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The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney

  • Rikke ZacharEmail author
  • Maiken K. Mikkelsen
  • Karsten Skjødt
  • Niels Marcussen
  • Reza Zamani
  • Boye L. Jensen
  • Per Svenningsen
Ion channels, receptors and transporters
Part of the following topical collections:
  1. Ion channels, receptors and transporters

Abstract

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65–70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90–100 kDa) and furin-cleaved (≈ 70–75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.

Keywords

ENaC Proteolysis Hypertension Prostasin Collecting ducts 

Notes

Acknowledgments

The author thanks Lars Vitved and Jette Hvelpelund (Department of Cancer and Inflammation) and Ann Sofie Madsen, Mohammed Ahmed, and Rugivan Sabaratnam (Department of Cardiovascular and Renal Research), University of Southern Denmark, for skillful technical assistance.

Author contributions

RiZ, KS, BLJ, and PS designed the study; RiZ, MKM, and PS carried out experiments; NM and ReZ provided human tissue; RiZ, PS, and BLJ analyzed the data; RiZ, BLJ, and PS interpreted results of experiments; RiZ prepared figures; RiZ, BLJ, and PS drafted manuscript. All authors edited and revised manuscript and approved final version.

Funding information

The work was supported by grants from the Danish Diabetes Academy funded by Novo Nordisk Foundation, Faculty of Health Sciences, University of Southern Denmark, Odense University Hospital, the Danish Research Council for Health and Disease; Innovationsfonden/The Strategic Research Council, the A.P. Møller Foundation, The Novo Nordisk Foundation and Leo Pharma.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

424_2019_2321_MOESM1_ESM.pdf (1014 kb)
ESM 1 (PDF 1014 kb)
424_2019_2321_MOESM2_ESM.docx (47 kb)
ESM 2 (DOCX 46 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cardiovascular and Renal Research, Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark
  2. 2.Department of Cancer and Inflammation, Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark
  3. 3.Department of Clinical Pathology, Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark
  4. 4.Department of Urology, Institute of Molecular MedicineUniversity of Southern DenmarkOdenseDenmark

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