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Urine exosomes from healthy and hypertensive pregnancies display elevated level of α-subunit and cleaved α- and γ-subunits of the epithelial sodium channel—ENaC

  • Molecular and Cellular Mechanisms of Disease
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Abstract

Preeclampsia is characterized by hypertension, proteinuria, suppression of plasma renin-angiotensin-aldosterone, and impaired urine sodium excretion. Aberrantly filtered plasmin in urine may activate proteolytically the γ-subunit of the epithelial sodium channel (ENaC) and promote Na+ reabsorption and urine K+ loss. Plasma and urine was sampled from patients with preeclampsia, healthy pregnant controls and non-pregnant women, and from patients with nephrostomy catheters. Aldosterone concentration, urine plasminogen, and protein were determined. Exosomes were isolated by ultracentrifugation. Immunoblotting was used to detect exosome markers; γ-ENaC (two different epitopes within the inhibitory peptide tract), α-ENaC, and renal outer medullary K-channel (ROMK) and compared with human kidney cortex homogenate. Urine total plasmin(ogen) was significantly increased in preeclampsia, plasma and urine aldosterone was higher in pregnancy compared to non-pregnancy, and the urine Na/K ratio was lower in preeclampsia compared to healthy pregnancy. Exosome markers ALIX and AQP-2 were stably associated with exosomes across groups. Exosomal α-ENaC-subunit migrated at 75 kDa and dominantly at 50 kDa and was significantly elevated in pregnancy. In human kidney cortex tissue and two of four pelvis catheter urine, ~90–100 kDa full-length γ-ENaC was detected while no full-length γ-ENaC but 75, 60, and 37 kDa variants dominated in voided urine exosomes. There was no difference in γ-ENaC protein abundances between healthy pregnancy and preeclampsia. ROMK was detected inconsistently in urine exosomes. Pregnancy and preeclampsia were associated with increased abundance of furin-cleaved α-ENaC subunit while γ-subunit appeared predominantly in cleaved form independently of conditions and with a significant contribution from post-renal cleavage.

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Acknowledgements

The authors thank Lis Teusch, Inger Nissen, and Susanne Hansen for skillful technical assistance.

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Authors and Affiliations

  1. The Research Unit, Department of Obstetrics and Gynaecology, Odense University Hospital, Odense, Denmark

    Maria R. Nielsen, Britta Frederiksen-Møller & Jan S. Jørgensen

  2. Department of Cardiovascular and Renal Research, Institute for Molecular Medicine, University of Southern Denmark, J. B. Winsloews Vej 21,3, 5000, Odense, Denmark

    Maria R. Nielsen, Britta Frederiksen-Møller, Rikke Zachar, Mie R. Hansen, Rikke Ydegaard, Per Svenningsen & Boye L. Jensen

  3. Department of Nephrology, Fredericia hospital, Fredericia, Denmark

    Kristian Buhl

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  1. Maria R. Nielsen
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Correspondence to Boye L. Jensen.

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This study was funded by the Lundbeck Foundation (R129-2012-10618, fellowship to Maria R. Nielsen), the Danish Strategic Research Council/Innovationsfonden (11-115861, fellowship to Britta Frederiksen-Møller Frederiksen), and the Region of Southern Denmark (10-15756, running costs and fellowship to Britta Frederiksen-Møller).

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Nielsen, M.R., Frederiksen-Møller, B., Zachar, R. et al. Urine exosomes from healthy and hypertensive pregnancies display elevated level of α-subunit and cleaved α- and γ-subunits of the epithelial sodium channel—ENaC. Pflugers Arch - Eur J Physiol 469, 1107–1119 (2017). https://doi.org/10.1007/s00424-017-1977-z

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