The Roles And Mechanisms Of Hyperglycemia And Endoplasmic Reticulum Stress In Kidney Repair

Background:Diabetes is a metabolic disease characterized by hyperglycemia,which seriously endangers health.Some studies showed that renal ischemia reperfusion(IR)injury induced more severe acute kidney injury(AKI)in diabetic rodents than in non-diabetic rodents.In addition,patients with diabetes had a higher risk of developing AKI than those without diabetes.Complete or maladaptive repair may occur after kidney injury.Tubulointerstitial fibrosis(TIF)is the main characteristic of maladaptive kidney repair.Meanwhile,it is the common and end pathological feature of almost all progressive and chronic kidney diseases(CKD).Endoplasmic reticulum stress(ERS)is a cellular stress state with the endoplasmic reticulum(ER)expansion because of the accumulation of misfolded proteins and/or unfolded proteins.ERS is related to the development of TIF.However,the roles and mechanisms of hyperglycemia and ERS in kidney repair remain largely unknown.Methods:The role and mechanisms of hyperglycemia in kidney repair:1.C57BL/6 male mice were intraperitoneally injected with 50mg/kg streptozotocin(STZ)or citric acid buffer daily for 5 consecutive days.Fasting blood glucose was monitored.2.C57BL/6 male mice were intraperitoneally injected with 50mg/kg STZ or citric acid buffer daily for 5 consecutive days.24 hours(h)post last STZ or citric acid buffer injection,left kidneys underwent 28 minutes of unilateral renal ischemia or sham followed by reperfusion for2 days.The right kidneys were then removed,and the left kidneys and blood samples were harvested 1 day after the right nephrectomy to test kidney injury.3.C57BL/6 male mice were intraperitoneally injected with 50mg/kg STZ or citric acid buffer daily for 5 consecutive days.24 h post last STZ or citric acid buffer injection,left kidneys underwent 28 minutes of unilateral renal ischemia or sham followed by reperfusion for2 weeks.The right kidneys were then removed,and the left kidneys were harvested 1 day after the right nephrectomy to test kidney repair.The role and mechanisms of ERS in kidney repair:In vivo:1.C57BL/6 male mice were intraperitoneally injected with different concentrations of tunicamycin(TM)or dimethyl sulfoxide(DMSO)to observe the survival rate of mice.2.C57BL/6 male mice were intraperitoneally injected with repeated low-dose of TM weekly for 2 weeks.Kidney tissues were collected on day 7 or 14 after drug injection.Changes in ERS,autophagy,fibrosis,and apoptosis were evaluated.In vitro:1.The human proximal tubular epithelial HK-2 cell line(HK-2)was treated with different concentrations of TM or transforming growth factor-β1(TGF-β1)at different times.The changes in ERS,autophagy,fibrosis,and apoptosis were observed.2.The HK-2 cells and the Boston University mouse proximal tubular cell line(BUMPT)were pretreated with ERS inhibitor4-phenylbutyric acid(4-PBA),PERK inhibitor GSK2656157,and autophagy inhibitor chloroquine(CQ)or 3-methyladenine(3-MA)for 1h,and then treated with 100 n M TM or 5 ng/m L TGF-β1 for 24 h to test changes in ERS,autophagy,fibrosis,and apoptosis.Results:1.All mice developed hyperglycemia and diabetes 6 days post the end of the STZ treatment.IR surgery performed 1 day after the end of the STZ injection did not affect the degree of the initial ischemic kidney injury compared to the citric acid buffer group.Hyperglycemia exacerbated TIF,renal inflammation,tubular atrophy,dedifferentiation,chronic kidney injury,interstitial cell proliferation,tubular senescence,peritubular capillary(PTC)rarefaction,the expression of profibrotic cytokines,and ERS during kidney repair after IR surgery.Meanwhile,it reduced tubular cell proliferation during kidney repair after IR surgery.2.In C57BL/6 mice,high dose of TM treatment resulted in the death of mice.Repeated low-dose of TM treatment activated ERS,autophagy,fibrosis,and apoptosis.In proximal tubular epithelial cells,TM and TGF-β1 sequentially induced ERS,autophagy,fibrosis,and apoptosis.Inhibition of autophagy by CQ attenuated TM or TGF-β1induced fibrosis and apoptosis.Inhibition of PERK with GSK2656157 mitigated TM or TGF-β1 induced autophagy,fibrosis,and apoptosis.Inhibition of ERS by 4-PBA alleviated TGF-β1 induced autophagy,fibrosis,and apoptosis.Inhibition of autophagy by CQ or 3-MA enhanced TM or TGF-β1 induced ERS.Conclusion:1.In diabetic and non-diabetic mice,kidney repair is worse in diabetic mice with similar levels of initial ischemic kidney injury.2.In mice and proximal tubular epithelial cells,ERS leads to the activation of autophagy via the PERK-e IF2α pathway.Activated autophagy contributes to renal fibrosis(maladaptive kidney repair)and apoptosis.In addition,autophagy inhibits ERS,which provides a negative feedback mechanism to reduce cellular stress.