The Epithelial Sodium Channel (ENaC) α‐ and γ‐subunits are cleaved by furin in human kidney

• Published in: The FASEB journal Vol. 31; no. S1
• Main Authors: Langkilde, Rikke Zachar, Mikkelsen, Maiken Krogsbæk, Skjødt, Karsten, Zamani, Reza, Marcussen, Niels, Jensen, Boye, Svenningsen, Per
• Format: Journal Article
• Language: English
• Published: 01-04-2017
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.31.1_supplement.856.3

Abstract only
The aldosterone‐sensitive Epithelial Sodium Channel (ENaC) plays an essential role in sodium homeostasis and blood pressure regulation.
In vitro
data suggest that during biosynthesis, the proprotein convertase furin cleaves the alpha and gamma subunit. Full activation takes place at the apical membrane by proteolytical excision of an inhibitory domain from the gamma subunit. The GPI‐anchored serine protease prostasin is a possible candidate to activate ENaC under physiological conditions, while aberrant filtrated plasmin cleaves under pathophysiological conditions. The aim of the study was to investigate proteolytical cleavage of ENaC in human kidney tissue and it was hypothesized that intracellular furin cleavage can be detected in the alpha and gamma subunit in human kidney; that apical plasmin/prostasin‐cleavage is associated with proteinuria and that cleaved products are detectable predominantly in human urinary exosomes.
Monoclonal antibodies directed against αENaC (AA 436–44, constant region at the c‐terminal side of putative furin cleavage) and against epitopes in the cleaved γENaC (AA 138–147 (furin‐cleavage site, C‐terminal) and AA 173–190 (overlapping prostasin‐cleavage site)) were developed in balb/c mice and tested on human kidney tissue from nephrectomized patients and on urinary exosomes from healthy persons. A furin cleavage site‐specific γENaC antibody revealed protein at 75 kDa compatible with furin‐cleavage in a human kidney cortex pool. An antibody overlapping and therefore requiring intact prostasin/plasmin cleavage site showed bands compatible with full‐length (100 kDa) and furin‐cleaved (75 kDa) γENaC. No difference was detected in patients with proteinuria. The furin‐cleaved part was mainly detected within the membrane. The αENaC antibody showed a dominant 50kDa product compatible with predominance of furin‐cleaved α‐subunit in the kidney tissue. All three antibodies revealed labeling associated with collecting ducts in kidney tissue sections when immunostained. 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. 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.
Support or Funding Information
Odense University Hospital PhD Fund, University of Southern Denmark ‐ Faculty of Health, The Danish Diabetes Academy – supported by the Novo Nordisk Foundation, Leo Pharma, A.P. Møller and Hustru Chastine Mc‐Kinney Møllers Foundation.