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.
|Number of pages||2|
|Publication status||Published - Jan 2015|
|Event||19th International Society for the Study of Hypertension in Pregnancy World Congress - Hilton New Orleans Riverside, New Orleans, United States|
Duration: 25. Oct 2014 → 29. Oct 2014
|Conference||19th International Society for the Study of Hypertension in Pregnancy World Congress|
|Location||Hilton New Orleans Riverside|
|Period||25/10/2014 → 29/10/2014|