Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Abstract

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC ␥ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

“…Thus, in both cases the loss of the segment between the two furin cleavage sites is not likely to be an obligatory mechanism of ENaC activation by ␣ cleavage. Such built-in redundancy into channel cleavage would certainly be consistent with the ability of multiple diverse proteases such as trypsin, subtilisin, elastase, prostasin, furin, and plasmin to activate ENaC in different systems with a similar final outcome (9,24,(31)(32)(33)(34)(35).…”

Physiological and Molecular Responses to Altered Sodium Intake in Rat Pregnancy

“…The changes toward a higher expression of ENaC subunits and a more pronounced cleavage with enhanced activity resemble ENaC expressional changes observed in nephrotic syndrome, where plasmin appears to have a causative role in activating the ENaC 27. Given the high levels of plasminogen activator inhibitor‐1 and ‐2 levels in pregnancy, other serine proteases might be involved.…”

Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

“…Soluble proteolytic activity is present in urine samples from nephrotic syndrome patients. 4 The major protease was identified as plasmin aberrantly filtrated from plasma as plasminogen. 4,5 In vitro, inward amiloride-sensitive current in collecting duct cells is enhanced by exposure to nephrotic urine containing plasmin and by purified plasmin.…”

“…4 The major protease was identified as plasmin aberrantly filtrated from plasma as plasminogen. 4,5 In vitro, inward amiloride-sensitive current in collecting duct cells is enhanced by exposure to nephrotic urine containing plasmin and by purified plasmin. 4,6 Plasmin was shown to release an inhibitory peptide tract from the exodomain of the γENaC subunit 4,6 either by direct cleavage at high concentrations or through Glycosylphophatidylinositol-anchored prostasin at low concentrations.…”

“…4,5 In vitro, inward amiloride-sensitive current in collecting duct cells is enhanced by exposure to nephrotic urine containing plasmin and by purified plasmin. 4,6 Plasmin was shown to release an inhibitory peptide tract from the exodomain of the γENaC subunit 4,6 either by direct cleavage at high concentrations or through Glycosylphophatidylinositol-anchored prostasin at low concentrations. 4,6,7 Addition of α 2 -antiplasmin and aprotinin inhibited the ability of nephrotic urine and plasmin to evoke current.…”