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

Objective: Our previous studies have confirmed that ALR has anti-apoptotic effect in ischemia-reperfusion(IR)renal injury,but its precise mechanism has not been fully elucidated.In this study,human kidney proximal tubular cells(HK-2)stably overexpressing of ALR was constructed by ALR-EGFP-3FLAG lentuvirus infection.The effects of hypoxia reoxygenation(HR)on mitochondrial dynamics-related proteins were observed.The effects of overexpression of ALR on mitochondrial dynamics and the possible mechanism were investigated in HK-2 cells after HR treatment.Methods: The ALR-containing lentiviral particles(Lv-ALR(ALR-EGFP-3FLAG))that specifically overexpress human 23 kD ALR were transfected into HK-2 cells,and HK-2 cells transfected with vector-only lentiviral particles(Lv-vector)were used as Control group,the successfully transduced cells were selected under a fluorescence microscope to observe GFP fluorescence,and the expression level of human 23 kD ALR was examined by western blot analysis using antiflag antibody;With the treatment of HR combined with Hank's balanced salt solution,renal ischemia reperfusion(IR)injury was simulated in HK-2 cells;Experiment was divided into Normal,IR,Lv-vector+IR,Lv-ALR+IR groups.After the treatment of hypoxia 6 hours and reoxygenation 12 hours,the apoptotic rate of HK-2 cells was evaluated with a flow cytometer.The protein–protein interaction was predicted using STRING v10(http://string-db.org/cgi/input.pl).The expression levels of Drp1,p-Drp1,and MTFP1,known as key proteins of mitochondrial fission,were detected by western blot analysis.And western blot analysis was also used to measure the related signaling pathway proteins mTOR,p-mTOR,4E-BP1,and p-4E-BP1.Furthermore,we tested the levels of the key mediators of mitochondrial fusion through Western blot,these include OPA1?Mfn1?and Mfn2.Results: After transduction,the successfully transduced cells were selected under a fluorescence microscope to observe GFP fluorescence,and cells with an infection efficiency of about 80% were included in the subsequent experiments.Compared to the Lv-vector group,Western blot showed that 23 kD ALR was expressed efficiently in Lv-ALR group(P<0.05).Additionally,the expression of 23 kD ALR after in vitro IR injury was also detected;The results showed that Drp1 expression in HK-2 cells was gradually upregulated at 6,12 and 24 hours after H/R treatment,peaking at 12 h,and the level was still maintained at a high level at 24h;We observed that ALR overexpression suppressed H/R-induced HK-2 cells apoptosis.Meanwhile,Drp1 and MTFP1 were both down-regulated in the Lv-ALR+IR group,as compared with the Lv-vector+IR group(P<0.05).And ALR overexpression also has an impact on phosphorylation of Drp1 Ser637 during AKI.Furthermore,the related signaling pathway proteins p-mTOR/mTOR and p-4E-BP1/4E-BP1 were more active in the Lv-ALR+IR group than in the Lv-vector+IR group(P<0.05).OPA1 was significantly increased(P<0.05),whereas Mfn1 and Mfn2 levels were not changed in the Lv-ALR+IR group(P>0.05),as compared with the Lv-vector+IR group.Conclusion: Overexpression of human 23 kD ALR alleviates renal I/R injury,probably by inhibiting mitochondrial fission and promoting fusion of mitochondrial inner membrane,both of which help to preserve mitochondrial functionality and reduce apoptosis.And fission processes potentially associate with activating the mTOR/4E-BP1 signaling pathway.