The Role Of PTEN-mediated PINK1/Parkin Signaling Pathway In Glomerular Podocyte Mitochondrial Dysfunction

Objective:?By constructing the podocyte model of PTEN gene overexpression and the model of PTEN gene silencing in vitro,To elucidate the mechanism of PTEN-mediated PINK1/Parkin signaling pathway in mitochondrial dysfunction of glomerular podocytes.?The model of glomerular podocyte injury was constructed in vitro to study the mechanism of autophagy in tacrolimus protecting glomerular podocyte injury induced by puromycin amino nucleotide.Methods:The stable PTEN overexpression cell line was constructed by transfection with lentiviral vector;the target gene PTEN knockout cell line was constructed by using Lipofectamine 2000 transfection kit.and the puromycin-injured podocyte model was constructed in vitro.The inverted microscope was used to observe the morphological and structural changes of the podocytes in each group;the apoptosis rate of each group was detected by flow cytometry;Indirect immunofluorescence was used to detect the expression and distribution of autophagy-related protein in each group;WB was used to detect the expression of autophagy-related protein in each group;Transmission electron microscopy was used to detect the changes of autophagosomes in each group.Results:?When PTEN gene was silenced,PINK1 protein expression was inhibited,and its downstream protein molecules Parkin and LC3-II were also decreased.Meanwhile,Podocyte apoptosis increased,mitochondrial JC-1 monomer gradually increased,namely mitochondrial depolarization increased,the difference was statistically significant(p<0.05);Transmission electron microscopy showed an increase in the number of mitochondria,swelling,mitochondrial cristae disorder,some vacuolization,isolated bilayer or multilayer envelope structure of the number of autophagosomes decreased significantly;On the contrary,after overexpression of PTEN gene in podocytes,the expression of PINK1 and Parkin increased,and the number of autophagosomes in podocyte cytoplasm increased significantly(P<0.05).?When the podocytes were injured by puromycin amino nucleotides,loose connection of podocytes,smaller cell bodies,and retraction or even disappearance of podocyte processes were observed;the expression of the podocyte autophagy-related protein decreases;the apoptosis rate increases significantly;and the number of autophagosomes decreases significantly.The above changes can be reversed after tacrolimus intervention in podocytes.Conclusion:?After PTEN gene silencing,PINK1 expression was decreased,and the activity of Parkin and LC3-?,the downstream proteins of PINK1 autophagy pathway,was significantly decreased.PINK1-mediated mitophagy level was decreased,and the ability to scavenge damaged mitochondria was decreased,resulting in an increase in the number of mitochondrial damage.After PTEN gene overexpression,PINK1 was expressed Increased activity of PINK1 autophagy pathway downstream proteins Parkin and LC3-?.Therefore,podocyte-specific overexpression of PTEN has a protective effect on glomerular podocytes,which can regulate mitochondrial autophagy dysfunction through PTEN-PINK1/Parkin signaling pathway.?Puromycin aminonucleotide can induce podocyte injury by inhibiting autophagy and promoting apoptosis;Tacrolimus plays a protective role by activating autophagy.