Date Published: September 24, 2018
Publisher: Public Library of Science
Author(s): Chih-Chiang Hu, Marina Katerelos, Suet-Wan Choy, Amy Crossthwaite, Susan P. Walker, Gabrielle Pell, Mardiana Lee, Natasha Cook, Peter F. Mount, Kathy Paizis, David A. Power, Diego Alvarez de la Rosa.
Pre-eclampsia is a hypertensive disorder of pregnancy characterised by hypertension and sodium retention by the kidneys. To identify changes in sodium uptake proteins in the tubules of the distal nephron, we studied their expression in urinary extracellular vesicles or exosomes (uEVs). Urine was collected from women with pre-eclampsia or during normal pregnancy, and from healthy non-pregnant controls. uEVs were isolated by centrifugation and analyzed by Western blot. Expression, proteolytic cleavage and phosphorylation was determined by densitometric analysis normalized to the exosome marker CD9. Results showed a significant increase in phosphorylation of the activating S130 site in NKCC2, the drug target for frusemide, in women with pre-eclampsia compared with normal pregnant women. Phosphorylation of the activating sites T101/105 in NKCC2 was similar but the activating T60 site in NCC, the drug target for thiazide diuretics, showed significantly less phosphorylation in pre-eclampsia compared with normal pregnancy. Expression of the larger forms of the α subunit of ENaC, the drug target for amiloride, was significantly greater in pre-eclampsia, with more fragmentation of theγ subunit. The differences observed are predicted to increase the activity of NKCC2 and ENaC while reducing that of NCC. This will increase sodium reabsorption, and so contribute to hypertension in pre-eclampsia.
Pre-eclampsia complicates 3–8% of pregnancies resulting in significant maternal, fetal and neonatal morbidity and mortality . The multisystem manifestations of pre-eclampsia occur after 20 weeks gestation with common clinical features including hypertension and proteinuria . The pathogenesis of pre-eclampsia involves placental release of soluble fms-like tyrosine kinase (sFlt-1), a non-membrane-associated circulating form of the receptor for vascular endothelial growth factor (VEGF), which inhibits endothelial VEGF signalling leading to reduced nitric oxide synthesis, endothelial injury, endotheliosis, glomerular dysfunction and proteinuria . Generalized edema is a common manifestation of pre-eclampsia, with proteinuric patients displaying avid sodium retention, which occurs despite suppression of the renin-angiotensin-aldosterone system and intravascular contraction [4, 5].
Greater phosphorylation of NKCC2 on S130 in the PE group might be predicted to lead to increased sodium reabsorption, particularly with increased total NKCC2 expression. However, phosphorylation of NKCC2 on the SPAK/OSR1 sites T101/105 was less in the PE group, so it becomes difficult to predict the overall effect on NKCC2 activity. Notwithstanding the complexity, a role for increased co-transporter activity by NKCC2 in the pathogenesis of PE appears consistent with the observation that in women with antenatal pre-eclampsia, postnatal frusemide is associated with a reduced requirement for antihypertensive therapy in hospital , although frusemide is not specific for NKCC2 alone.
This is the first study to identify changes in expression of ENaC and phosphorylation of NCC and NKCC2 in PE. NKCC2 phosphorylation on Ser130 increases its co-transporter activity. Phosphorylation of NCC was less in the PE group, and this is predicted to reduce its activity. The effect might be due to reduced sodium flowing into the distal convoluted tubule due to the increased upstream activity of NKCC2 in the PE group. The changes in expression of the α and γ subunits of ENaC are novel, with the presence of truncated forms of γ-ENaC and greater expression of α-ENaC predicted to increase sodium uptake. This study, therefore, identifies NKCC2 as a major contributor to sodium retention in pre-eclampsia, with ENaC likely to have a lesser role because of its smaller capacity.