Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA136

Poster Communications

Intact and Furin-cleaved Epithelial Sodium Channel (ENaC) γ- and α-subunit is detected in Human Kidney and Urinary Exosomes

R. Z. Langkilde1, M. K. Mikkelsen1, K. Skjødt2, N. Marcussen3, R. Zamani4, B. L. Jensen1, P. Svenningsen1

1. Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense C, Denmark. 2. Department of Cancer and Inflammation, University of Southern Denmark, Odense, Denmark. 3. Departments of Clinical Pathology, Odense University Hospital, Odense, Denmark. 4. Department of Urology, Odense University Hospital, Odense, Denmark.

The amiloride-sensitive epithelial sodium channel ENaC, located at the apical membrane of the aldosterone-sensitive distal nephron segments and the collecting ducts establishes the "fine-tuning" of sodium reabsorption and is essential for sodium homeostasis and blood pressure regulation. The channel consists of three homologue subunits: α, β and γ(1). ENaC activity is regulated by proteolytic activation during biosynthesis by cleavage of the α and γ-subunits by furin(2). Full activation is believed to occur extracellularly at the luminal membrane by a second cleavage of the γ-subunit by the GPI-anchored serine protease prostasin(3-5). The relevance of these proteolytical activation events in vitro is not fully elucidated in human. It was hypothesized that intracellular furin cleavage of the α and γ subunit of ENaC can be detected in human kidney; that apical plasmin/prostasin-cleavage is associated with proteinuria and that predominantly cleaved products are detectable in human urinary exosomes. Three sets of monoclonal antibodies were generated by immunizing balb/c mice with the relevant peptide synthesized sequence; end-specific against the γ-ENaC furin cleavage-site (mAb-furin, AA 138-147/EAESWNSVSC), overlying the non-cleaved γ-ENaC prostasin cleavage site (mAb-intactRKRK, AA 173-190/CDFFTGRKRKVGGSIIHKA) and against the α-ENaC-subunit (mAb-alpha, AA436-445/PRPQNVEYC) and tested on human kidney cortex tissue pool from nephrectomized patients and on urinary exosomes from healthy persons (all patients gave informed written consent and the use of the tissue was approved by the Institutional Review Board Ethics Committee). A furin cleavage site-specific γENaC antibody revealed a protein at ≈70 kDa compatible with furin-cleavage in a human kidney cortex pool (n=4). An antibody overlapping and therefore requiring intact prostasin/plasmin cleavage site showed bands compatible with full-length (≈90 kDa) and furin-cleaved (≈70 kDa) γENaC. Deglycosylation resulted in a shift in these bands to 75 and 50 kDa respectively. The furin-cleaved protein was enriched within membrane fractions. The α-ENaC antibody showed a dominant ≈50kDa product compatible with predominance of furin-cleaved α-ENaC in tissue. In urinary exosomes furin-cleaved, but not intact γENaC were detected. αENaC was detected in both its intact (≈80kDa) and furin-cleaved form in exosomes. No difference was detected with predicted low aldosterone (patients receiving ACE-inhibitor, n=4), predicted high aldosterone (patients receiving diuretics, n=3) or in patients with proteinuria (n=5) compared to controls (n=4). The results indicate that proteolytic processing by furin of both alpha and gamma ENaC occurs in human kidney. The presence of prostasin-cleavage was not clearly present. Cleavage products of both γENaC and αENaC and intact αENaC were detectable in urinary exosomes.

Where applicable, experiments conform with Society ethical requirements