Placenta-derived Mesenchymal Stem Cells Alleviate Podocyte Injury In Diabetic Kidney Disease By Modulating Mitophagy Via The SIRT1-PGC-1α-TFAM Pathway

Background and aims:With the increasing prevalence of diabetes mellitus,the number of patients with diabetic kidney disease(DKD)is also increasing.DKD has become a major cause of chronic kidney disease and end-stage renal disease(ESRD)worldwide.After progression to ESRD,kidney transplantation or renal replacement therapy is the final choice for the patients with DKD,and cardiovascular risk and mortality risk are significantly increased.DKD has become a serious social health problem worldwide.However,its pathogenesis is still unclear and effective treatment methods are lacking.Mesenchymal stem cells(MSCs)have the characteristics of high proliferation,multidirectional differentiation,regenerative repair and self-renewal,and can also regulate the disorder of glucose metabolism,thus may become a new method for the treatment of DKD.However,the specific mechanism of MSCs in the treatment of DKD is still unclear,and there is still a lack of foreign literature on the effects of placental MSCs(P-MSCs)on DKD.In recent years,mitophagy has been the focus of research on the mechanism of MSCs repairing damaged cells or tissues,and mitophagy dysfunction is one of the important pathogenesis of DKD.Therefore,the purpose of this study was to investigate the therapeutic effect and molecular mechanism of P-MSCs on DKD from the perspective of mitophagy.Methods:Firstly,high glucose(HG)induced podocyte cell line was used to establish a cell model of DKD.In this study,western blotting,reverse transcription polymerase chain reaction,and immunofluorescence were used to detect the expression of PINK1/Parkin-mediated mitophagy-related markers(Beclin1,LC3,P62,PINK1,Parkin,Tom20),podocyte injury-related markers(Podocin,Desmin),SIRT1,PGC-1α,and TFAM.The structure of autophagosomes,lysosome and mitochondria were observed by transmission electron microscopy.Mitochondrial membrane potential and reactive oxygen species were detected by flow cytometry,and ATP content was detected by ATP kit.Then,by constructing SIRT1 si RNA,PGC-1α si RNA and SIRT1 overexpression plasmids and transfecting them into podocytes,the expression of mitophagy-related markers,podocyte injury-related markers,SIRT1,PGC-1α,and TFAM were detected.Hence,the molecular mechanism of P-MSCs improving mitophagy inhibition and podocyte injury induced by HG was investigated.Finally,streptozotocin-induced DKD rats were constructed,and P-MSCs were injected into DKD rats through tail vein injection.The effect of P-MSCs on the mitophagy,podocyte injury,SIRT1,PGC-1α,and TFAM in DKD rats were verified at the animal level by immunohistochemical staining.Results:Firstly,The results in vitro experiments showed that as compared with the control group,exposing podocytes to HG aggravated podocyte injury,represented by a decreased expression of Podocin along with increased expression of Desmin,and inhibited PINK1/Parkin-mediated mitophagy,manifested as a decreased expression of Beclin1,the LC3 II,Parkin,PINK1 and Tom20 associated with an increased expression of P62.Importantly,these indicators were reversed by P-MSCs.In addition,P-MSCs improved the mitochondrial function of podocytes induced by HG,which was manifested by the increase of ATP content,mitochondrial membrane potential,and the decrease of reactive oxygen species.After the intervention of P-MSCs,the nuclear damage of podocytes was reduced,the autophagosomes and lysosomes were increased significantly,and the mitochondrial damage was alleviated.Then,the results showed that P-MSCs up-regulated the expressions of SIRT1,PGC-1α and TFAM.Next,SIRT1 si RNA,PGC-1α si RNA and SIRT1 overexpression plasmids were constructed and transfected into podocytes,respectively.The results showed that knockdown/overexpression of SIRT1 could prevent/promote P-MSCs to improve mitophagy inhibition and podocyte injury,and decreased/increased the expression of PGC-1α and TFAM.Interestingly,mitophagy inhibition and podocyte injury were reversed when SIRT1 was overexpressed and PGC-1α was inhibited at the same time.Thus,P-MSCs may play an important role in improving mitophagy inhibition and podocyte injury through SIRT1-PGC-1α-TFAM pathway.Finally,The results from animal studies showed that P-MSCs could improve mitophagy inhibition and podocyte injury,manifested as an increased expression of Beclin1,LC3 II,Parkin,PINK1 and Tom20 associated with a decreased expression of P62 and Desmin after injection of P-MSCs.In addition,P-MSCs can up-regulate the expression of SIRT1,PGC-1α and TFAM.Therefore,P-MSCs may ameliorate mitophagy inhibition and podocyte injury of DKD through SIRT1/PGC-1α/TFAM pathway.Conclusion:Our results suggest that P-MSCs alleviate podocyte injury in DKD by modulating mitophagy via the SIRT1-PGC-1alpha-TFAM pathway.Targeting PINK1/parkin-mediated mitophagy and SIRT1-PGC-1α-TFAM signaling pathways may provide new potential therapeutic approaches for P-MSCs in DKD,which will provide new experimental data for stem cell therapy of DKD.