Renoprotection And Mechanisms Of Adipose-derived Mesenchymal Stem Cell-derived Exosomes On Sepsis-Induced Acute Kidney Injury

Objective:To study the renoprotection of adipose-derived mesenchymal stem cellderived exosomes(AMSCs-Exos)on sepsis-induced acute kidney injury(SAKI)and its possible molecular mechanism.Methods:Human adipose tissue was obtained by liposuction,AMSCs was isolated and purified and identified.Exosomes in AMSCs culture medium were extracted and identified by ultracentrifugation.Human umbilical vein endothelial cells(HUVECs);were stimulated by bacterial lipopolysaccharide(LPS).The sepsis cell model in vitro was established and cocultured with AMSCs-Exos.Western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and immunohistochemistry were used to detect the changes of silent mating type information regulation2 homolog1 synthase(SIRT1),nuclear factor kappa B p65 protein(NF-κB p65),inflammatory and apoptosis-related cytokines in each group before and after AMSCs-Exos intervention.And the changes of SIRT1,NF-κB p65,inflammatory and apoptosis-related cytokines in the cell model were detected again after the cell model was transformed with SIRT1 siRNA,a selective small interfering RNA(siRNA).The animal model of sepsis in vivo was established by cecal ligation and puncture(CLP),and we examined and evaluated the liver,kidney function,and renal tissue by HE staining.Then the CLP model was intervened with AMSCs-Exos,and the changes of SIRT1,NF-κB p65,inflammatory and apoptosis-related cytokines in animal models were detected and observed before and after the intervention.After pretreatment with EX527,an inhibitor of SIRT1 activity,AMSCs-Exos was given to detect the changes of SIRT1,NF-κB p65,inflammatory and apoptosis-related cytokines in mice.Results:1.AMSCs adhered to the wall and grew in a long spindle shape,similar to fibroblasts in shape,which was identified by flow cytometry in accordance with the characteristics of stem cells.The results of AMSCs-Exos identification showed that it was doubly concave disc-shaped under fluoroscopic electron microscope,and its diameter was about 100nm by nanoparticle tracking analysis(NTA).CD63 and CD81 were positive andβ-actin was negative by WB.2.After LPS stimulated HUVECs culture for 24 hours,the level of SIRT1 decreased.Compared with LPS group,after intervention with AMSCs-Exos,the levels of SIRT1 and Bcl-2 increased,while NF-κB p65,TNF-α,IL-6,Bax and Cleaved Caspase-3 decreased(P<0.001).When SIRT1 siRNA was added to LPS group,the effect of AMSCs-Exos disappeared.3.With the extension of modeling time,the general condition of CLP model mice gradually became worse,and the mortality increased.The indexes of liver and kidney function increased gradually with the passage of time.After the establishment of the model,the levels of serum inflammatory cytokines TNF-α,IL-6 and MCP-1 increased gradually.HE staining showed that acute kidney injury appeared in the model group,which reached the peak at 24 hours.4.The general condition,survival rate,and HE staining of kidney tissue were not significantly changed in the Sham+Exos group compared with the Sham group 24 hours after model establishment.The general condition of mice in the CLP group became worse,survival rate decreased,serum liver and kidney function index increased,after treatment with AMSCs-Exos,and there was a significant improvement.The results of ELISA showed that the concentration of TNF-α,IL-6,and MCP-1 in the kidney of the CLP group was higher than that of the Sham group.In CLP+Exos group,the levels of inflammatory factors in renal tissue were decreased significantly(P<0.05).HE staining showed that renal morphology was damaged in the CLP group,which was significantly improved after intervention with AMSCs-Exos.The results of immunohistochemistry showed that the infiltration of inflammatory factors in renal tissue of the CLP group was more severe than that of the Sham group,and was significantly improved after intervention with AMSCs-Exos.WB results showed that the levels of SIRT1 and Bcl-2 protein were significantly decreased in the CLP group.NF-κB p65,TNF-α,Bax,Cleaved Caspase-3/Caspase-9 were up-regulated.The levels of SIRT1 and Bcl-2 proteins were elevated after AMSCs-Exos administration,NF-κB p65,TNF-α,Bax,Cleaved Caspase-3/Caspase-9 were down-regulated,and the difference was statistically significant(P<0.05).After EX527 pretreatment,CLP model was made,and the effect of AMSCs-Exos disappeared.Conclusion:1.AMSCs-Exos were successfully obtained.2.AMSCs-Exos can inhibit the expression of inflammatory and apoptotic cytokines in vascular endothelial cells by activating the SIRT1/NF-κB signaling pathway and improve endothelial cell injury.3.The mouse model of SAKI was successfully established by CLP operation.4.AMSCs-Exos can inhibit the inflammatory reaction and apoptosis of SAKI by activating SIRT1/NF-κB signaling pathway and alleviate acute kidney injury in mice.