Exosomal miR-20a-5p derived from renal tubular epithelial cells regulates podocyte cytoskeletal remodeling via targeting myosin X in diabetic kidney disease.

Abstract

Background: Renal tubular epithelial cells are increasingly recognized as active participants in the pathogenesis of diabetic kidney disease, where tubular injury often precedes glomerular dysfunction. Exosomes, as critical mediators of intercellular communication, may transmit signals between renal tubules with glomeruli. However, the specific role of exosomes derived from renal tubular epithelial cells (RTECs) in modulating podocyte function, particularly during the early stages of diabetic kidney disease, remains unclear.

Methods: Exosomes derived from RTECs cultured under high glucose and palmitic acid (HG + PA) conditions were isolated and administered to wild-type mice or incubated with cultured podocytes to evaluate their biological impact. In parallel, plasma exosomes from diabetic kidney disease patients were isolated to assess their biological effects. Exosomes derived from HK-2 cells cultured under HG + Pa conditions were isolated and subjected to miRNA sequencing, followed by target screening via miRDB prediction. The functional role of miR-20a-5p was assessed in vivo using adeno-associated virus (AAV) mediated overexpression and knockdown in db/m and db/db mice, respectively. Furthermore, an in vitro co-culture system of HK-2 cells and podocytes was established to mimic tubule-to-podocyte crosstalk. The molecular interaction between myosin X and F-actin was interrogated using dual-luciferase reporter assays, co-immunoprecipitation, and molecular dynamics simulations.

Results: Exosomes derived from HG + PA-treated RTECs induced podocyte foot process effacement and downregulated key cytoskeleton-associated proteins including nephrin, CD2AP, and myosin X. Exosomal miRNA sequencing identified miR-20a-5p as the most significantly upregulated miRNA under diabetic conditions. Overexpression of miR-20a-5p in db/m mice recapitulated podocyte injury, whereas knockdown in db/db mice mitigated foot process effacement. Dual-luciferase assays confirmed that miR-20a-5p directly targets the 3' untranslated region of myo10. The knockdown of myo10 disrupted its binding to F-actin and decreased the expression of cytoskeletal regulatory proteins. Molecular dynamics simulations were employed to assess the structural stability and interaction dynamics between myosin X and F-actin. In co-culture systems, miR-20a-5p modified HK-2 cells significantly altered podocyte morphology and F-actin integrity, confirming its regulatory role via exosome-mediated signaling.

Conclusion: This study identifies miR-20a-5p as a key exosomal mediator released by RTECs under diabetic conditions, contributing to podocyte cytoskeletal remodeling by targeting myo10. These findings offer new insights into the pathogenic crosstalk between tubules and glomeruli, indicating exosome-mediated miRNA signaling as a potential target in early diabetic kidney disease.