Objective: Proteolytic processing of the epithelial sodium channel (ENaC) is thought to contribute to its activation. In nephrotic syndrome, filtered plasminogen may be converted to plasmin by urokinase-type plasminogen activator. Plasmin can activate ENaC and therefore may contribute to renal sodium retention in nephrotic syndrome (Svenningsen et al. J Am Soc Nephrol 20: 299, 2009). Additional proteases and/or mediators in the urine may affect ENaC activity in the context of renal disease. We investigated whether urine samples of adult (n=19) and pediatric (n=4) patients with proteinuria caused by a range of renal diseases can modify the activity of heterologously expressed human ENaC. Methods: Xenopus laevis oocytes were injected with cRNA for human abg-ENaC and amiloride-sensitive ENaC whole-cell currents (DI_ami) were determined by two-electrode voltage-clamp technique after pre-incubation of oocytes in chymotrypsin, activated human plasmin, or in urine samples of patients with proteinuria. Urinary protease activities were examined by zymography. Results: We demonstrated that plasmin increases DI_ami in human ENaC expressing oocytes by ~3.9-fold. In contrast, most urine samples of patients with proteinuria failed to stimulate ENaC. Only 2 out of 19 adult urine samples and none of the 4 pediatric urine samples had a modest (~1.5-fold) stimulatory effect. Protease activity could be detected in all urine samples of patients with proteinuria (n=23) but not in those of healthy controls (n=3). For some urine samples we detected an inhibitory effect on ENaC. The finding that inhibitory urine reduced the stimulatory effect of chymotrypsin on ENaC suggests that it may contain protease inhibitors. Conclusion: The effect of urine samples of patients with proteinuria on ENaC activity is variable and may depend on the type and duration of the underlying renal disease and on the balance of proteases and protease inhibitors present.