Proteinuria is associated with a high abundance of urine extracellular vesicles from collecting duct principal cells.

Proteinuria promotes renal Na+ retention through aldosterone-independent activation of the epithelial Na+ channel (ENaC). While this mechanism is well documented in rodent models, its relevance and mechanistic basis in human disease remain poorly defined. Using a cross-sectional design, we isolated urinary extracellular vesicles (uEVs) from healthy subjects (control, n=20), patients with proteinuria (2.5±1.7g albumin/g creatinine, n=20) and nephrotic syndrome (5.5±3.4g albumin/g creatinine, edema, hypoalbuminemia and hyperlipidemia, n=20) to test the hypothesis that, independent of aldosterone, proteinuria severity correlates with uEV-ENaC abundance and cleavage. uEVs were analyzed by nanoparticle tracking analysis and immunoblotting for the EV markers ALIX and CD63, along with proximal tubular-aquaporin (AQP) 1, distal convoluted tubular Na+,Cl--contransporter (NCC), collecting duct-AQP2, γENaC, and total and phosphorylated extracellular signal-regulated kinase (ERK). Aldosterone and oxidative stress marker 8-isoprostane were measured in urine by ELISA. Nephrotic samples exhibited higher uEV and CD63 abundances. uEV-AQP1 abundance did not differ between groups, but uEV-NCC abundance was lower in patients. uEV-AQP2 abundance was significantly higher in nephrotic samples and correlated with proteinuria. Despite similar urine aldosterone levels, total and cleaved uEV-γENaC abundances were significantly higher in patient samples and correlated with the uEV concentration, CD63, and AQP2 levels. The higher abundance of collecting duct-derived uEVs was not associated with oxidative stress marker 8-isoprostane, but patient samples contained higher levels of total and phosphorylated ERK. Collectively, proteinuria in humans is associated with an increased abundance of collecting duct-derived uEVs containing cleaved γENaC, supporting an aldosterone-independent mechanism of ENaC activation in proteinuric kidney disease.