C-C HU3, M KATERELOS4, PF MOUNT1,3,4, N COOK1,3, S-W CHOY1,2,3, A CROSTHWAITE1,2,3, SP WALKER2,3, G PELL2, K PAIZIS1,2, DA POWER1,3,4
1Department of Nephrology, Austin Health, Heidelberg, VIC; 2Mercy Hospital for Women, Heidelberg, VIC; 3University of Melbourne, Parkville, VIC; 4Kidney Laboratory, IBAS at Austin Health, Heidelberg, VIC
Aim: To characterise expression and phosphorylation of renal tubular sodium transporters in urinary exosomes from women with pre-eclampsia (PE) compared to normotensive pregnant (NP) and non-pregnant controls (NC).
Background: PE is characterised by hypertension, systemic vasoconstriction, proteinuria and renal sodium retention. To better understand the cause of sodium retention, we examined expression and phosphorylation of the major distal renal tubular salt transporters (bumetanide-sensitive cotransporter NKCC2, thiazide-sensitive cotransporter NCC, epithelial sodium channel ENaC) in women with PE, using urinary exosomes.
Methods: A cross-sectional study of 18 PE patients, 22 NP patients and 20 NC women was performed. Exosomes were isolated from urine by ultracentrifugation. Expression of sodium transporters was analysed by Western Blot corrected for expression of the exosome marker CD9. Statistical comparisons were made using the Kruskal-Wallis test.
Results: Expression of NKCC2 was increased 1.6-fold in PE compared to NP (p=0.046). Phosphorylation on the SPAK/OSR1 activation site T101/105 was reduced 1.9-fold (p=0.030) while phosphorylation of the activating PKA S130 site was increased 2.8-fold (p<0.001) in PE compared to NP. There was no difference in expression of NCC but phosphorylation of the SPAK/OSR1 site T60 was reduced 2.5-fold (p=0.008). Expression of the alpha (p<0.001) and full-length gamma (p<0.001) subunits of ENaC was increased 6.0-fold and 2.7-fold, respectively, in PE compared to NP. There was a non-significant trend to increased expression of the cleaved 50 kD form of gamma ENaC (3.0; p=0.06).
Conclusions: This data suggests a role for increased activity of ENaC and NKCC2 in mediating sodium-retention in PE, occurring despite reduced signalling through the WNK/SPAK/OSR1 pathway. Factors responsible for up-regulation of ENaC and NKCC2 expression and phosphorylation of NKCC2 on S130 remain to be identified.