Palmitic Acid-induced Podocyte Apoptosis Via The Reactive Oxygen Species-dependent Mitochondrial Pathway

Background/Aims:Chronic kidney disease(CKD)is often accompanied by hyperlipidemia,which accelerates progression of CKD.Podocytes play a key role in glomerular selective filtration function,which are highly specialized and terminally differentiated visceral epithelial cells with a very limited ability to regenerate.Podocyte injury or dysfunction plays a pivotal role in the development of proteinuria in all forms of glomerular nephritis and DN.Our previous studies demonstrated that palmitic acid(PA)can induce podocyte apoptosis;however,the underlying mechanisms are unclear.In the present study,we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes.Methods:We cultured mouse podocytes and treated them with PA.Then,cell viability was measured using the Cell Counting Kit-8 colorimetric assay,lipid uptake was assessed by Oil Red O staining,boron-dipyrromethene staining and transmission electron microscopy(TEM),apoptosis was measured by flow cytometry and TEM,mitochondrial injury was assessed by JC-1 staining,MitoTracker Deep Red and TEM.The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8,cleaved caspase-9,cleaved caspase-3,cleaved poly(ADP-ribose)polymerase(PARP),B-cell lymphoma 2(Bcl-2),Bax,Bid,cytochrome c,and Fas-associated protein with death domain(FADD)using western blotting.The translocation of Bax and cytochrome c were detected by immunofluorescence.Differentiated podocytes were pre-incubated with the mitochondrial antioxidant,mitoTEMPO(100 nM)for 1h,the mitochondrial production of reactive oxygen species(ROS)was evaluated by fluorescence microscopy using the MitoSOX Red reagent,and apoptosis was measured by flow cytometry and western blotting.Results:PA treatment significantly increased lipid accumulation and induced podocyte apoptosis.We investigated whether the two primary apoptosis signaling pathways(death receptor-mediated pathway and mitochondria-mediated pathway)were involved in the execution of PA-induced podocyte apoptosis,and found that the levels of FADD,Caspase-8,and Bid did not significantly change during this process.Meanwhile,PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression,with Bax translocation to the mitochondria.Furthermore,PA treatment induced mitochondrial impairment,and triggered the release of cytochrome c from the mitochondria to cytosol,with a concomitant dose-dependent increase in the levels of Cleaved caspase-9,Cleaved caspase-3,and PARP.Meanwhile,PA treatment increased mitochondrial production of ROS,and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis.Conclusion:Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway rather than the death receptor pathway,and mitochondrial ROS production participated in this process.