Role Of Autophagy In Pathogenesis Of Diabetic Nephropathy And Intervention Study Of Autophagy Reinforcing Agents

Objective: In recent years，the incidence rate of diabetes, especially type2diabetes,gradually increases. If blood glucose is not effectively controlled,25％～40％type2diabetes will become diabetic nephropathy. Autophagy is a highly regulated intracellularprocess for the degradation of cytoplasmic components, especially protein aggregatesand damaged organelles. It is essential for maintaining healthy cells. Autophagy isbelieved to cause or contribute to acute kidney injury such as renal ischemia reperfusioninjury and aging and age-related diseases. However, little is known about therelationship between autophagy and diabetic nephropathy. To investigate the effects andmechanism of autophagy in diabetic nephropathy, we suppress or enhance autophagicfunction by treating the diabetic rats with rapamycin and metformin.Methods:160～180g male rats were selected to establish type2diabetic nephropathymodel, and the rats were killed after28weeks after diabetic model. To examine theeffects of autophagy on diabetic nephropathy, we also used rapamycin and metformin togavage the diabetic rats. LC3, p62/SQSTM1, polyubiquitin aggregates andmitochondrial autophagy markers such as Parkin, PINK1, Bnip3, Ambra1and theautophagy pathway related factors such as AMPK, Phospho-AMPK, mTOR andPhospho-mTOR were detected by Western blot analysis. Western blot analysis was alsoperformed to assess oxidative damage makers such as nitrotyrosine and4-hydroxynonenal.Results:(1) With the progress of diabetes, diabetic nephropathy gradually occurred.Compared with control group, there is a significantly increased expression of theoxidative damage and mitochondrial autophagy in diabetic rats. We found that the LC3-Ⅱ was markedly decreased, and meanwhile, p62and polyubiquitin aggregates weresignificantly increased. The expression of phospho-mTOR was increased and phospho-AMPK was decreased.(2) We gave the diabetic rats gavage administrationwith rapamycin and metformin. Compared with diabetic group, we found that thediabetic renal injury was alleviated in metformin group. Enhanced mitochondrialautophagy, an increased expression of LC3-Ⅱ and and decreased expression ofp62/SQSTM1, polyubiquitin aggregates were observed in metformin group. The proteinexpression level of phospho-mTOR was decreased and phospho-AMPK was increased.In rapamycin group, the protein expression changes of LC3-Ⅱ, p62/SQSTM1,polyubiquitin aggregates and phospho-mTOR were in the same with the metformingroup. But, the expression of phospho-AMPK was decreased and mitochondrialautophagy was not obvious in rapamycin group.Conclusion: The results suggest that autophagy function decreases in the diabeticnephropathy. The possible pathogenesis is to activate mTOR and inhibit AMPK. Theincrease of oxidative damage resulted in enhanced mitochondrial autophagy to alleviatethe diabetic renal injury. However, the function of mitochondrial autophagy function isnot enough to degrade p62and polyubiquitin aggregate, resulting in the development ofdiabetic nephropathy. Metformin can enhance autophagy and attenuate oxidativedamage to put off the development of diabetic nephropathy. However, the function ofrapamycin was not obvious. These studies may provide a new insight into thepathogenesis of diabetic nephropathy and provide new directions for research.