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Identification of plasmin as an ENaC activating protein in nephrotic urine

Per Svenningsen


Nephrotic syndrome is characterized by proteinuria, sodium retention, and edema formation. Recent evidence suggests that sodium retention originates in the renal cortical collecting duct and is mediated by hyper-activity of the epithelial sodium channel (ENaC). 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. Firstly, we found that urine from rats with puromycin aminonucleoside induced nephrotic syndrome and human nephrotic 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 α2-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 ascites.
Secondly, we went on to investigate the mechanism by which plasmin-stimlate ENaC activity. Cy3-labeled plasmin was found to bind to the surface of M-1 cells, and this binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein and prostasin expression. Biotin-label transfer showed that plasmin interacted with GPI-anchored prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the γ-subunit, leading to release of an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasminstimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 μg/ml). At a high plasmin concentration of 30 μg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin blocked plasmin-stimulated ENaC activity in single M-1 cells as measured by wholecell patch clamp technique. In M-1 cells expressing heterologous FLAG-tagged prostasin, we found that prostasin and γENaC were co-localized. A monoclonal antibody directed against the inhibitory peptide of γENaC produced specific immunofluorescence labeling of M-1 cells and pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way.
Thirdly, in monolayers of M-1 cells mounted in Ussing chambers we found that after proteolytic activation of ENaC activity, a subsequent decay in short-circuit current was detected. The decay in short-circuit current was attenuated by the AMPactivated kinase (AMPK) inhibitor Compound C in a dose-dependent manner, reaching significance at a dose of 80 μM. To test if trypsin-stimulation of ENaC activity in M-1 cells leads to activation of AMPK, we generated a genetically encoded fluorescence resonance energy transfer (FRET)-based reporter of AMPK activity. In agreement with the electrophysiological experiments, we found that trypsin stimulated AMPK acitivity in M-1 cells expression the AMPK reporter and this activation was significantly reduced in cells incubated with the ENaC inhibitor amiloride.
In conclusion, 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 plasmin-stimulated ENaC activity is dependent on prostasin expression at low plamin concentration. After proteolytic activation of ENaC, AMPK activity is stimulated, which leads to a subsequent decay in ENaC activity. Thus, we suggest a novel concept by which a defective glomerular filtration barrier allows filtration of substances that activate ENaC and sodium reabsorption. A concept that may be expanded to other disease state, such as preeclampsia or diabetes mellitus, characterized by proteinuria and sodium retention