Sodium reabsorption in the distal nephron is tightly regulated in part by epithelial sodium channel (ENaC) and sodium chloride cotransporter (NCC), although non-invasive measure of these proteins in humans has not previously been feasible. We recently analyzed the urinary exosomal proteome and identified candidate targets for quantification of ENaC and NCC using targeted mass spectrometry.
To test the hypothesis that urinary exosomal ENaC and NCC are altered during renin-angiotensin-aldosterone system activation, we activated the endogenous RAAS using a low sodium diet (LS) in two separate studies. We provided 8 subjects LS diet (10mmol/day for 7days) to assess urinary protein excretion at 7 days (study 1) and longitudinally over the course of 1 week (study 2). Daily 24-hour urine was collected to monitor sodium balance, and spot urine samples were obtained each morning on days 0, 2, 4, and 6 of LS diet. Urinary exosomal ENaC-α, ENaC-γ, and NCC peptides were analyzed using targeted multiple-reaction-monitoring analysis quantified with stable-isotope peptide standards, and results were normalized to urine creatinine concentration.
In study 1, urinary ENaCγ increased after 8 days of LS diet (Figure A). In study 2, urinary exosomal ENaCγ (Figure B) and NCC peptides (Figure C) increased in a time-dependent manner during LS diet. These measures of urinary sodium channel expression may provide further insight into distal sodium reabsorption in human hypertension.
Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC