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Objective:To observe the expression of sirtuin 3 (Sirt3) and mitochondrial damage-associated proteins in lipopolysaccharide (LPS)-induced acute kidney injury mouse model and renal tubular epithelial cells, and to explore the role of Sirt3 in LPS-induced abnormal mitochondrial dynamics in renal tubular epithelial cells.Methods:Eighteen specific pathogen free (SPF) male C57BL/6 mice were randomly assigned to control group, LPS 24 h group and LPS 48 h group. The control group was intraperitoneally injected with physiological saline (0.1 ml/10 g), and LPS 24 h group and LPS 48 h group were intraperitoneally injected with LPS (10 mg/kg) solution. Renal functional indexes of mice were analyzed by automatic biochemical analyzer. The pathological change of the kidney was observed by HE staining, and the expressions of dynamin-related protein-1 (Drp1), optic atrophy type 1 (Opa1) and Sirt3 were evaluated by Western blotting. Expression and distribution of Sirt3 in kidney was assessed by immunohistochemistry. Human renal tubular epithelial cells (HK-2) were exposed to 10 μg/ml LPS for 24 h, and the expression of Drp1, Opa1 and Sirt3 were detected by Western blotting. Cell apoptosis was assessed by Hoechst-33342 staining. After transfection to HK-2 cells with pcDNA3.1-Sirt3 recombinant plasmid, the expressions of Sirt3, Drp1, Opa1 and cell apoptosis were detected by the same methods as above.Results:(1) The levels of blood urea nitrogen and serum creatinine in LPS group were significantly higher than those in control group (both P<0.05), and the pathological changes of kidney were obvious. (2) Compared with the control group, the expression of mitochondrial fission-associated protein Drp1 in renal tissue of LPS group was significantly higher ( P<0.05), and the expression of mitochondrial fusion associated protein Opa1 was significantly lower ( P<0.05). (3) Compared with the control group, the expression of Sirt3 in LPS group was significantly lower ( P<0.05), and immunohistochemistry results showed that Sirt3 was mainly expressed in glomerular vascular endothelial cells and renal tubular epithelial cells. (4) In vitro, LPS stimulation induced increased Drp1 expression in HK-2 cells ( P<0.05), decreased Opa1 and Sirt3 expression (both P<0.05), and increased apoptosis ( P<0.05). (5) LPS-induced mitochondrial dynamics disturbance and apoptosis were alleviated by pcDNA3.1-Sirt3 recombinant plasmid transfection. Conclusions:LPS can induce down-regulation of Sirt3 expression and disturbance of mitochondrial dynamics, and Sirt3 may play a protective role in LPS-induced acute kidney injury by regulating mitochondrial dynamics.
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Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes.Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide,50 mg/L)、rapamycin (Rap,autophagy enhancer,1 ng/L),3-methyladenine (3-MA,autophagy inhibitor,2 mmol/L) for 2 hours.The expression of autophagy protein LC3-Ⅱ and Beclin-1 as well as oxidative stress-related proteins Catalase,MnSOD were detected by Western blot.The formations of autophagy were observed by MDC staining,and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining.Cell activity was evaluated by CCK8 assay.Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media,the expression of LC3-Ⅱ and Beclin-1 were enhanced,Catalase and MnSOD were inhibited (all P ＜ 0.05).Rapamycin increased the expression of Catalase,MnSOD and cell activity of podocytes,reduced the generation of ROS (all P ＜ 0.05),but in Rap group,cell activity showed no significant difference (P ＞ 0.05).3-MA decreased the expression of Catalase 、MnSOD and inhibited the cell activity of podocyte,increased the generation of ROS (all P ＜ 0.05).Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.