Distal tubular sodium retention is a potent driver of hypertension, with the thiazide sensitive sodium-chloride cotransporter (NCC) a key player. The upstream modulators of NCC are unclear, but recent evidence has revealed the kinases ‘with-no-lysine kinase 4’ (WNK4) and ‘STE20/SPS1-related, proline alanine-rich kinase’ (SPAK) to be involved. The wider role of mineralocorticoids is poorly understood, but animal models implicate aldosterone as a potent regulator, possibly via effects on plasma potassium. We therefore studied the effects of fludrocortisone on NCC, pNCC, WNK4 and SPAK in human urinary exosomes.Methods:
Daily morning urine samples were collected from 26 patients undergoing fludrocortisone suppression testing (100mcg q6h) to diagnose or exclude primary aldosteronism (PA). Exosomes were isolated by ultracentrifugation and NCC, pNCC, WNK4 and SPAK expression quantified by immunoblot.Results:
We observed a rise in NCC (3.68 fold increase, P < 0.01) and median pNCC (2.73 fold increase, P < 0.01) during fludrocortisone administration. The ratio of pNCC/NCC however dropped by 48% (P < 0.01). Abundance of WNK4 also increased by 3.23 fold (P < 0.01) but changes in SPAK were not significant once corrected for changes in exosome marker abundance. Patients without PA (n = 6) had lower baseline aldosterone levels (144 vs 643 pmol/l, P < 0.01) than those with PA (n = 20) and a lower abundance of NCC (p = 0.03), pNCC (p = 0.027) and WNK4 (p = 0.002). Plasma potassium was unchanged during the test, but at baseline there was a very strong negative correlation between NCC, pNCC and WNK4 abundance (P < 0.01 for all), which was stronger than the relationship with aldosterone.Conclusions:
Mineralocorticoid administration causes a rapid and progressive increase in abundance and phosphorylation of NCC in humans. NCC abundance is stimulated to a greater extent than phosphorylation, possibly via stimulation of the WNK4 pathway. These effects may be driven by changes in plasma potassium.