WNK1-related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron

Abstract

Large deletions in the first intron of the With No lysine (K) 1 (WNK1) gene are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare form of human hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. We generated a mouse model of WNK1-associated FHHt to explore the consequences of this intronic deletion. WNK1(+/FHHt) mice display all clinical and biological signs of FHHt. This phenotype results from increased expression of long WNK1 (L-WNK1), the ubiquitous kinase isoform of WNK1, in the distal convoluted tubule, which in turn, stimulates the activity of the Na-Cl cotransporter. We also show that the activity of the epithelial sodium channel is not altered in FHHt mice, suggesting that other mechanisms are responsible for the hyperkalemia and acidosis in this model. Finally, we observe a decreased expression of the renal outer medullary potassium channel in the late distal convoluted tubule of WNK1(+/FHHt) mice, which could contribute to the hyperkalemia. In summary, our study provides insights into the in vivo mechanisms underlying the pathogenesis of WNK1-mediated FHHt and further corroborates the importance of WNK1 in ion homeostasis and blood pressure.

Keywords: potassium balance; sodium transport; transgenic mouse.

Fig. 1.

WNK1^+/FHHt mice display signs of hypervolemia. (A) Quantification of the expression level of Renin transcripts in the kidney cortex of mice WNK1^+/FHHt (n = 7) and control littermates (WNK1^+/i1lox; n = 6) by real-time RT-qPCR showed that expression of Renin is significantly decreased in WNK1^+/FHHt males (**P < 0.01). Results (mean ± SEM) are expressed in arbitrary units relative to the expression of ubc, and the expression level in WNK1^+/i1lox mice was arbitrarily set to one. (B) Profiles over 24 h in SBP under a 12:12-h day:night (7:00 AM to 7:00 PM and 7:00 PM to 7:00 AM) schedule in WNK1^+/i1lox (n = 7) and WNK1^+/FHHt (n = 6) mice kept under a control diet (NNa; 0.3% of Na⁺) instrumented with a telemetric system under basal condition. Data are mean ± SEM, and each point corresponds to a 1-h average. (C) SBP is increased in WNK1^+/FHHt mice (n = 6) compared with WNK1^+/i1lox littermates (n = 7). Data correspond to the 12-h night period mean ± SEM. *P < 0.05 (repeated measures two-way ANOVA). (D–F) Twelve-hour average SBP (night period) was slightly and transiently increased by administration of a high salt diet (HNa; 3% NaCl) in WNK1^+/FHHt mice, whereas the SBP of WNK1^+/i1lox mice was not affected (E). Data are mean ± SEM. *P < 0.05 compared with WNK1^+/FHHt kept under NNa (Dunnett multiple comparisons test after a one-way repeated measures ANOVA). (F) Twelve-hour average SBP (night period) was normalized in WNK1^+/FHHt mice by a hydrochlorothiazide treatment during 3 d, whereas the SBP of WNK1^+/i1lox mice was not modified. **P < 0.01 compared with WNK1^+/FHHt mice kept under a HNa (paired Student t test). For all panels, data correspond to the 12-h night period mean ± SEM.

Fig. 2.

The deletion of WNK1 first intron leads to increased expression of L-WNK1 in the DCT and CNT. Quantification of the expression levels of (A) L-WNK1 and (B) KS-WNK1 transcripts in the cortical segments of the nephron of WNK1^+/i1lox (n = 6) and WNK1^+/FHHt (n = 6) mice by real-time RT-qPCR showed that expression of L-WNK1 was increased in the DCT and CNT of WNK1^+/FHHt males. Results (mean ± SEM) are expressed in arbitrary units relative to the expression of ubc. *P < 0.05; ***P < 0.001 (unpaired Student t test).

Fig. 3.

The expression, phosphorylation, and transcriptional expression of the NCC are increased in WNK1^+/FHHt mice. (Left) Representative immunoblots performed on the membrane-enriched fractions of the renal cortex of control and WNK1^+/FHHt males (n = 6 per group) incubated with anti-NCC and antiphospho-NCC (pNCC) antibodies. (Right) Densitometric analysis showed that the abundance and phosphorylation of NCC were significantly increased in WNK1^+/FHHt mice (black bars) compared with controls (open bars). Data are mean ± SEM. *P < 0.05; ****P < 0.0001 (unpaired Student t test).

Fig. 4.

The abundance of the phosphorylated form of SPAK is not affected in homogenates of kidney cortex from WNK1^+/FHHt mice. (Left) Representative immunoblots performed on renal cortex homogenates from control and WNK1^+/FHHt males (n = 7 per group) incubated with antibodies raised against SPAK and the phosphorylated SPAK/OSR1 S motif. (Right) Densitometric analysis showed that the abundance of SPAK and its phosphorylated form on Ser343 is not modified in homogenates of cortex isolated from WNK1^+/FHHt kidneys compared with control kidneys. Data are mean ± SEM.

Fig. 5.

The expression and activity of ENaC are similar in WNK1^+/i1lox and WNK1^+/FHHt mice. (A, Upper) Representative immunoblots performed on membrane-enriched fractions of the renal cortex from control and WNK1^+/FHHt males (n = 5 per group) incubated with antibodies raised against the α-, β-, or γ-subunit of the channel. (A, Lower) Densitometric analysis showed that the abundance of the three ENaC subunits is not modified in WNK1^+/FHHt mice compared with control littermates. Data are mean ± SEM. (B) Effects of amiloride on urinary excretion of (Left) Na⁺ and (Right) K⁺ in WNK1^+/i1lox and WNK1^+/FHHt mice. One single dose of amiloride (12.5 mg/kg body weight; black bars) or vehicle (white bars) was administered i.p. to WNK1^+/i1lox (n = 8) and WNK1^+/FHHt (n = 8) mice. Urine samples were collected from 0 to 6 h after injection to measure urinary Na⁺ and K⁺ excretion. Results (mean ± SEM) are expressed as the ratio to urinary creatinine. **P < 0.01, ****P < 0.001 vs. vehicle (Bonferroni multiple comparisons test after one-way ANOVA). Ns, not significant.

Fig. 6.

The expression of the potassium channel ROMK is decreased in the late DCT of WNK1^+/FHHt. Representative images of WNK1^+/i1lox and WNK1^+/FHHt kidneys after double labeling for ROMK and the water channel aquaporin 2 (AQP2). (Scale bar: 50 µm.) Thick ascending limb was identified by the typical strong expression of ROMK (arrows), late DCT was identified by presence of intercalated cells but weak to absent AQP2 signal, and collecting duct was identified by presence of significant AQP2 signal. Note substantially weaker ROMK signal in the late DCT and CNT of WNK1^+/FHHt kidneys compared with the controls (asterisks). Note also that the late DCT of WNK1^+/i1lox kidneys shows prominent apical ROMK signal, whereas the late DCT of WNK1^+FHHtx kidneys shows rather diffused and dotted cytoplasmic ROMK signal.