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). Although 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 ± 1.7 g albumin/g creatinine, n = 20), and patients with nephrotic syndrome (5.5 ± 3.4 g albumin/g creatinine, edema, hypoalbuminemia, and hyperlipidemia, n = 20) to test the hypothesis that, independent of aldosterone, proteinuria severity correlates with uEV-γENaC abundance and cleavage. uEVs were analyzed by nanoparticle tracking analysis and immunoblotting for the extracellular vesicle (EV) markers ALIX and CD63, along with proximal tubular aquaporin (AQP) 1, distal convoluted tubular Na+,Cl--cotransporter (NCC), collecting duct AQP2, γENaC, and total and phosphorylated extracellular signal-regulated kinase (ERK). Aldosterone and the 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 the 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.NEW & NOTEWORTHY Human data on proteinuria-driven ENaC activation is limited. Using urinary extracellular vesicles (uEVs), we show that proteinuria increases the uEV concentration. In contrast to the proximal tubular AQP1 and distal convoluted tubular NCC, proteinuria was associated with higher uEV levels of CD63, AQP2, and γENaC, which correlated with albuminuria, independent of aldosterone. These findings imply that proteinuria enhances the kidney's uEV secretion in a segment-specific manner and support an aldosterone-independent mechanism of ENaC activation.