The Role Of Smad3 In Regulating Fatty Acid Oxidation Through NDRG1 In Cisplatin Induced Acute Kidney Injury

Objective:Bioinformatics was used to analyze key differentially expressed proteins and the activation pathways in cisplatin-induced AKI mice before and after the intervention of SIS3 inhibitors based on i TRAQ-PRM proteomics.In combination with in vitro experiments,potential molecules involved in the regulation of fatty acid oxidation by Smad3 were searched for,and the mechanism of Smad3 regulating fatty acid oxidation and promoting cisplatin-induced acute renal injury was preliminarily studied.Methods:1、The Cisplatin induced AKI mouse model was established and SIS3 intervention was given.The experiment was divided into 4 groups: Control group,Smad3 inhibitor group(SIS3),Cisplatin group and Cisplatin +Smad3 inhibitor group(Cisplatin+SIS3).Kidney samples were collected for protein extraction.i TRAQ technology was used to search for differentially expressed proteins.PRM technology was used to verify key differentially expressed proteins(DEPs).2、Cisplatin was used to stimulate mouse renal tubular epithelial cells.After treatment with SIS3,the expression changes of fatty acid metabolism related proteins,intracellular lipid droplet deposition,and oxidative stress were observed,and the expression changes of key proteins identified by proteomics were verified.Results:1、Through proteomic quantitative technology,a total of 4787 proteins were identified.A total of 87 DEPs were detected in the cisplatin group compared with the control group,among which 26 proteins were up-regulated and 61 proteins were down-regulated.Compared with the control group,a total of 125 DEPs were found in SIS3 group,among which 53 proteins were up-regulated and 72 proteins were down-regulated.A total of 130 DEPs were detected in cisplatin +SIS3 group compared with cisplatin group,among which 68 proteins were up-regulated and 62 proteins were down-regulated.Bioinformatics analysis shows that these DEPs are mainly enriched in energy metabolism pathways,with lipid metabolism being the most critical.Combined with PRM technology,the key differential protein NDRG1 was screened out.2、In vitro experiments,it was found that compared with the control group,the intracellular phosphorylation level of Smad3 protein increased,the expression of NDRG1 decreased,the production of malondialdehyde(MDA)and reactive oxygen species(ROS)increased,and the expressions of CPT1 A and PPARα,key proteins of fatty acid oxidation,decreased in cisplatin group.However,the expressions of SREBF1 and SCD1 related to lipid synthesis were increased,and lipid droplet deposition was obvious.Compared with the cisplatin group,the intracellular phosphorylation level of Smad3 protein decreased,the expression of NDRG1 increased,the production of MDA and ROS decreased,and the fatty acid oxidation key proteins CPT1 A and PPAR in the cisplatin+SIS3 group α The expression of SREBF1 and SCD1 increased,while the expression of SREBF1 and SCD1 related to lipid synthesis decreased,and lipid droplet formation decreased.Conclusion:In acute renal injury induced by cisplatin,cisplatin acts on renal tubular epithelial cells,downregulating the expression of NDRG1,promoting the phosphorylation of Smad3,causing oxidative damage to fatty acids,leading to lipid accumulation in renal tubular epithelial cells,resulting in lipotoxicity,increased oxidative stress,and promoting the progression of AKI.After treatment with Smad3 inhibitors,their fatty acid oxidation disorders and oxidative stress were improved.In cisplatin induced acute renal injury,Smad3 may regulate fatty acid oxidation through NDRG1.