Study On The Mechanism Of CLS Down-regulated Expression By High Glucose Inhibits CL-mediated Mitophagy In Renal Tubular Epithelial Cells And The Protective Effect Of SS-31

Objectives: Diabetic nephropathy(DN)is the main cause of disability and death in diabetic patients,and it is also one of the main causes of end-stage renal disease.The role of mitophagy disorders in the pathogenesis of DN has become a hot issue in research today.Our team has found that the expression level of cardiolipin(CL)in renal tubular epithelial cells was significantly decreased under high glucose culture.Combined with previous studies,in mitochondria,CL can cross the outer membrane and bind to microtubule-associated protein-1 light chain 3(LC3)to enhance mitophagy.In this study,we aim to investigate in renal tubular epithelial cells whether the alteration of CL synthesis pathway proteins under the influence of high glucose affects mitophagy by altering CL expression level,and to provide new ideas for the elucidation of the pathogenesis of DN.At the same time,we aim to investigate whether SS-31,a mitochondria-targeted antioxidant,protects the structure and function of mitochondria in renal tubular epithelial cells under the effect of high glucose by activating mitophagy,and provide a theoretical basis for finding new therapeutic drugs for DN.Methods: 1.HK-2 cells were given high glucose stimulation(30 mmol/L glucose concentration),and we used Western blot and RT-PCR to measure the effects of high glucose on the key enzymes of cardiolipin synthesis pathway,including cytidine diphosphate diacylglycerol synthase(CDS),phosphatidylglycerol synthase(PGS),cardiolipin synthase(CLS)and tafazzin(TAZ).2.HK-2 cells were transfected with CLS expression plasmid and CLS-si RNA,and the effects of overexpression and knockdown were verified by Western blot and RT-PCR.3.ELISA was applied to detect the changes of CL expression in each group with CLS changes and the SS-31 treated group.4.The mitochondria of cells in normal glucose group(NG),high glucose group(HG),CLS expression plasmid group,CLS-si RNA group,and SS-31 treatment group were isolated by differential centrifugation,and the mitochondrial proteins were extracted.Western blot analysis was used to measure the expression levels of mitochondrial autophagy-related proteins LC3II/I and p62 in each group.5.The morphological changes were examined using transmission electron microscopy.6.JC-1 assay was used to measure mitochondrial membrane potential,and ATP concentration was measured by luciferase assay to observe mitochondrial function.Results: 1.The protein expression level of CLS decreased significantly in HG group,while CDS,PGS,and TAZ showed no statistical differences between HG and NG groups;the m RNA expression levels of PGS,CLS,and TAZ decreased significantly in HG group,while the m RNA expression level of CDS was not significantly different between the two groups.2.The overexpression and knockdown effects of CLS were successfully verified,and the knockdown effect of si RNA3 was the most significant.3.Under the condition of high glucose and low expression of CLS,the synthesis of CL was inhibited and the expression level of CL in HK-2 cells was reduced.4.The expression of mitochondrial autophagy-related proteins LC3II/I was significantly decreased in HG group,and the CLS overexpression group and SS-31 treatment group were significantly increased compared with HG group and CLS knock-down group;there was a significant increase in p62 expression in the HG group,and meanwhile there was a significant decrease in p62 expression in the CLS overexpression group.5.As shown by transmission electron microscopy,mitochondrial swelling,fragmentation and disappearance of cristae appeared in the HG group.In HK-2 cells treated with CLS overexpression and SS-31,mitochondrial morphology remained largely intact and mitochondrial autophagosomes were visible.6.Compared with NG group,mitochondrial membrane potential and ATP concentration were significantly decreased in HG group,while mitochondrial membrane potential and ATP concentration in both CLS overexpression group and SS-31 treatment group were higher than those in HG group and CLS knockdown group.Conclusions: 1.The expression of CLS,the key enzyme in cardiolipin synthesis pathway,decreases in renal tubular epithelial cells under high glucose stimulation.2.Downregulation of CLS resulted in decreased CL synthesis in renal tubular epithelial cells,which in turn inhibited mitophagy,resulting in functional and structural abnormalities of the mitochondria,which could be reversed by overexpression of CLS.3.CL-targeted conjugate SS-31 can improve the abnormal structure and function of mitochondria when renal tubular epithelial cells are exposed to high glucose levels by activating mitophagy.