| 【Objective】Urinary protein is an important driving factor for accelerating the progression of chronic kidney disease.Albumin is the major component of urinary protein and can induce renal tubular epithelial cells damage through a variety of mechanisms.A large number of studies have shown that mitochondrial damage and dysfunction are one of the important reasons for albumin-induced renal tubular epithelial cell damage.Mitophagy can selectively remove dysfunctional or damaged mitochondria and plays an important role in maintaining mitochondrial homeostasis.Our previous studies have confirmed that albumin overload enhances mitophagy in renal proximal tubular epithelial cells(TECs)via the PINK1/Parkin pathway.However,the specific role of mitophagy in the albumin overload model remains unclear.Therefore,in this study,we used Parkin siRNA and Parkin overexpression plasmid transfection to regulate mitophagy in albumin overload model,and then detect the changes in mitochondrial function,oxidative stress and cell damage in TECs.Further explore the role and mechanism of PINK1/Parkin pathway-mediated mitophagy in proximal renal proximal tubular epithelial cell injury induced by albumin overload.【Methods】(1)Human renal proximal tubular epithelial cell line(HK-2).(2)Urinary albumin overload model: HK-2 cells cultured in vitro were treated with human serum albumin(8 mg/ml)for 8h.(3)siRNA transfection and grouping: Parkin siRNA transfection was used to intervene mitophagy.We established control group,ALB group,scrambled siRNA+ALB group,Parkin siRNA+ ALB group.(4)Overexpression plasmid transfection and grouping: Parkin overexpression plasmid transfection was used to intervene mitophagy.We established control group,ALB group,empty Vector+ALB group,Parkin + ALB group.(5)Detection index: the changes of mitophagy in each group were detected by fluorescent probe double staining.The expression of Parkin,Pink1,LC3,p62,cleaved-caspase 3 and cytoplasmic cytochrome-c in HK-2 cells were detected by immunoblotting.TUNEL assay was applied to measure the apoptosis rate of HK-2 cells.CCK-8 was used to assay viability of HK-2 cells.Mitochondrial membrane potential was tested by JC-1 kit staining.ROS levels were examined by DCF immunofluorescence.Mitochondrial ROS were examined by MitoSOXTM Red fluorescent staining technique.【Results】(1)Changes of mitophagy in albumin overload model after Parkin siRNA transfection or Parkin overexpression plasmid transfection: double staining with fluorescent probe showed that compared with the control group,the mitochondrial autophagy positive points of ALB group,scrambled siRNA+ALB group and empty vector+ALB group increased significantly.The mitochondrial autophagy positive points in Parkin siRNA+ALB group were significantly decreased compared with scrambled siRNA+ALB group;the mitochondrial autophagy positive points in Parkin+ALB group further increased than that in the empty vector+ALB group.Western blotting showed that the levels of Parkin,PINK1 and LC3-II in ALB group,scrambled siRNA+ALB group and empty vector+ALB group were higher than that in the control group(P<0.05),and the expression of p62 was lower than that in the control group(P<0.05).The expressions of Parkin,PINK1 and LC3-II in Parkin siRNA+ALB group were lower than that in scrambled siRNA+ALB group(P<0.05),and the expression of p62 was significantly higher than that in scrambled siRNA+ALB group(P<0.05).The expressions of Parkin,PINK1 and LC3-II in Parkin+ALB group were higher than that in empty vector+ALB group(P<0.05),and the expression of p62 was significantly lower than that in empty vector+ALB group(P<0.05).(2)The effect of inhibited or enhanced mitophagy on mitochondrial function in albumin overload model: JC-1 staining showed that the mitochondrial membrane potential in ALB group,scrambled siRNA+ALB group and empty vector+ALB group decreased significantly compared with the control group.The mitochondrial membrane potential in Parkin siRNA+ALB group decreased significantly compared with scrambled siRNA(P<0.05).The mitochondrial membrane potential in Parkin+ALB group increased significantly compared with empty vector+ALB group(P<0.05).Western blotting showed that the levels of cytoplasmic cytochrome-c in ALB group,scrambled siRNA+ALB group and empty vector+ALB group were higher than that in control group(P<0.05).The levels of cytoplasmic cytochrome-c in Parkin siRNA+ALB group were higher than that in scrambled siRNA+ALB group(P<0.05).The levels of cytoplasmic cytochrome-c in Parkin+ALB group were lower than that in empty vector+ALB group(P<0.05).(3)The effect of inhibited or enhanced mitophagy on oxidative stress in the albumin overload model: the results of DCF and MitoSOXTM fluorescence staining showed that the expression of ROS in ALB group,scrambled siRNA+ALB group and empty vector+ALB group was higher than that in the control group(P<0.05).The expression of ROS in Parkin siRNA+ALB group was higher than that in scrambled siRNA+ALB group(P<0.05).The expression of ROS in Parkin+ALB group was lower than that in empty vector+ALB group(P<0.05).(4)The effect of inhibited or enhanced mitophagy on HK-2 cell injury in the albumin overload model: The results of TUNEL,Western blot and CCK-8 showed that compared with the control group,the apoptosis rate and the expression of cleaved-caspase 3 protein of ALB group,scrambled siRNA+ALB group and empty vector+ALB group were significantly increased(P<0.05),while the viability was significantly decreased(P<0.05).The apoptosis rate and the expression of cleaved-caspase 3 of Parkin siRNA+ALB group was higher than that of scrambled siRNA+ALB group(P<0.05),while the cell viability was further attenuated(P<0.05).The apoptosis rate and the expression of cleaved-caspase 3 of Parkin+ALB group were significantly lower than that of empty vector+ALB group(P<0.05),while the cell viability was significantly enhanced(P<0.05).【Conclusion】PINK1/Parkin-mediated mitophagy plays a protective role in albumin overload induced renal tubular epithelial cell damage by improving mitochondrial function,reducing cellular and mitochondrial oxidative stress.Therefore,intervention in mitophagy may be an important strategy for preventing and treating urinary protein-induced renal tubular epithelial cell damage. |
