The Expression Of Autophagy Related Proteins In Renal Tubular Epithelium Of Diabetic Nephropathy

Background:Diabetic nephropathy(DN) is a serious complication of diabetes mellitus, and its prevalence has been increasing worldwide. Therefore, there is an urgent need to identify a new therapeutic target to prevent diabetic nephropathy.Recently, utrients in excess and intracellular stress associated with renal hypoxia,mitochondrial reactive oxygen species (ROS), and endoplasmic reticulum (ER) stress has recently been proposed and focused as new pathogenesis of diabetic nephropathy. Among these, Autophagy may play an important role in DN. Autophagy is an adaptive responder of cells to various stress. cell injury or accumulation of damaged organelles/protein aggregates may activate the autophagic pathway to maintain intracellular homeostasis. It has been shown that autophagy is closely related to other complication of diabetes and that autophagy increases in insulin resistance, thus protecting theβcell of pancreas againstcell damage, suggesting that autophagy has been associated with the pathogenesis and progression of diabetic complication. so, we hypothesized that autophagy may play an important role in the pathogenesis of diabetic nephropathy and have the protective potential of autophagy in diabetic nephropathy.LC3is classified as LC3-Iand LC3-Ⅱ.LC3-Ⅱ formation is recognized as a marker of existence of autophagosomes in cell or animal experiments and positively related with the sum of phagophores.we can judge the state of autophage by the change of LC3-II in the cell. The protein p62, also known as sequestosome1(SQSTM1), is known to localize to autophagosomes via LC3interaction and to be constantly degraded by the autophagy-lysosome system. The accumulation of p62is observed in autophagy-deficient cells.In this study, we provide in vivo evidence that the abnormality on ATG expression including p62and LC-3exist in DN, indicating that autophagy dysfunction may induce DN.Objective:1.To observe the expression of protein LC3and P62in the renal tissues of type I diabetes nephrology models.2. To study the effect of different glucose concentrations stimulation on the expression of LC3and P62in hunman proximal tubular cells(HK-2).Methods:1) In vivo experiment:①Establishment of STZ induced type I diabetes nephrology models:Sprague-Dawley rats were randomly divided into control(n=10), diabetic nephropathy (DN)(n=10). The type I DN model was induced by tail intravenous injection of STZ(50mg/kg). The rats in the control group were injected with0.1mol/L sodium citrate solution. Diabetic model was considered to be successful established when the blood glucose was>16.7mmol/L and the urine glucose was+++～++++after three days of the injection. Rats were sacrificed in the the8th week after Diabetic model was successful established and the kidneys were harvested and subjected to the studies.②Renal pathological changes were observed by HE.③The expression and distribution of LC3and P62was tested by immunohistochemistry.④Pearson correlation analysis was applied to detect the correlation between the LC3, P62and proteinuria, creatinine.2) In vitro experiment:Human proximal tubular cells were treated with different concentration of glucose for48hours, the expression of protein LC3and p62was detected by Western Blot analysis.Results:1) In vivo experiment result:①The biochemical test results of STZ induced type I diabetes nephrology models:Blood glucose fluctuated between19.2mmol/1and23.3mmol/l, creatinine fluctuated between72.5umol/1and122.7umol/l, proteinuria fluctuated between4.63g/24h and9.19g/24h.②Renal pathological changes:Compared with the control group, the type I diabetic nephropathy model showed glomerular mesangial expansion,basement membrane (GBM) thickening and degeneration and compensatory expansion of renal tubular cells.③Immunohistochemistry results:immunohistochemistry showed that the expression of p62was significantly up-regulated in the proximal tubular cells of DN model, about2.5times as much as control group, LC3was down-regulated in the proximal tubular cells, about35percent of the control group(P<0.01).④Pearson correlation analysis:LC3is negatively related with proteinuria (P<0.05), creatinine, p62is positively related with proteinuria, creatinine (P<0.01)2) In vitro experiment results:Stimulated by highglucose in HK-2 cells, the expression of p62protein increased, the expression of LC3protein decreased, both of them occurred in a dose-dependent manner. Mannitol had no influence on the expression of p62and LC3which indicated that there was no relationship between Osmotic pressure and Autophagy.Conclusion:(1) Expression of p62increases in the renal tissues of STZ rats, and LC3decreases in STZ rats. Serum creatinine and proteinuria were both positively correlated with P62,but negatively correlated with LC3.(2) High glucose up-regulates the expression of P62but down-regulates the expression of LC3in human proximal tubular cells in a dose-dependent manner.(3)The abnormal expression of autophagy related proteins in STZ induced type I diabetes nephrology models and highglucose induced HK-2indicated decreased autophage in renal tubular epithelium of diabetic nephropathy.