Humanin Improves Mitochondrial Function And Alleviates Renal Tubular Injury In LPS-induced AKI

BackgroundSepsis-associated acute kidney injury is the most common type of AKI in the intensive care unit with high clinical morbidity and mortality.The pathogenesis of SA-AKI is different from AKI caused by ischemia-reperfusion factors,has not been fully elucidated so far.Previous studies have shown that mitochondrial damage in renal tubular epithelial cells is an important mechanism leading to SA-AKI.Intervention of mitochondrial damage and protection of the mitochondria may be effective means to treat SA-AKI.Humanin,a mitochondrial polypeptide containing 24 amino acids,is an endogenous mitochondrial protective protein and was discovered in the brain tissue of patients with Alzheimer’s disease in 2001.Humanin is abundantly expressed in normal kidney of mice and humans.Studies have shown that Humanin has mitochondrial protective effect in ischemia-reperfusion injury and diseases such as the heart and brain.Its mechanism may be related to anti-apoptosis,anti-inflammatory,autophagy regulation and maintenance of mitochondrial homeostasis.Whether Humanin plays a protective role in AKI has not yet been reported.Peroxisome proliferator-activated receptorγ-coactivator1-α（PGC1-α）is a key factor regulating mitochondrial homeostasis.It can promote mitochondrial biogenesis and protect it by regulating the transcription and expression of mitochondrial transcription factor A（TFAM）.Studies have confirmed that Humanin can upregulate AMPK/PGC1-αsignaling pathway in pancreaticβcells,alleviate mitochondrial damage,and play a protective role in diabetes and insulin resistance.This study aims to explore whether Humanin can protect renal tubular epithelial cells by improving mitochondrial function in the animal and cell models of SA-AKI,providing a new theoretical basis for the prevention and treatment of SA-AKI.Objective（1）Clarify the protective effect of Humanin on renal tubular epithelial cells during SA-AKI.（2）To explore the possible mechanism of Humanin’s protective effect on renal tubular epithelial cells during SA-AKI.Methods（1）In vivo:LPS（10mg/kg）was given to 6～8 weeks C57BL/6 mice intraperitoneally to induce sepsis-associated acute kidney injury,and Humanin（1mg/kg）was injected 15 minutes later.Mice were euthanized at 24 hours after LPS injection.Serum creatinine and blood urea nitrogen used evaluate the renal function.Renal inflammation（IL-1β,IL-6 and HMGB1）was evaluated by real-time PCR.The expression of PGC1-αand TFAM,which can promote mitochondrial biogenesis,was measured by real-time PCR,western blot and immunohistochemistry.Detection of changes in the expression of the mitochondrial outer membrane proteins mitofusin1（MFN1）and mitofusin2（MFN2）,which are related molecules that promote mitochondrial fusion.The mitochondrial damage of renal tubular cells and autophagy were observed under electron microscope,and the expression changes of autophagy-related proteins LC3-II and p62 were detected by Western Blot.（2）In vitro:Cultured human proximal tubular epithelial cell line（HK-2）was stimulated by LPS（160μg/ml）to induce endotoxemia renal tubular epithelial cell injury model 15minutes after LPS stimulation,add 5μmol/L Humanin solution.MTT was used to detect cell viability;mitochondrial membrane potential was detected by JC-1;Mitotracker was used to mark mitochondrial morphology;mitochondrial superoxide content was detected by Mito SOX^TM staining.The protein expression level of PGC1-α,TFAM,MFN1,MFN2,LC3-II and p62 was measured by Western Blot analysis.Results（1）In vivo:Compared with the control group,the serum creatinine and urea nitrogen were significantly increased in the LPS treatment group.Humanin significantly reduced serum levels of Scr and BUN.Humanin also attenuated serum levels of kidney injury molecule 1,a biomarker of proximal tubular injury.The PAS staining showed that compared with the control group,necrosis and apoptosis were not obvious and just mild vacuolar degeneration was observed in the proximal renal tubular epithelial cells in LPS-induced AKI mice.There is no significantly difference between the LPS+HN group and the control group.Immunofluorescence staining revealed that Humanin was down-regulated in renal tubular epithelial cells of mice in the LPS group,and the expression of mitochondrial biogenesis-related proteins PGC1-α,TFAM,mitochondrial fusion proteins MFN1 and MFN2was down-regulated.Humanin injection could restore their expression.Under the electron microscope,we observed that compared with the control group and Humanin group,the mitochondria in the renal tubular epithelial cells of the LPS group were significantly damaged,the number of mitochondria was reduced,the arrangement was disordered,and they were obviously swollen.In some renal tubular epithelial cells,mitochondrial cristae were broken.The autophagic vesicles in renal tubular epithelial cells increased significantly,and the autophagy-related protein LC3-II and autophagy substrate p62 were significantly increased.Intraperitoneal injection of Humanin can significantly reduce the accumulation of autophagic vesicles in renal tubular epithelial cells,and the expression of LC3-II and p62 protein is obvious downregulated.（2）In vitro:Compared with the control group,HK-2 cell viability was decreased,mitochondria membrane potential decreased,mitochondria become fragmented,and mitochondrial superoxide accumulated in LPS stimulated HK-2 cells.The expressions of PGC1-α,TFAM,MFN1 and MFN2 were down-regulated,and the autophagy-related protein LC3-II and autophagy substrate p62 were significantly increased.Co-culture of Humanin and HK-2 cells stimulated by LPS can increase the activity of mitochondrial in HK-2 cells,reduce mitochondrial fragmentation,and improve oxidative respiratory damage.Humanin can also restore the normal expression of PGC1-α,TFAM,MFN1,MFN2,LC3-II,and p62.ConclusionThrough animal experiments and in vitro cell experiments,our data have shown a protective effect of Humanin on the kidney in sepsis-associated acute kidney injury.The related mechanism is further studied,and it is found that Humanin can improve the autophagy flow of renal tubular epithelial cells and promote the biogenesis and fusion of mitochondria,reduce the damage of mitochondria in renal tubular epithelial cells during sepsis-associated acute kidney injury,providing new theoretical foundation for the prevention and treatment of sepsis-associated acute kidney injury.