MiR-196b-5p-enriched Extracellular Vesicles Mediated Aldosterone-induced Renal Fibrosis In Diabetic Mice

Objective: Aldosterone is a mediator of progressive renal disease,but the mechanisms for aldosterone-mediated renal impairment in diabetic mice are not fully defined.We therefore sought to investigate the specific role of aldosterone in diabetic kidney disease(DKD)and to elucidate the mechanisms involved.Methods: Aldosterone and/or mineralocorticoid receptor antagonist(MRA)eplerenone were used to treat the db/db diabetic mice.Proximal tubule epithelial cells(PTECs)and fibroblasts were cultured.Blood and kidney samples from diabetic patients with or without DKD were used to verify the findings from animals and cultured cells.Results: We found that aldosterone promoted proteinuria and tubulointerstitial extracellular matrix(ECM)accumulation in db/db diabetic mice while eplerenone mitigated the adverse effect of aldosterone.Unexpectedly,the fibroblast,main source of ECM,did not directly respond to aldosterone in vitro.However,coculture of proximal tubule epithelial cells(PTECs)and fibroblasts found that when PTECs derived extracellular vesicles(EVs)were taken up by fibroblasts,ECM production increased remarkably.Moreover,C57BL/6 mice injected with EVs from renal cortex of Aldosterone treated db/db mice showed increased ECM accumulation.Function of the ingredients of PTECs derived-EVs were analyzed,and RNAs were identified to be responsible for the EVs-induced fibroblast dysfunction.Further miRNA array analysis revealed that miR-196b-5p was the most remarkably increased miRNA in PTECs-derived EVs with aldosterone stimulation.Overexpression of miR-196b-5p in fibroblasts increased ECM production,accompanied with inhibition of the SOCS2 expression and enhanced STAT3 phosphorylation.In addition,plasma levels of miR-196b-5p was higher in patients with DKD as compared with patients without DKD and miR-196b-5p levels positively correlated with the albuminuria concentration.In kidney specimens from diabetic patients,expression of miR-196b-5p,locating mainly in PTECs,increased in patients with DKD as compared with the non-DKD.Conclusion: This study demonstrates the involvement of miR-196b-5p-EVs pathway as a novel mechanism in aldosterone-induced renal fibrosis in diabetes.EVs rich in miR-196b-5p mediate the crosstalk between PTECs and fibroblast during the development of renal fibrosis,which might be associated with STAT3/SOCS2 signaling pathway.In addition,miR-196b-5p might be a potential marker for DKD progression.