Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

Autor:	Wörn M; Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, Tübingen, Baden-wurttemberg, Germany., Kalbacher H; Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Baden-wurttemberg, Germany.; Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Baden-wurttemberg, Germany., Artunc F; Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, Tübingen, Baden-wurttemberg, Germany.; Institute of Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich at the University Tübingen, Tübingen, Baden- wurttemberg, Germany.; German Center for Diabetes Research (DZD) at the University Tübingen, Tübingen, Baden-wurttemberg, Germany.
Jazyk:	angličtina
Zdroj:	Current medicinal chemistry [Curr Med Chem] 2022; Vol. 29 (42), pp. 6433-6445.
DOI:	10.2174/0929867329666220608162256
Abstrakt:	Background: Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). Objective: We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. Methods: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. Results: No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. Conclusion: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria. (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
Databáze:	MEDLINE
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