Effects And Possible Mechanism Of Augmenter Of Liver Regeneration On Mitochondrial Biogenesis In Renal Ischemia Reperfusion Injury

Objective: Our previous study found that augmenter of liver regeneration(ALR)could attenuate ischemia-reperfusion(IR)renal injury,but the protective mechanism is remains unknown.In this study,human renal tubular epithelial cell(HK-2)with stable down-regulation of ALR was constructed by lentuvirus infection and purine screening.The effects of hypoxia reoxygenation(HR)on ALR expression and mitochondrial biogenesis was observed.The effects of down-regulation ALR on mitochondrial biogenesis and the possible mechanism was investigated in HK-2 cells after treating with HR.Methods: The lentiviral siRNA/ALR that specifically interferes with human 23 kD ALR was transfected into HK-2 cells,and HK-2 cells transfected with lentiviral siRNA/control were used as control group,the expression of 23 KD ALR were detected by real-time quantitative PCR and Western blot;With the treatment of HR combined with sugar-free and serum-free medium,renal ischemia reperfusion(IR)injury was simulatedin HK-2 cells.Experiment was divided into normal,IR,IR+siRNA/ALR,siRNA/Control groups.After the treatment of hypoxia 6 hours and reoxygenation 12 hours,the levels of reactive oxygen species(ROS)and apoptosis were analyzed by flow cytometry.The changes of mitochondria morphology were detected by transmission electron microscope.The expression of mitochondrial biogenesis-related gene such as COX-1,NDUFB 8 and ATP synthase ? were detected by real-time quantitative PCR,and the expression of regulators of mitochondrial biogenesis such as SIRT 1,PGC-1? and NRF-1 were detected by Western blot.Results: Real-time PCR and Western blot analysis showed that the expression of ALR mRNA and protein in the siRNA/ALR group was obviously lower than the siRNA/control group(P<0.05).Compared with the Normal group,the levels of ROS and cell apoptosis increased in the IR group(P<0.05),and morphological abnormalities such as mitochondrial pyknosis,matrix deepening and cristae disorder were observed by transmission electron microscopy,the expression of COX-1,NDUFB 8 and ATP synthase ? mRNA levels decreased significantly(P<0.05),the expression of PGC-1?,SIRT 1,NRF-1 Protein levels increased significantly(P<0.05);Compared with the IR+siRNA/Control group,the levels of ROS and apoptosis were increased(P<0.05),and the mitochondrial morphology injury were aggravated,the expression of COX-1,NDUFB 8,ATP synthase ? mRNA levels significantly decreased(P<0.05),the expression of SIRT 1,PGC-1? and NRF-1 Protein levels decreased(P<0.05).Conclusion: 23 kD ALR participated in the mitochondrial biogenesis of HK-2 cells.Down-regulation of 23 kD ALR in HK-2 cells inhibited mitochondrial biogenesis and aggravated mitochondrial injury,and its possible mechanism was related to inhibition of SIRT1/PGC-1?/NRF1 expression.