Basic ResearchThe Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human KidneyZachar, Rikke M.*; Skjødt, Karsten†; Marcussen, Niels‡; Walter, Steen§; Toft, Anja§; Nielsen, Maria R.*; Jensen, Boye L.*; Svenningsen, Per* Author Information *Departments of Cardiovascular and Renal Research and †Cancer and Inflammation, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark; and ‡Departments of Clinical Pathology and §Urology, Odense University Hospital, Odense, Denmark Correspondence: Dr. Per Svenningsen, Department of Cardiovascular and Renal Research, University of Southern Denmark, Winsløwparken 21, 3, DK-5000, Odense C, Denmark. Email: [email protected] Received November 12, 2013 Accepted April 22, 2014 Journal of the American Society of Nephrology 26(1):p 95-106, January 2015. | DOI: 10.1681/ASN.2013111173 Buy Metrics Abstract The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved–to–full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved–to–full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAbinhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC. Copyright © 2015 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.