Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells

Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na+ excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the gamma subunit. In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of gamma ENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact gamma ENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly. DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na+ excretion and hypertension.