Effects Of Mfn2 On ER Stress And Apoptosis Of Renal Tubular Epithelial Cells Injured By Hypoxia Reoxygenation

Objective: In this study,we established a hypoxic reoxygenation injury model to simulate ischemia and reperfusion injury in the epithelium of the rat proximal renal tubule cells.to observe the effects of Mfn2 on the stress-coupling structure of the mitochondrial endoplasmic reticulum and the endoplasmic reticulum during reoxygenation under hypoxia.We hope that investigation of its regulatory relationship will lead to new insights for the clinical treatment of renal ischemia reperfusion injury.Methods:(1)In this study,adenoviral plasmids with Mfn2 gene silencing and overexpression were first constructed and transfected into the tail vein of rats to establish ischemia-reperfusion rat models;(2)The renal tubular epithelium treated with hypoxia reoxygenation was divided into normal control group,(H/R)group,H/R+Adv Mfn2 group,and H/R+Adv NC group.The proliferation of NRK-52 E cells was detected by CCK-8 method;Apoptosis was measured by flow cytometry and mitochondrial membrane potential by JC-1;LDH and intracellular ROS,ATP in upregulation were determined by flow assays.Concentrations of intracellular calcium ions were measured by Fluo-3AM flow,intracellular calcium ions were measured by Rhod-AM flow,and intracellular calcium ions were measured by Mag-Fluo-AM flow.Finally,Mfn2 expression levels were measured by Western blot.the activation of the key proteins GRP78,Perk,P-PERK related to endoplasmic reticulum stress,and the mitochondrial calcium channel protein MCU MICU1 VDAC1 IP3 RI GRP75,to verify the effect of Mfn2 on apoptosis of renal tubular epithelial cells in ischemia-reperfusion AKI;Results(1)Compared with the control group,the OD value of H/R group decreased and the level of apoptosis increased significantly;The OD value of H/R+adv-mfn2 group was higher than that of H/R group,and the level of apoptosis was significantly lower than that of H/R group(P<0.05);(2)In terms of cell damage assessment,the ratio of JC-1 monomer in H/R group was significantly higher than that in control group,while that in H/R+adv-mfn2 group was significantly lower than that in H/R group(P<0.05);LDH and ROS in H/R group were higher than those in normal group,while those in H/R+adv-mfn2 group were significantly lower than those in H/R group(P<0.05);ATP value in H/R group was lower than that in normal group,while that in H/R+adv-mfn2 group was significantly higher than that in H/R group(P<0.05);(3)In H/R group,the expression level of Mfn2 in the main endoplasmic reticulum pressure protein was significantly decreased(P<0.05);Compared with H/R+adv-mfn2,the levels of GRP78 and p-PERK were significantly lower than those of H/R group(P<0.05);(4)After injury,the intracellular calcium level in H/R group increased as a whole,the mitochondrial calcium level increased,and the endoplasmic reticulum calcium level decreased(P;the levels of MCU,MICU1,VDAC1,IP3R1 and GRP75 in H/R+adv-mfn2 group decreased significantly(P<0.05).Conclusion:(1)Mfn2 protein expression was decreased in rat renal tubule epithelial cells following H/R injury.and calcium ions in endoplasmic reticulum transfer to mitochondria,leading to calcium overload in mitochondria;ERS-associated protein and mitochondria-associated calcium channel protein MCU,MICU1 VDAC1,IP3R1,GRP75 increased significantly.(2)After overexpression of Mfn2 transfection of H / R-damaged renal tubular epithelial cells,the calcium overload in mitochondria,endoplasmic reticulum stress and mitochondrial calcium channel protein levels were improved,suggesting that Mfn2 may reduce the apoptosis of renal tubular epithelial cells by reducing the calcium overload in mitochondria,Inhibition of ERS reduces apoptosis of renal tubule epithelial cells.