Activation of the renal Na+:Cl- cotransporter by angiotensin II is a WNK4-dependent process

Abstract

Pseudohypoaldosteronism type II is a salt-sensitive form of hypertension with hyperkalemia in humans caused by mutations in the with-no-lysine kinase 4 (WNK4). Several studies have shown that WNK4 modulates the activity of the renal Na(+)Cl(-) cotransporter, NCC. Because the renal consequences of WNK4 carrying pseudoaldosteronism type II mutations resemble the response to intravascular volume depletion (promotion of salt reabsorption without K(+) secretion), a condition that is associated with high angiotensin II (AngII) levels, it has been proposed that AngII signaling might affect WNK4 modulation of the NCC. In Xenopus laevis oocytes, WNK4 is required for modulation of NCC activity by AngII. To demonstrate that WNK4 is required in the AngII-mediated regulation of NCC in vivo, we used a total WNK4-knockout mouse strain (WNK4(-/-)). WNK4 mRNA and protein expression were absent in WNK4(-/-) mice, which exhibited a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation at T-58. Immunohistochemistry revealed normal morphology of the distal convoluted tubule with reduced NCC expression. Low-salt diet or infusion of AngII for 4 d induced phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and of NCC at S-383 and T-58, respectively, in WNK4(+/+) but not WNK4(-/-) mice. Thus, the absence of WNK4 in vivo precludes NCC and SPAK phosphorylation promoted by a low-salt diet or AngII infusion, suggesting that AngII action on the NCC occurs via a WNK4-SPAK-dependent signaling pathway. Additionally, stimulation of aldosterone secretion by AngII, but not by a high-K(+) diet, was impaired in WNK4(-/-) mice.

Fig. 1.

Normal systolic blood pressure (BP) (A) with increased PRA (B) and normal aldosterone (C) in WNK4^−/− mice. Values are presented as the mean ± SE; *P < 0.001 vs. wild type.

Fig. 2.

Effects of WNK4 absence on distal nephron Na⁺ transport pathways. (A) Western blot analysis of kidney protein samples of WNK4^+/+ and WNK4^−/− mice on a normal diet. Representative blots are shown for each protein analyzed. The numbers displayed are the results of the densitometric analysis of at least two blots per assay, expressed as a percentage ± SE of wild type (100%). *P < 0.0001 vs. wild type. (B) NCC expression at the mRNA level measured through real-time PCR. RNA samples extracted from WNK^+/+ (n = 12) or WNK4^−/− (n = 10) kidneys were used. Values shown are the relative abundance of NCC mRNA to 18S mRNA. (C) Immunohistochemical analysis of NCC and phosphorylated NCC (pNCC). Kidney slices from WNK4^+/+ and WNK4^−/− mice were treated simultaneously with the same antibody and detection solutions. (Magnification: 400×.) (D and E) Effect of (D) hydrochlorothiazide (HCTZ) and (E) amiloride on Na⁺ excretion. Urinary excretion of Na⁺ before (days −1 and 0) and after a single administration at time 0 of HCTZ (50 mg/kg body weight, i.p.) (D) or amiloride (5 mg/kg body weight, i.p.) (E) to WNK4^+/+ mice (open bars) and WNK4^−/− mice (gray bars). n = 6 mice per group except in the WNK4^+/+ amiloride group (n = 9), *P < 0.05 and **P < 0.005 vs. WNK4^+/+; ^$P < 0.005 and ^$$P < 0.001 vs. control. (F) CCDs from WNK4^−/− mice display amiloride-sensitive Na⁺ reabsorption. Sodium flux (JNa⁺) in microperfused CCDs from WNK^+/+ mice (n = 6) and WNK4^−/− mice (n = 4) treated or not treated with 10 μM amiloride (amilo). *P < 0.05 vs. CCDs not treated with amiloride. All data are presented as the mean ± SE.

Fig. 3.

NCC expression and phosphorylation are not stimulated in WNK4^−/− mice on a low-salt diet. (A) Representative Western blots of kidney proteins from WNK4^+/+ and WNK4^−/− mice on a normal or low-salt diet (4 d). The results from the densitometric analysis in which at least three blots were included per assay and at least six samples from different mice were analyzed per group are shown above each blot as the mean ± SE. P < 0.0005 vs. normal diet. Two bands are seen using the SPAK antibodies. These bands were reported previously in kidney (36) and represent isoforms of SPAK (34). Thus, both bands were used for the densitometric analysis. (B) WNK4^+/+ and WNK4^−/− mice were studied separately to allow longer exposure times during the film scan for the WNK4^−/− blots so that NCC expression could be detected. No increase with a low-salt diet was observed. NCC phosphorylation (at T58) could not be detected even on a low-salt diet.

Fig. 4.

Response to AngII infusion is altered in WNK4^−/− mice. (A) Representative Western blots of kidney proteins from WNK4^+/+ and WNK4^−/− mice infused with vehicle or AngII (400 μg⋅kg⁻¹⋅d⁻¹). The results from densitometric analysis are expressed as percentages of wild-type levels above each blot (100%). At least two blots were included per assay, and at least six samples from different mice were analyzed per group. *P < 0.05 vs. vehicle. (B) NCC expression and phosphorylation (T58) in wild-type mice treated with spironolactone and infused with vehicle or AngII. For densitometric data shown above the blot, two blots were used for a total of six samples from per group. *P < 0.05 vs. vehicle. (C) Urinary Na⁺ and Cl⁻ excretion of WNK4^+/+ (n = 6) and WNK4^−/− (n = 6) mice at day 1 of infusion with vehicle (open bars) or AngII (black bars). Values are presented as the mean ± SE; *P < 0.05 vs. vehicle. (D) Kidney AT1 expression in WNK4^+/+ and WNK4^−/− mice. (E) Plasma aldosterone concentration (mean ± SE) of WNK4^+/+ and WNK4^−/− mice infused with vehicle (open bars) or AngII (black bars). *P < 0.05 vs. vehicle. Measurements were performed in duplicate. Samples from six different animals per group were studied.

Fig. 5.

Response of WNK4^−/− mice to changes in dietary K⁺ content. (A) Plasma K⁺ concentration in WNK4^+/+ (open bars) and WNK4^−/− (gray bars) mice kept on a normal, low-, or high-K⁺ diet. Samples from nine different animals were studied per group, except for the WNK4^+/+ normal-diet group (n = 8) and WNK4^−/− low-K⁺ group (n = 10). Values are presented as the mean ± SE; *P < 0.005 and **P < 0.00005 vs. WNK4^+/+. (B) Plasma aldosterone concentration of WNK4^+/+ (open bars) and WNK4^−/− (gray bars) mice subjected to a normal or high-K⁺ diet. From left to right, n = 7; n = 10; n = 8; n = 9; *P < 0.005 vs. normal diet.