cyanine-dye-3 and Nephrotic-Syndrome

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 gamma-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 microg/ml). At a high plasmin concentration of 30 microg/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, gammaENaC and prostasin were colocalized. 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. We conclude that, 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.

Topics: Animals; Biotinylation; Carbocyanines; Cell Line; Disease Models, Animal; Epithelial Sodium Channels; Fibrinolysin; Fluorescent Antibody Technique; Fluorescent Dyes; Ion Channel Gating; Kidney Tubules, Collecting; Membrane Potentials; Mice; Nephrotic Syndrome; Oligopeptides; Patch-Clamp Techniques; Peptides; Phosphoinositide Phospholipase C; Protein Binding; Rats; Recombinant Fusion Proteins; RNA Interference; Serine Endopeptidases; Time Factors