Down-regulation Of XBP1 Protects Against Renal Ischemia-reperfusion Injury Via Reducing Ubiquitylation And Degradation Of NRF2 Mediated By HRD1

PartⅠ: Renal ischemia-reperfusion injury and hypoxia/reoxygenation of renal tubular epithelial cells induce endoplasmic reticulum stress associated with the XBP1 pathwayObjective: To explore the relationship between the damage caused by renal ischemia-reperfusion(IR)and hypoxia/reoxygenation(H/R)and endoplasmic reticulum stress(ERS)pathways.Methods: 6-8w male C57BL/6 mice were divided into 2 groups(9 mice for each group):(1)sham group;(2)Renal ischemia-reperfusion injury group(IRI group): the left kidney was clamped with a microvascular clamp for 45 minutes,and the right kidney was removed.6 mice in each group were observed for survival rate within 14 days after surgery.The blood and left kidney tissue of 3 mice in each group were collected 24 hours after surgery.After centrifugation,serum creatinine(CR)and neutrophil gelatinase-associated lipocalin(Ngal)concentration were measured.The renal tissue was stained with Hematoxylin and Eosin(H&E)and DHE staining.The expression of P53/ATM in kidney cells was detected by immunohistochemistry.Renal cell apoptosis was observed by TUNEL immunohistochemistry.The endothelium ultrastructural changes of renal tubular epithelial cells were observed by electron microscopy.Bi P and CHOP were detected by immunofluorescence staining.Unfolded protein response(UPR)-related protein were measured by Western blot analysis.The mouse renal tubular epithelial cell line(TCMK1) was divided into two groups:(1)normal control group(normal control group): cells receiving no treatment;(2)hypoxia/reoxygenation(H/R)group: cells undergoing H/R.The TCMK1 cells proliferation was detected by CCK8.The production of ROS and apoptosis proporation were detected by flow cytometry.Bi P and CHOP were stained by immunofluorescent.Results: All mice in the IRI group died within 3 days,and the survival rate of the sham group was 100% at 14 days after IRI.the blood CR and Ngal level of IRI group were significantly higher than sham group.Meanwhile,the pathological damage of the kidney,the production of ROS,the expression of P53/ATM and the apoptosis proporation in the IRI group were also significantly increased.The endoplasmic reticulum of renal tubular epithelial cells in sham group showed no degenerative changes such as swelling,deformation and degranulation.In contrast,the endoplasmic reticulum of renal tubular epithelial cells in IRI group was significantly swollen,deformed and degranulated.The fluorescence density of Bi P/CHOP and UPR-related proteins relative quantity in IRI group were much larger than sham group,and XBP1 expression was prominently increased.IRI significantly prompted ROS production in the IRI group.Compared with the normal control group,the cells proliferation in H/R group decreased significantly with the prolongation of hypoxia,and the ROS production and apoptosis proporation were also increased significantly in H/R group.Conclusion: IRI and H/R can cause severe damage in vivo and in vitro,increase ROS and apoptosis,destroy the body function and down-regulate the survival rate.Besides,IRI induces strong ERS and significantly increase XBP1 expression,These data suggest that XBP1 may play a key role in renal IRI.PartⅡ Effect of regulation of XBP1 on renal ischemia-reperfusion injury and renal tubular epithelial cells hypoxia/reoxygenationAims: IRI is closely related to ERS and oxidative stress.We have found that XBP1 may play a role in the process of renal IRI and TCMK1 H/R.Hence,this study investigated the effect of regulating XBP1 on IRI kidney and H/R treatment of TCMK1 cells.Methods: TCMK1 cells were grouped according to the ways of XBP1 regulation:(1)normal control group(normal control group): cells receiving no treatment;(2)empty lentivirus control group(lenti-control group): cells were transduced with empty lentiviruses;(3)XBP1 overexpression lentivirus group(lenti-XBP1 group): t cells were transduced with lenti-XBP1;(4)XBP1 expression interfered with lentivirus group(lenti-sh RNA-XBP1 group): cells were transduced with lenti-sh RNA-XBP1.After transduction of the corresponding viruses,the expression of XBP1 m RNA and protein was detected by RT-PCR and Western blot.After H/R treatment of TCMK1 cells,TCMK1 cells were grouped according to the ways of XBP1 regulation:(1)H/R control group;(2)empty lentivirus H/R group(lenti-control H/R group): cells transduced with empty lentiviruses undergoing H/R;(3)XBP1 overexpression lentivirus H/R group(lenti-XBP1 H/R group): t cells transduced with lenti-XBP1 undergoing H/R;(4)XBP1 expression interference Lentiviral H/R group(lenti-sh RNA-XBP1 H/R group): cells transduced with lenti-sh RNA-XBP1 undergoing H/R.The cells proliferation of each group was detected by CCK8 assay,and the ROS and apoptotic proporation of each group were detected by flow cytometry.6-8w male C57BL/6 mice were divided into 5 groups according to the regulation of XBP1:(1)WT group(n=9);(2)XBP1+/-group(n=9): XBP1+/-mice receiving IRI.6 mice in each group were observed for survival rate within 14 days after surgery.The blood and left kidney tissue of 3 mice in each group were collected 24 hours after surgery.After centrifugation,serum creatinine(CR)and neutrophil gelatinase-associated lipocalin(Ngal)concentration were measured.The renal tissue was stained with Hematoxylin and Eosin(H&E)and DHE staining.Renal damage apoptosis were observed by TUNEL.Results: Compared with the normal control group,the expression of XBP1 mRNA and protein was not significantly different between the lenti-control group and normal control group.The XBP1 m RNA and protein relative quantity in lenti-XBP1 group was significantly increased,while the lenti-sh RNA-XBP1 group was significantly decreased.The proliferation of TCMK1 cells in lenti-XBP1 H/R group decreased,and the increasement of ROS production and apoptosis proporation was more obvious than that in H/R control group.The cells in the lenti-sh RNA-XBP1 H/R group significantly had higher proliferation,less ROS and lower apoptosis rates than those in the H/R control group.The 14-day survival rate of the AAV9-sh RNA-XBP1 group reached 33.33%,while the 14-day survival rate of the XBP1+/-group reached 66.66%.The rise of CR and Ngal was significantly inhibited in the XBP1+/-group.Renal pathological damage,ROS production,cells apoptosis proporation were also significantly lower in the XBP1+/-group than in the WT group.Conclusion: After IRI or H/R,up-regulation of XBP1 aggravated tissue cell damage,promoted oxidative stress,decreased renal function,and increased apoptosis significantly.In contrast,down-regulation of XBP1 reduced damage,increased survival,inhibited oxidative stress,and protected kidneys and TCMK1 cells against IRI and H/R treatment.PartⅢ Down-regulation of XBP1 protects against renal ischemia-reperfusion injury via reducing ubiquitylation and degradation of NRF2 mediated by HRD1Aims: XBP1 and HRD1 are key ER stress signaling molecules.NRF2 is a major transcription factor that is resistant to oxidative stress.We found that down-regulation of XBP1-HRD1 and upregulation of NRF2 are consistent in reducing apoptosis,maintaining proliferation,and reducing ROS after H/R treatment.Therefore,this experiment investigated the anti-IRI effect and potential mechanism of regulating XBP1,HRD1 and NRF2.Methods: TCMK1 cells were grouped according to the direction of XBP1 regulation:(1)H/R control group;(2)empty lentivirus H/R group(lenti-control H/R group): cells transduced with empty lentiviruses undergoing H/R;(3)XBP1 overexpression lentivirus H/R group(lenti-XBP1 H/R group): t cells transduced with lenti-XBP1 undergoing H/R;(4)XBP1 expression interference Lentiviral H/R group(lenti-sh RNA-XBP1 H/R group): cells transduced with lenti-sh RNA-XBP1 undergoing H/R.TCMK1 cells were grouped according to the direction of HRD1 regulation:(1)H/R control group;(2)negative si RNA H/R group(si RNA control H/R group): cells transfected negative si RNA undergoing H/R;(3)negative plasmid H/R group(plasmid control H/R group): cells transfected negative plasmid undergoing H/R;(4)HRD1 expression interfered with H/R group(si RNA-HRD1 H/R group): cells transfected si RNA-HRD1 undergoing H/R;(5)HRD1 overexpression H/R group(plasmid-HRD1 H/R group): cells transfected plasmid-HRD1 undergoing H/R.TCMK1 cells were grouped according to the orientation of NRF2:(1)H/R control group;(2)DMSO H/R group(DMSO H/R group): cells incubated with 0.01% DMSO undergoing H/R;(3)NRF2 expression inhibition H/R group(ML385 H/R group): cells supplemented with NRF2 inhibitor(ML385)undergoing H/R;(4)NRF2 overexpression H/R group(CDDO-Me H/R group): cells supplemented with NRF2 agonist(CDDO-Me)undergoing H/R.The kidney tissues of the IRI group,the AAV9-vector control group,the XBP1+/-group were collected as described above.The m RNA and protein expression changes of XBP1,HRD1,NRF2 and HO-1 in each group were detected by RT-PCR and Western blot.The colocalization of HRD1 and NRF2 molecules after H/R treatment was detected by immunofluorescence staining and laser confocal.HEK-293 T cells were co-immunoprecipitated to study the interaction of HRD1 and NRF2 molecules after H/R treatment.Finally,bioinformatics,co-immunoprecipitatin and ubiquitylation assays were performed using HEK-293 T cells to determine the interaction mechanism between HRD1 and NRF2.Results: XBP1 positively regulated HRD1 expression.HRD1 positively regulates NRF2 expression at the transcriptional level and reversely regulates NRF2 expression at the post-transcriptional level.The binding of HRD1 and NRF2 to each other were detected by the immunological fluorescence,laser confocal and immunoprecipitation after H/R.Ubiquitylation assays showed that XBP1-HRD1 promoted ubiquitylation of NRF2 and thus accelerated its degradation via QSLVPDI amino acid sequence of NRF2.Conclusion: XBP1-HRD1-NRF2 shows a complete signaling pathway during IRI or H/R.Down-regulation of XBP1 can reduce the ubiquitylation degradation of NRF2 by reducing the expression of HRD1,thereby protecting the IR kidney and providing a new target for the prevention and treatment for renal IRI.