Changes in urinary excretion of water and sodium transporters during amiloride and bendroflumethiazide treatment

Abstract

Aim: To quantify changes in urinary excretion of aquaporin2 water channels (u-AQP2), the sodium-potassium-chloride co-transporter (u-NKCC2) and the epithelial sodium channels (u-ENaC) during treatment with bendroflumethiazide (BFTZ), amiloride and placebo.

Methods: In a randomized, double-blinded, placebo-controlled, 3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake. The subjects were treated with amiloride 5 mg, BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day. On the examination day, glomerular filtration rate was measured by the constant infusion clearance technique with (51)Cr-EDTA as reference substance. To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENaC, u-NKCC2, the gamma fraction of ENaC (u-ENaCγ), and u-AQP2 were measured at baseline and after infusion with 3% hypertonic saline. U-NKCC2, u-ENaCγ, u-AQP2 and plasma concentrations of vasopressin (p-AVP), renin (PRC), angiotensin II (p-ANG II) and aldosterone (p-Aldo) were measured, by radioimmunoassay. Central blood pressure was estimated by applanation tonometry and body fluid volumes were estimated by bio-impedance spectroscopy. General linear model with repeated measures or related samples Friedman's two-way analysis was used to compare differences. Post hoc Bonferroni correction was used for multiple comparisons of post infusion periods to baseline within each treatment group.

Results: At baseline there were no differences in u-NKCC2, u-ENaCγ and u-AQP2. PRC, p-Ang II and p-Aldo were increased during active treatments (P < 0.001). After hypertonic saline, u-NKCC2 increased during amiloride (6% ± 34%; P = 0.081) and increased significantly during placebo (17% ± 24%; P = 0.010). U-AQP2 increased significantly during amiloride (31% ± 22%; P < 0.001) and placebo (34% ± 27%; P < 0.001), while u-NKCC2 and u-AQP2 did not change significantly during BFTZ (-7% ± 28%; P = 0.257 and 5% ± 16%; P = 0.261). U- ENaCγ increased in all three groups (P < 0.050). PRC, AngII and p-Aldo decreased to the same extent, while AVP increased, but to a smaller degree during BFTZ (P = 0.048). cDBP decreased significantly during BFTZ (P < 0.001), but not during amiloride or placebo. There were no significant differences in body fluid volumes.

Conclusion: After hypertonic saline, u-NKCC2 and u-AQP2 increased during amiloride, but not during BFTZ. Lower p-AVP during BFTZ potentially caused less stimulation of NKCC2 and AQP2 and subsequent lower reabsorption of water and sodium.

Keywords: Amiloride; Aquaporin2; Epithelial sodium channels; Hypertonic saline; Sodium; Sodium transporters; Sodium-potassium-chloride co-transporter; Thiazide; Urine; Water.

Figure 1

Flow chart.

Figure 2

Effects of 3% hypertonic saline on urinary excretion of sodium-potassium-2chloride co-transporter (A), gamma fraction of epithelial sodium channels (B) and aquaporin2 (C) in 22 healthy subjects treated with bendroflumethiazide, amiloride or placebo. Values are means ± SEM. General linear model with repeated measurements (GLM-RM) was performed to test for differences between groups. A: U-NKCC2 increased during amiloride (P = 0.081) and placebo (P = 0.010) treatments. The increase in u-NKCC2 was however only significant during placebo. U-NKCC2 did not change during BFTZ; B: U-ENaCγ increased significantly and to the same extent during all three treatments; C: U-AQP2 increased significantly during amiloride and placebo (P < 0.001), but not during BFTZ. Paired t-test was used for comparison of post-infusion periods vs baseline. ^aP < 0.050; ^bP < 0.001. AQP2: Aquaporin2; U-NKCC2: Urinary excretion of sodium-potassium-2chloride co-transporter; ENaC: Epithelial sodium channels; BFTZ: Bendroflumethiazide.

Figure 3

Effects of 3% hypertonic saline on plasma concentrations of renin (A), angiotensin II (B), aldosterone (C) and arginine vasopressin (D) in 23 healthy subjects pre-treated with bendroflumethiazide, amiloride or placebo. A-C: There was a significant difference between PRC, p-AngII and p-Aldo plasma levels throughout the study day. Values are means ± SEM. One-way ANOVA was used to test for differences between treatments; D: P-AVP increased significantly at 150 min with a borderline significant difference between treatments (^aP = 0.048). Values are medians with upper and lower limits. Friedman’s test was used to test for differences between treatments. BFTZ: Bendroflumethiazide; PRC: Plasma renin concentration.

Figure 4

Effects of 3% hypertonic saline on (A) intracellular and (B) extracellular volume and (C) total body water in 23 healthy subjects pretreated with bendroflumethiazide, amiloride or placebo. Values are means ± SEM. General linear model with repeated measures was non-significant between treatments. Paired t-test was used for comparison of post infusion periods vs baseline ^bP < 0.001. BFTZ: Bendroflumethiazide; ECV: Extracellular volume; ICV: Intracellular volume; TBW: Total body water.