Parkin-Mediated Mitophagy Ameliorated Fatty Acid-Induced Mitochondria Dysfunction And Apoptosis In Tubular Epithelial Cells Via Oxidative Stress

Objective:To detect the role of Parkin in the HK-2 cells apoptosis stimulated by palmitic acid.Methods:(1) Human experimentation: Collection of Chronic kidney disease kidney specimens, inclusion criteria: renal damage but normalor mildly abnormal renal function, with hyperlipidemia. Renal pathological types: Membranous kidney, IgA nephropathy.Collection of patients having trauma operations to remove kidney tissue trauma without kidney disease and hyperlipidemia as control group. The Beclin1, LC3 and Parkin expression were measured in paraffin-embedded human kidney tissue samples(Ctr=18, CKD=20) by immunohistochemistry and immunofluorescence staining.(2) Animal experiment: 20 male Sprague–Dawley rats were randomized divided into High Fat Diet group(HFD=10) and Low Fat Diet group(LFD =10), after 12 weeks, the Serum cholesterol and triglyceride levels were detected. Kidneys were isolated,and the Beclin1, LC3 and Parkin expression in rat kidney were detected by immunohistochemistry staining, immunofluorescence staining and Western blot analysis.(3) Cell experiment: We cultured immortalized human proximal tubular epithelial cell line(HK-2). HK-2 cells were transfected with Lentiviruse GFP-LC3. HK-2 were treated with palmitic acid(PA), a saturated FFA, then measured beclin1, LC3 and Parkin expression by immunofluorescence and Western blot analysis, acid in the cell vacuole by monodansylcadervarine(MDC) staining, mitochondrial transmembrane potential(ΔΨM) by JC-1 staining, mitochondrial morphology by Mitochondria tracer, co-localization of mitochondria with GFP-LC3 and Parkin by immunofluorescence staining, co-localization of Parkin with GFP-LC3 by immunofluorescence staining, ROS production by DCFH-DA fluorescence staining and apoptosis by TUNEL assay. After knockdown of Parkin using Parkin siRNA, ROS,TUNEL,JC-1 and cleaved caspase3 in HK-2 induced by PA were detected. We also measured apoptosis in HK-2induced by PA after inhibition of ROS overproduction by antioxidant. All statistical analyses were perforned using SPSS 21.0 statistical software.Numeric data were analyzed by student,s t-test and one-way ANOVA. The difference was statistically significant(p<0.05).Results:(1) The expression of Parkin, Beclin1 and LC3 in kidney tissue were increased in CKD patients with hyperlipidemia than control patients without renal disease.(2) Increased expression of Parkin, Beclin1 and LC3 in HFD rats kidney.(3) The expression cholesterol and triglycerides were increased in HFD than LFD(p<0.05).(4) PA increased autophagy, mitophagy and Parkin expression in HK-2 cells. PA increased lipid deposition in HK-2 cells than group. PA increased the confocal of GFP-LC3 and mitochondria. PA increased the confocal of GFP-LC3 and Parkin.(5) siRNA-mediated knockdown of Parkin exacerbated PA-inducedΔΨM reduction, ROS production and apoptosis.(6) Antioxidant inhibited the overproduction of ROS and apoptosis in HK-2 induced by palmitic acid.Conclusion : Parkin-mediated mitophagy ameliorated fatty acid induced mitochondria dysfunction and apoptosis in tubular epithelial cells via oxidative stress.