We aimed to identify possible mechanisms by which renal sodium retention could be activated during proteinuria. By using single cell patch clamp technique we found, surprisingly, that urine from the rat Puromycin Aminonucleoside model of Nephrotic syndromel and from nephrotic patients activate the epithelial sodium channel (ENaC) in model systems – mainly mouse collecting duct M1 cells. The activation depended on a serine protease activity, which was identified by MALDI-TOF mass spectrometry to be plasmin. Consistent with this, purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability of nephrotic urine to activate ENaC. The activation by plasmin involved cleavage of an inhibitory peptide from the ENaC gamma subunit ectodomain. Plasmin was found to bind to the surface of the M-1 cells, in a way which depended on the presence of a GPI-anchored protein, which was identified by plasmin biotin-label transfer to be the channel-activating protease prostasin. Removal of GPI-anchored proteins led to inhibition of the ability of low concentrations of plasmin (1–4 mg/ml) to stimulate ENaC activity. At higher concentrations activation of ENaC did not depend on GPI-anchored proteins. Consistent with this, knockdown of prostasin blocked plasmin-stimulated ENaC activity. In nephrotic urine plasmin was likely to be converted from filtered plasminogen by tubular urokinase-type plasminogen activator (uPA). Thus, the uPA-inhbitor amiloride blocked production of plasmin in nephrotic rat urine in vivo. In conclusion, a defective glomerular filtration barrier allows passage of proteolytic enzymes that activate ENaC directly, or indirectly. This mechanism provides a simple coupling between proteinuria and sodium retention, and is currently under investigation in proteinuric patient groups.