Effects of urine composition on epithelial Na⁺ channel-targeted protease activity

We examined human urinary proteolytic activity toward the Epithelial Sodium Channel (ENaC). We focused on two sites in each of alpha and gamma ENaC that are targets of endogenous and exogenous proteases. We examined the effects of ionic strength, pH and urinary H⁺-buffers, metabolic intermediates, redox molecules, and large urinary proteins. Monoatomic cations caused the largest effect, with sodium inhibiting activity in the 15–515 mEq range. Multivalent cations zinc and copper inhibited urinary proteolytic activity at concentrations below 100 μmol/L. Similar to sodium, urea caused a 30% inhibition in the 0–500 mmol/L range. This was not observed with acetone and ethanol. Modulating urinary redox status modified activity with H₂O₂ stimulated and ascorbate inhibited activity. Minimal effects (<10%) were observed with caffeine, glucose, several TCA cycle intermediates, salicylic acid, inorganic phosphate, albumin, creatinine, and Tamm–Horsfall protein. The cumulative activity of ENaC-cleaving proteases was highest at neutral pH, however, alpha and gamma proteases exhibited an inverse dependence with alpha stimulated at acidic and gamma stimulated at alkaline pH. These data indicate that ENaC-targeting urinary proteolytic activity is sensitive to sodium, urea and pH and changes in these components can modify channel cleavage and activation status, and likely downstream sodium absorption unrelated to changes in protein or channel density.