Umbilical Cord Mesenchymal Stem-derived Exosomes Regulate Autophagy To Improve Chronic Kidney Disease Through MiR-146a-5p/TRAF6 Pathway

Objective: To study the effect of human umbilical cord mesenchymal stem cell-derived exosome(huc MSC-Ex)on the pathophysiology and function of the kidney in rats with chronic kidney disease,and to explore the potential mechanisms.Methods: 1.After isolation and culture,the surface markers CD73,CD90 and CD105 of huc MSCs were detected by flow cytometry,and huc MSCs were induced to differentiate into adipose and osteogenesis.The huc MSC-Ex was purified by ultracentrifugation,the morphology of huc MSC-Ex was observed by transmission electron microscope,and the exosomal protein markers CD9,CD63 and CD81 were detected by western blot.2.In vivo,unilateral ureteral obstruction was used to build a rat model of chronic kidney disease.SD rats were randomly divided into 4 groups:sham operation group(Control group),sham operation + exosomes group(Control+Ex group),model group(UUO group)and model + exosomes group(UUO+Ex group).Serum and kidney specimens were collected after successful modeling.Serum creatinine(Scr)and blood urea nitrogen(BUN)of rats in each group were tested.The pathological changes of the kidneys in each group were observed under light microscope.The epithelial-mesenchymal transition(EMT)markers CK18 and TGF-?1,?-SMA and Collagen-I were detected by immunohistochemistry,autophagosomes were observed under electron microscope,the expression of renal tubular epithelial autophagy-related protein LC3 was observed by immunofluorescence,and the expression of mi R-146a-5p in the kidney was detected by q PCR.After regulating the expression of mi R-146a-5p,the expression of downstream TRAF6 and autophagy-related proteins LC3,Beclin-1 and p62 were detected.3.In vitro,rat renal tubular epithelial cells(NRK-52E)were induced by TGF-?1 to induce epithelial-mesenchymal transition(EMT)and co-cultured with huc MSC-Ex.NRK-52 E cells were randomly divided into4 groups:(1)Control group: normal NRK-52 E cells;(2)Control+Ex group:normal NRK-52 E cells were co-cultured with 100 ?g huc MSC-Ex;(3)TGF-?1 Group: 5ng/ml TGF-?1 was added to NRK-52 E cells to induce epithelial-mesenchymal transition;(4)TGF-?1+Ex group: 5ng/ml TGF-?1was added to NRK-52 E cells,and 100 ?g huc MSC-Ex was added at the same time.The morphology of NRK-52 E cells was observed under light microscope,the expression of EMT marker protein ?-SMA was observed by immunofluorescence,autophagosomes were observed under electron microscope,the expression of mi R-146a-5p in cells was detected by q PCR.NRK-52 E cells were transfected with mi R-146a-5p mimic,inhibitor and negative control respectively,bioinformatics predicted the binding site of mi R-146a-5p and TRAF6,luciferase reporter gene was used for structural verification,and western blot was used to detect the expression of downstream TRAF6 and autophagy-related proteins LC3,Beclin-1 and p62.Result: 1.Under the light microscope,huc MSCs showed a long spindle shape.Flow cytometry showed that huc MSCs highly expressed CD73,CD90,CD105,but did not express CD34,CD45 and HLA-DR.and could be induced to differentiate into adipose and osteogenesis.Furthermore,exosomes can be extracted from the supernatant of huc MSCs.The huc MSC-Ex were revealed as round-shaped vesicles with double-layer membrane under transmission electron microscope.2.In vivo,after huc MSC-Ex enters the kidney tissue of rats with chronic kidney disease,the level of mi R-146a-5p increased,the expression of TRAF6 decreased,the formation of autophagosomes and the expression of autophagy-related proteins LC3 and Beclin-1 increased,and the expression of autophagy substrate protein p62 decreased.In addition,the expression of epithelial-mesenchymal transition related protein CK18 increased,while the expression of TGF-?1,?-SMA and Collagen-I decreased,renal function and renal pathological changes were improved,the levels of Scr and BUN decreased,and at the same time,the degree of renal tubular damage and the area of renal interstitial fibrosis reduced.3.In vitro,after huc MSC-Ex co-cultured with TGF-?1 induced NRK-52 E cells,the cell morphology was partially restored,the expression of EMT marker protein ?-SMA decreased,and the number of intracellular autophagosomes increased,the level of mi R-146a-5p was also up-regulated.After NRK-52 E cells were transfected with mi R-146a-5p mimic,the level of mi R-146a-5p in the cells increased,the level of TRAF6 decreased,the expression of autophagy-related proteins LC3 and Beclin-1 increased,and the expression of autophagy substrate protein p62 decreased.After transfected with mi R-146a-5p inhibitor,the level of mi R-146a-5p in the cell decreased,the level of TRAF6 increased,the expression of autophagy-related proteins LC3 and Beclin-1 decreased,and the expression of autophagy substrate protein p62 increased.The database predicts that mi R-146a-5p and Beclin-1 have binding sites,and the results of the luciferase reporter gene show that mi R-146a-5p can directly inhibit TRAF6 by binding to TRAF6.Conclusion: 1.The ultracentrifugation method can efficiently extract huc MSC-Ex,thereby providing the possibility for the treatment of chronic kidney disease and the repair of kidney damage.2.In vivo,huc MSC-Ex regulates autophagy in rats with unilateral ureteral obstruction through mi R-146a-5p/TRAF6,reduces renal tissue fibrosis,and effectively improves kidney function.3.In vitro,huc MSC-Ex carries and releases mi R-146a-5p to injured NRK-52 E cells,which negatively regulates the expression of TRAF6,increases the level of autophagy,and improves the epithelial mesenchymal transition of NRK-52 E cells.