249-POS

• Published in: Pregnancy hypertension 2015-01, Vol.5 (1), p.125-126
• Main Authors: Nielsen, Maria Ravn, Rytz, Mie, Frederiksen-Møller, Britta, Svenningsen, Per, Zachar, Rikke M, Jørgensen, Jan Stener, Jensen, Boye Lagerbon
• Format: Article
• Language: English
• Subjects: Cardiovascular; Obstetrics and Gynecology
• ISSN: 2210-7789
• DOI: 10.1016/j.preghy.2014.10.255

Objectives Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity of ENaC by proteolytic cleavage of the γ -subunit ectodomain and release of a 43-aminoacid inhibitory tract from the channel. Exosomes are membrane vesicles released into the urine from apical membranes of the kidney epithelial cells. Objectives: (1) To investigate if the proteolytic state of the ENaC γ -subnit can be studied in urine exosomes from pregnant women. (2) To investigate if the ENaC γ -subunit ectodomain is abnormally activated by proteolysis in preeclamptic women. Methods 100 mL spot urine samples from 14 preeclamptic women, 17 pregnant women and 9 non-pregnant women were collected with protease inhibitors. Plasmin/plasminogen was measured. Exosomes were recovered by ultracentrifugation at 220,000× g at 4 °C for 100 min. The exosome fraction was used for western blotting with a newly developed monoclonal antibody, mAb3C7, directed against the “inhibitory” tract in γ -ENaC. Aquaporin-2 (AQP2) was used as a positive control for the presence of collecting duct membrane. Results Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group ( p < 0.001). Plasma aldosterone was increased in pregnancy compared to non-pregnant women ( p < 0.001). Creatinine-normalized urine exosome fractions were positive for AQP2. No intact, full-length ENaC γ -subunit was detected in the exosome fractions. In some samples the inhibitory tract was not detected while AQP2 was. Others displayed bands corresponding to ENaC cleaved by furin and/or by prostasin/plasmin, but with no systematic difference between normal pregnancy and preeclampsia Conclusions It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ -ENaC, 2) Urine exosome fraction displays a variable pattern of γ -ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women. Disclosures M.R. Nielsen: None. M. Rytz: None. B. Frederiksen-Møller: None. P. Svenningsen: None. R.M. Zachar: None. J.S. Jørgensen: None. B.L. Jensen: None.