Nephrotic syndrome is characterized by massive proteinuria, renal sodium retention, and edema formation. In the puromycin aminonucleoside (PAN) rat nephrosis model there is increased reabsorption of NaCl mediated by the epithelial sodium channel (ENaC) situated in the cortical collecting duct. The stimulus for increased ENaC activity does not involve any of the known sodium-retaining hormones. Extracellular serine protease activity can stimulate ENaC activity. We hypothesized that proteases from the plasma could be filtered through the defective glomerular barrier and subsequently activate ENaC. We found that urine from PAN nephrotic rats and human patients increased sodium currents significantly in the mouse cortical collecting duct (M-1) cell line, as assessed by whole-cell patch clamp. Nephrotic urine displayed serine protease activity and purification by aprotinin affinity precipitation and ion exchange chromatography led to the isolation of an 80-kDa ENaC-activating protein, which was identified as plasmin(ogen) by MALDI-TOF mass spectrometry. Addition to nephrotic urine of the plasmin inhibitors 2-antiplasmin and Pefabloc PL abolished serine protease activity and ENaC activation. Tubular urokinase-type plasminogen activator (uPA) converts filtered plasminogen to plasmin. Consistent with this, treatment of nephrotic rats with the uPA (and ENaC) inhibitor amiloride inhibited plasmin formation in urine and alleviated edema and ascites. We suggest a novel concept by which a defective glomerular filtration barrier allows filtration of substances that activate ENaC and sodium reabsorption. We identify plasmin formed from filtered plasminogen as the major urinary ENaC activator and show that uPA is a relevant additional pharmacological target in nephrotic syndrome. The mechanism may contribute to sodium retention in other conditions with proteinuria.