In Primary Aldosteronism, Mineralocorticoids Influence Exosomal Sodium-Chloride Cotransporter Abundance

Abstract

Distal tubular sodium retention is a potent driver of hypertension, and the thiazide-sensitive sodium-chloride cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 μg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.

Keywords: Exosomes; NCC; Na transport; aldosterone.

Figure 1.

Increases in NCC, pNCC and WNK4 during FST. (A) Representative immunoblot from a single patient of basal day (D0), D1, D3, and D4 (additional summary blots available in Supplemental Appendix). (B) Box plot of NCC and pNCC levels by day of fludrocortisone suppression test. The upper and lower hinges correspond to the first and third quartiles, and the whiskers extend to the highest and lowest values within 1.5× the interquartile range, with outliers indicated by dots. Levels are measured as OD per milligram of creatinine in the sample (not adjusted for changes in ALIX), where results have been standardized to the respective median day 0 (prefludrocortisone) values. (C) Box plot of WNK4 and SPAK levels by day of fludrocortisone suppression test. The upper and lower hinges correspond to the first and third quartiles, and the whiskers extend to the highest and lowest values within 1.5× the interquartile range, with outliers indicated by dots. Levels measured as OD per milligram of creatinine in the sample (not adjusted for changes in ALIX), where results have been standardized to the respective median day 0 (prefludrocortisone) values.

Figure 2.

Correlations with plasma potassium at baseline (pre-fludrocortisone). Relative abundance of pNCC, WNK4, and SPAK (normalized to control sample across all blots and ALIX abundance) at baseline before administration of fludrocortisone versus plasma potassium. Plotted on log10 y axis with linear regression lines.