies are required to lower acute mortality and to prevent chronic kidney failure progression.The endoplasmic reticulum(ER)is the key intracellular organelle responsible for the synthesis and folding of membrane and secretory proteins and Ca2+ reservoir.Unfavorable local environmental abnormalities,such as Ca2+ homeostatic destruction,hypoxia,viral or bacterial infections,can lead to protein folding mutations,which interfere with the normal folding of proteins,result in the accumulation of unfolded proteins in the ER and induce endoplasmic reticulum stress(ERS).The accumulation of unfolded proteins in the ER under conditions of ER stress results in an evolutionarily conserved cellular response referred to as the unfolded protein response(UPR).In mammalian cells,there are three key UPR pathways,activated via protein sensors located in the ER membrane,including activating transcription factor-6(ATF6),inositol-requiring la(IREla),and protein kinase RNA-like ER kinase(PERK).In resting cells,the three sensors are inactive,and are believed to be in association with the ER chaperone BiP.Upon accumulation of misfolded proteins in the ER,BiP dissociates from the sensors,allowing activation;then three major signaling pathways of PERK/Eif2?,IRE1?/XBPls and ATF6/ERSE are generated.Then ATF6 moves to the Golgi,and is cleaved by SI and S2 proteases.The cleaved cytosolic fragment(ATF6n),which has a DNA-binding domain,migrates to the nucleus to activate the transcription of genes encoding ER chaperones,enzymes that promote protein folding,maturation,secretion,ER-associated degradation(ERAD)components,and others.Therefore,the up-regulation of GRP78/BiP expression is a marker protein that confirms the occurrence of UPR.So induction of the UPR serves to maintain ER function,facilitates recovery from stress,and may be protective toadditional 'adaptive'responses.However,sustained/prolonged ER stress may be cytotoxic,for example,lead to apoptosis.The apoptotic pathways include PERK,leading to the induction of C/EBP homologous protein(CHOP),as well as IRE1a kinase/RNase activity.The latter canlead to association with tumor necrosis factor receptor-associated factor 2(TRAF2),activation of apoptosis signal-regulating kinase-1 and c-jun N-terminal kinase(JNK).A lot of research has confirmed that ERS is one of the mechanisms of Acute Kidney Injury(AKI)induced by Ischemia/reperfusion(I/R).I/R injury has been shown to induce ER stress in the kidney,including induction of XBP-1 splicing followed by upregulation of GRP78,predominantly in the proximal tubule epithelium.IRE1/X Binding Protein 1(XBP1)pathway is one of the most important pathway of ERS,which is mainly involved in lipid synthesis,ERAD,protein folding,transport,and secretion.IRE1a remains inactive under normal conditions;when ERS is present,IRE1 is activated by dimerizes and autophosphorylation.IRE1 is an upstream regulator of XBP1 that mediates splicing of pre-XBP1 mRNA.XBP1 has two forms of XBPls and XBPlu.It has been shown that only the spliced form of XBP1(XBPls)can induce the UPR efficiently.Studies have shown that sepsis induces the development of ERS that activates the IRE1-NF-?B pathway and accelerates sepsis through the increase of pro-inflammatory cytokines(TNF-a,IL-1?,IL-6)in proximal tubular epithelial cells,which accelerate the occurrence of AKI.This shows that ERS can activate NF-?B.Nuclear factor-?B(NF-?B)is a key transcriptional regulator of inflammatory genes,including TNF-a,IL-1?and IL-6,etc.Accumulating evidences suggest that the activation of NF-?B can promote the transcription of these pro-inflammatory genes,trigger the inflammation cascade and play an important role in the renal ischemia reperfusion injury(IRI).Recently,several groups have identified small molecule inhibitors that selectively block IRE1?-XBP1 activation.Two major sites on IRE1? have been identified as targets for developing inhibitors:the catalytic core of the RNase domain and the ATP binding site of the kinase domain.Small molecules targeting the RNase domain include salicylaldehydes,4?8C,MKC-3946,STF-083010.A new type of IRE1-XBP1 inhibitor,STF083010,inhibits only IRE1 RNase activity but does not alter the phosphorylation process,thus decreasing XBP1s protein level.STF083010 has the potential function to inhibit the long-term ERS and prevent further cell damage.The study confirmed that STF83010 has significant anti-myeloma effect,reconstructs the sensitivity of breast cancer MCF7-TAMR cells to tamoxifen treatment,and reduces neuronal apoptosis of locus coeruleus in post-traumatic stress rats.Therefore,we applied the XBP1 inhibitor STF083010 to renal IRI,observed its effect on renal IRI,and initially explored its possible mechanism.Objective:To investigate the role and possible mechanism of XBP1 inhibitor STF083010 in renal ischemia reperfusion injury.Methods:Twenty-four Male Sprague Dawley rats weight(150-200)g were randomly divided into sham group,ischemia reperfusion group(Model),STF083010 group,Mock group,6 rats in each group.Sham group:After laparotomy,only bilateral renal arteries were isolated without clipping.Ischemia-reperfusion group:After laparotomy,the bilateral renal arteries were isolated.Then non-invasive vascular clamps simultaneously clip the renal artery for 40 min and remove the non-invasive vascular clamp for reperfusion.STF083010 group:DMSO was used to completely dissolve the STF083010 reagent,diluted with physiological saline to a concentration of 3 mg/ml,and intraperitoneal injection was performed at a dose of 15 mg/kg 1 hour before surgery.Mock group:intraperitoneal injection of 0.5 ml DMSO 1 h before surgery.The time of reperfusion was used as the standard in each group of SD rats.After 24 hours,the rats were sacrificed.Intravenous aortic blood was placed in a coagulation tube with a indwelling needle.The blood was centrifuged at 3000 rpm/min for 20 minutes,and then the serum was stored in a-80? low temperature freezer.Immediately pulse the kidneys with 0.9%sodium chloride injection through the abdomen until the color of the kidneys turns from red to white,remove the two kidneys and treat them at-80 ? in a low-temperature refrigerator,then store them.Part I:To observe the changes of renal morphology and biochemical indicators in each group.HE staining,serum creatinine(Scr)and blood urea nitrogen(BUN)were detected in each group.Part II:To observe the effect of STF083010 on the inflammatory pathway of renal IRI.MPO activity was detected in each group of renal tissues;the level of serum TNF-aand IL-1? was detected by ELISA;XBP1s and NF-?B p65 protein were detected by immunohistochemistry;RT-PCR was used to detect the expression of XBP1s mRNA and XBP1u mRNA in renal tissues;the expression of XBP1s,GRP78,IRE1 and NF-?B p65 protein was detected by Western blot in renal tissues.Part III:To observe the effect of STF083010 on the apoptosis pathway of renal IRI.TUNEL method was used to detect apoptosis in each group of renal tissues;CHOP protein was detected by immunohistochemistry;Western blot was used to detect the expression of CHOP and cleaved Caspase 3 protein in renal tissues.Results:1.This study showed that the levels of Scr(136.48±28.70)umol/l and BUN(38.59±7.79)mmol/l in the Model group were significantly higher than the level of Scr(61.48±11.67)umol/l and BUN(7.88±2.30)mmol/l in the Sham group(p<0.05).In the Model group,the kidney volume increased,the medulla was bruised,the color of the medulla was dark,and the color of the cortex was pale by the naked eyes.HE staining showed slight glomerular damage,mainly renal tubular injury,tubular epithelial cells swelling,watery degeneration,necrosis of varying degrees,more exfoliated cells,tube type;interstitial congestion,edema and inflammatory cell infiltration.The renal tubular necrosis score in the Model group was significantly higher than that in the Sham group(p<0.05).The biochemical indicators and pathological morphology confirmed the successful establishment of the ischemia/reperfusion model.2.Application of STF083010 before modeling of renal I/R,the levels of Scr(78.971±14.349)umol/1 and BUN(17.453±3.724)mmol/1 in STF083010 group were significantly lower than the level of Scr(136.483±28.704)umol/l and BUN(38.588±7.786)mmol/l in the Model group(p<0.05).Compared with the Model group,under the light microscope the STF083010 group showed a significant reduction in tubular epithelial cell swelling,occasionally tubular epithelial cell necrosis and cell shedding,interstitial edema and inflammatory cell infiltration reduced.The renal tubular necrosis score was significantly lower(p<0.05).It demonstrated that STF083010 could alleviate renal function damage and improved kidney tissue morphological damage.3.The immunohistochemical results showed that the NF-?B p65 and XBP1s positive cells in the Model group were mainly distributed in the border region of cortex and medulla,especially in the proximal renal tubules.ELISA results showed that compared with the Sham group,serum TNF-a and IL-1? levels in the Model group were significantly lower(p<0.05).MPO results showed that compared with the Sham group,the MPO activity in the Model group was significantly lower(p<0.05).The results of RT-PCR showed that the XBP1s mRNA level in the Model group was.significantly increased.The ratio of XBP1s/XBP1u mRNA was 8.69,which was 7.69 times higher than that in the Sham group.Western blot results showed that the expression levels of XBP1s,GRP78,pIRE1 and NF-?B p65(in the nucleus)in the Model group were significantly higher than those in the Sham group,and the differences were significant(p<0.05).This indicates that the inflammatory mediators increased after I/R injury,induced ERS,and initiated UPR.At the time of IRI,the expression of NF-?B p65 protein in the cytoplasm was decreased,while the expression of NF-?B p65 protein in the nucleus was increased.NF-?B p65 activation was confirmed.Application of STF083010 before modeling of renal I/R,Compared with the Model group,the number of XBPls and NF-?B p65 positive cells in the STF083010 group was significantly lower(p<0.05).Serum TNF-a and IL-1? levels in the STF083010 group were significantly lower than those in the Model group(p<0.05).The MPO activity in the STF083010 group was significantly lower than that in the Model group(p<0.05).The ratio of XBPls/XBPlu mRNA in STF083010 group was decreased,and the ratio of the two was 2.74.The expression levels of XBP1s,GRP78,pIREl and NF-kB p65(in the nucleus)were significantly decreased(p<0.05).It was confirmed that STF083010 can alleviate the renal I/R induced inflammatory response by inhibiting ERS.4.Compared with the Sham group,the apoptosis index of the Model group was significantly higher than that of STF083010 group.Apoptotic cells mainly concentrated in proximal tubule epithelial cells and distal convoluted tubule epithelial cells.The apoptotic index in the STF083010 group was significantly lower(p<0.05).Therefore,it was confirmed that renal I/R resulted in apoptosis,and STF083010 had the reduction of apoptosis.Compared with the Sham group,Immunohistochemical results showed that the number of CHOP-positive cells in the Model group increased significantly,mainly distributed in the renal tubular epithelial cells in the cortical area.The number of CHOP-positive cells in the STF083010 group were significantly lower(p<0.05).Western blot showed that the expression of CHOP and cleaved Caspase 3 protein in STF083010 group was significantly lower than that in the Model group(p<0.05),which was consistent with the TUNEL results.It is suggested that STF083010 has the effect of improving renal I/R induced apoptosis.Conclusions:1.STF083010 could improve the renal function of ischemia/reperfusion SD rats,reduce renal tubular epithelial cell swelling,interstitial edema and inflammatory cell infiltration;renal tubular necrosis score was significantly reduced.It was confirmed that STF083010 had a protective effect on renal IRI.2.During the time of renal IRI,the levels of XBPls,pIRE1 and GRP78 increased.We speculated that I/R activated ERS and initiated UPR..3,STF083010 can reduce the ratio of XPB1s/XBP1u mRNA level,and decrease the level of pIRE1.It was presumed that it blocked the splicing of XBP1 induced by IRE1,thereby decreased the level of XBP1s,reduced the expression of inflammatory factors NF-?B,TNF-a,IL-1?,Which could exert its anti-inflammatory effect.The protective effect of STF083010 on renal IRI in rat was related to its anti-inflammatory mechanism.4.STF083010 inhibits apoptosis related to apoptotic factor CHOP and cleaved Caspase 3 by blocking the splicing of XBP1 induced by IRE1 and thus exerts its anti apoptosis effect.The protective effect of STF083010 on renal IRI in rats was related to its anti-apoptotic mechanism.