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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

  • Ion channels, receptors and transporters
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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.

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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.

Funding

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.

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  1. Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, J.B. Winsløvsvej 21, 3, 5000, Odense, Denmark

    Rikke Zachar, Maiken K. Mikkelsen, Boye L. Jensen & Per Svenningsen

  2. Department of Cancer and Inflammation, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Karsten Skjødt

  3. Department of Clinical Pathology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Niels Marcussen

  4. Department of Urology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

    Reza Zamani

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  1. Rikke Zachar
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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.

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Zachar, R., Mikkelsen, M.K., Skjødt, K. et al. The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney. Pflugers Arch - Eur J Physiol 471, 1383–1396 (2019). https://doi.org/10.1007/s00424-019-02321-z

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