Acute intravenous NaCl and volume expansion reduces NCC abundance and phosphorylation in urinary extracellular vesicles

Background:NaCl loading and volume expansion suppress the RAAS to reduce renal tubular reabsorption of NaCl and water, but effects on NCC and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. Methods:We used uEVs as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 hypertensive patients with repeatedly raised aldosterone-to-renin ratios undergoing infusion of 2L of 0.9% saline over 4 hours. Results:When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change (FC)=0.70), pendrin (FC=0.84), AQP2 (FC=0.62) and uEV markers including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52) and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC) (FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, while the significant decrease in AQP2 persisted (FC=0.62). Conclusions:We conclude that (1) decreases in NCC and pNCC induced by acute NaCl loading and volume expansion may be due to diluted post-test urines, (2) the lack of change of NCC and pNCC when corrected for ALIX despite a fall in plasma aldosterone may be due to the lack of change in plasma K+ and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion.