The epithelial Na+ channel alpha- and gamma-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney

摘要

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney alpha- and gamma-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved gamma-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in gamma-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in gamma-ENaC (mAb-intactRKRK), and (3) the alpha-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a approximate to 65-70 kDa protein, compatible with furin-cleaved gamma-ENaC; mAb-intactRKRK detected full-length (approximate to 90-100 kDa) and furin-cleaved (approximate to 70-75 kDa) gamma-ENaC. mAb-alpha detected a approximate to 50 kDa protein compatible with furin-cleaved alpha-subunit. Furin-cleaved gamma-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length gamma-ENaC migration 20 kDa. While gamma-ENaC uEV levels were below the detection limit, alpha-ENaC migrated as intact (approximate to 75 kDa) and furin-cleaved (approximate to 50 kDa) in uEVs. Kidney levels of alpha- and gamma-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved gamma-ENaC as controls (n = 4). Cleaved alpha-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.