The appropriate regulation of the amiloride-sensitive epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is essential for the maintenance of body sodium balance and the long term regulation of arterial blood pressure. Recent evidence suggests that proteolytic processing of ENaC contributes to ENaC activation. However, (patho-) physiologically relevant proteases remain to be identified. Here, we used the M-1 mouse renal collecting duct cell line as a system to investigate the effects of candidate proteases and protease inhibitors on ENaC-mediated transepithelial sodium transport. To prevent ENaC activation by endogenous proteases, the confluent M-1 cells grown on filters were pre-treated with furin inhibitor.

We tested tissue kallikrein since it is known to be expressed in the aldosterone-sensitive distal nephron and is secreted into the urinary fluid. So far, the function of tissue kallikrein in this nephron segment is largely unknown. Exposing M-1 cells to kallikrein increased the amiloride-sensitive short circuit current (I_SC-Ami) in a concentration-dependent manner. This suggests a physiological role of urinary tissue kallikrein in the regulation of tubular sodium absorption. In patients with crescentic glomerulonephritis and other inflammatory kidney diseases, the urinary concentration of neutrophil elastase is significantly higher than in healthy controls. Additionally, the presence of urinary protease inhibitors (e.g. uristatin) is increased in infection and during acute and chronic inflammation. We demonstrated that neutrophil elastase stimulated I_SC-Ami in M-1 cells. This stimulation was diminished in the presence of uristatin but not by the broad range protease inhibitor aprotinin.

We also hypothesized that ENaC activation by urinary proteases may contribute to renal sodium retention in nephrotic syndrome. In nephrotic syndrome circulating plasminogen is filtered and may be converted to plasmin by urokinase expressed in the distal nephron. Therefore, we tested the effect urokinase and plasminogen on ENaC in M 1 cells. A combined application of urokinase and plasminogen increased I_SC-Ami. In contrast, application of plasminogen or urokinase alone had no effect.

Taken together, our findings indicate that urinary proteases may be important (patho)-physiological modulators of ENaC activity in the distal nephron. Moreover, the balance between endogenous proteases and protease inhibitors may be critical for determining the level of renal ENaC activity.