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This Article Full Text Full Text (PDF) Supplemental Figure and Video All Versions of this Article: 297/6/R1733    most recent 00321.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Svenningsen, P. Articles by Skøtt, O. PubMed PubMed Citation Articles by Svenningsen, P. Articles by Skøtt, O.

Articles

Prostasin-dependent activation of epithelial Na⁺ channels by low plasmin concentrations

Departments of ¹ Physiology and Pharmacology and ²Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark

Submitted June 9, 2009 ; accepted in final form September 29, 2009

Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na⁺ channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the -subunit, which releases 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 plasmin-stimulated 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 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, ENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of ENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the -subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.

serine protease; sodium retention; kidney; M-1 cells; nephrotic syndrome