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BBB Seminar: Ole Skøtt

Proteinuria and sodium retention: a novel mechanism

Ole Skøtt
Institute of Medical Biology, University of Southern Denmark, Odense, Denmark

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. We have shown 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 gamma subunit ectodomain. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a GPI-anchored protein, and biotin-label transfer showed that the interaction partner was the GPI-anchored protein prostasin. Removal of GPI-anchored proteins from the M-1 cells with PI-PLC inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration, while at high plasmin concentrations, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were co-localized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes, such that plasminogen is converted into plasmin in the tubule and it activates ENaC. At low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.

Selected references:

Svenningsen et al. J Am Soc Nephrol. 2009, 20(2):299-310
Svenningsen et al. Am J Physiol Regul Integr Comp Physiol. 2009 [Epub ahead of print]


Host: Rolf Kåre Reed, Department of Biomedicine