Introduction

Essential hypertension is one of the most common diseases of the modern world. High blood pressure or hypertension contributes to morbidity and mortality from stroke, coronary heart disease and end-stage renal disease.1 Both family and epidemiological studies have suggested that 20–50% of blood pressure variation that exists within a population is of genetic origin;2, 3 however, the genes responsible for this variation in blood pressure have not been fully elucidated.

Blood pressure is maintained by a complex network, including renal, neuronal, endocrine and vascular mechanisms. Therefore, blood pressure is determined by complex interactions among many different genes, and is strongly influenced by environmental factors. Clinical and epidemiological studies have shown that dietary sodium intake is positively associated, and potassium intake is inversely associated, with blood pressure.4, 5 In addition, the dietary sodium to potassium (Na/K) intake ratio correlates positively with blood pressure more strongly than either sodium or potassium intake alone.4 Therefore, the effects of sodium and potassium intake on blood pressure are most likely to be biologically interdependent. Furthermore, moderate sodium reduction or potassium supplementation is significantly associated with reduced blood pressure. However, the blood pressure response to dietary sodium and potassium intake seems to vary considerably among individuals.6

The WNK lysine-deficient protein kinase 1 (WNK1) and WNK4 genes are expressed in the distal nephron, specifically in the distal convoluted tubule and the connecting tubule and collecting duct.7 WNK1 and WNK4 have been implicated as important modulators of salt homeostasis, regulating the balance between renal sodium reabsorption and potassium excretion.8, 9, 10 Mutations in WNK1 or WNK4 genes cause a familial disorder, pseudohypoaldosteronism type II, which is a rare, autosomal dominant disease characterized by hypertension and elevated serum potassium levels with normal renal function.7 Therefore, the possibility has been proposed that common genetic variants in both WNK1 and WNK4 affect blood pressure variations and/or susceptibility to essential hypertension.11, 12 Indeed, several studies have indicated associations between the common variants of WNK1 and WNK4 genes and blood pressure levels.11, 13, 14, 15, 16 However, the functional common variants that directly affect the activity of WNK1 or WNK4 have not been identified.

WNK1 is expressed in multiple splicing variants. At least two transcripts, a kidney-specific short form of WNK1 (KS-WNK1) and a more ubiquitous long form (L-WNK1), have been produced by alternative splicing of the WNK1 gene in the kidney.17, 18 Recent studies have suggested that the ratio of L-WNK1 to KS-WNK1 is important for the regulation of renal sodium reabsorption and potassium secretion.12 Furthermore, it has been proposed that the molecular switch for the splicing of L-WNK1 and KS-WNK1 is affected by the amount of dietary potassium intake.19, 20, 21 In this study, we investigated whether the common variations in the WNK1 gene are potential contributors to individual variations in blood pressure. Furthermore, we analyzed the interactions between the common WNK1 variants and dietary potassium and sodium intake for determining inter-individual variations in blood pressure.

Materials and methods

Participants

The participants were 691 randomly selected, apparently healthy Japanese men (mean age±s.d., 53.7±5.1 years; age range, 45–65 years), who visited a medical center for routine medical checkups. All participants provided written informed consent, and the study was approved by the Ethics Committee of the University of Shizuoka. Men taking antihypertensive drugs were excluded from the study. After overnight fasting, blood samples were collected from each participant. Resting blood pressure level was measured using a mercury sphygmomanometer in participants who were in the sitting position after at least 10 min of rest. Of the 691 men, 97 were diagnosed with hypertension according to the standard WHO (World Health Organization) criteria (systolic blood pressure (SBS) ⩾140 mm Hg and/or diastolic blood pressure (DBP) ⩾90 mm Hg) by a physician. The clinical characteristics of the participants are shown in Table 1.

Table 1 Characteristics of the study participants
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Sodium and potassium intake assessment

The levels of dietary sodium, potassium and alcohol intake were estimated using a brief-type self-administered diet history questionnaire (BDHQ).22, 23 The BDHQ was developed on the basis of the self-administered diet history questionnaire (DHQ), which has been validated using three different standard methods for dietary assessment.24 The BDHQ and DHQ were designed to obtain dietary habits for the previous month for total energy and 38 nutrients including sodium and potassium.22, 23, 24 Dietary data were available for 615 participants (89.0%) in this study.

DNA analysis

Genomic DNA was isolated from peripheral blood leukocytes using the phenol extraction method. We have determined the genotypes of five single-nucleotide polymorphisms (SNPs) of the WNK1 gene (rs2286007, rs880054, rs956868, rs12828016 and rs2255390) by PCR–restriction fragment length polymorphism analysis. We have selected these SNPs on the basis of the public NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/). At first, we have analyzed the heterozygosity and genotyping success rate for 12 SNPs (rs11554421, rs2369402, rs2240282, rs2286006, rs2286007, rs880054, rs9804992, rs16928108, rs956868, rs12301299, rs12828016 and rs2255390) in a pilot study comprising 96 participants, and selected 3 non-synonymous SNPs (rs2286007, rs956868 and rs12828016) with a relatively high heterozygosity (minor allele frequency (MAF)>0.10) and two intronic SNPs (rs880054 and rs2255390) with high heterozygosity (MAF >0.45). The genotyping success rate for each of these five SNPs is >98%. The genomic structure of the WNK1 gene and locations of these five SNPs are shown in Figure 1.

Figure 1
figure 1

Genomic structure of the WNK1 gene and location of the genotyped single-nucleotide polymorphisms (SNPs). Exons are indicated by the vertical bars and alternatively spliced exons by the white boxes. The locations of the five genotyped SNPs are indicated by arrows.

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Statistical analysis

The relationships between blood pressure levels and each genotype or haplotype of the WNK1 gene were analyzed by multiple linear regression analysis incorporating age, body mass index (BMI), smoking and alcohol consumption as covariates. Statistical analyses were carried out using the JMP software package (SAS Institute, Cary, NC, USA). P<0.05 was considered statistically significant. No adjustment for multiple testing was made. The coefficients of linkage disequilibrium (LD) (∣D′∣ and r2) were calculated using the SNPAlyze program (Dynacom, Yokohama, Japan). The haplotypes and their frequencies were estimated by the maximum-likelihood method with an EM-based algorithm using the SNPAlyze program.

Results

We analyzed the relationships between the genotypes of five SNPs (rs2286007, rs880054, rs956868, rs12828016 and rs2255390) in the WNK1 gene and blood pressure levels. Statistically significant associations were observed between the genotypes of three SNPs (rs880054, rs956868 and rs12828016) and SBP and/or DBP levels in the multiple linear regression analysis adjusted for age, BMI, smoking and alcohol consumption as covariates. The A allele (rs880054, A>G polymorphism in intron 10), the Thr allele (rs956868, Pro1056Thr polymorphism in exon 13) and the Met allele (rs12828016, Met1808Ile polymorphism in exon 21) were associated with increased blood pressure with gene dosage effects. On the other hand, the genotypes of the remaining two SNPs (rs2286007 and rs2255390) were not associated with blood pressure levels (Table 2).

Table 2 Relations between genotypes of five SNPs in WNK1 and the SBP and DBP levels
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These five SNPs were in LD with each other (∣D′∣>0.85∣), in particular the genotypes of the A>G polymorphism in intron 10 (rs880054) and the Met1808Ile polymorphism (rs12828016) were identical in almost all participants (98.0%). The pairwise LD values of ∣D′∣ and r2 are shown in Table 3. There are four common haplotypes with frequencies of >5% that account for 97% of all chromosomes in our participants (Table 4).

Table 3 The pairwise linkage disequilibrium (LD) values of ∣D′∣ (upper) and r2 (lower)
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Table 4 Estimated WNK1 haplotypes and frequencies in the general Japanese population
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The SBP level in carriers of haplotype 3 (Thr-A-Thr-Met-G) was higher than that in noncarriers of this haplotype (P=0.025) (Table 5). On the other hand, the SBP and DBP levels of carriers of haplotype 2 (Thr-G-Pro-Ile-G) were lower than those in noncarriers of this haplotype (P=0.017, P=0.0083, respectively) (Table 5). These data indicate that haplotype 3 is associated with increased blood pressure levels, whereas haplotype 2 is associated with decreased blood pressure levels.

Table 5 Comparison of blood pressure levels between carriers and non-carriers of each haplotype
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Next, to examine the interaction between the haplotypes of the WNK1 gene and dietary sodium and potassium intake for determining blood pressure levels, we classified the participants into two groups according to population median for the Na/K intake ratio. As expected, of the total participants, the SBP and DBP levels in the group with a high Na/K intake ratio were higher than those of the group with a low Na/K intake ratio (P=0.0085, P=0.013, respectively) (Table 6). These significant differences in blood pressure levels caused by the dietary Na/K intake ratio were observed in carriers of both haplotype 1 (Thr-A-Pro-Met-A) and haplotype 2. Among the carriers of haplotype 1 and haplotype 2, the SBP or DBP levels in the group with a high Na/K intake ratio were significantly higher than those in the group with a low Na/K intake ratio (P=0.019, P=0.025, respectively) (Table 6). On the other hand, the blood pressure levels in carriers with haplotype 3 were similar among both the high and low Na/K intake ratio groups. These findings suggest that the genetic differences in the WNK1 gene influence the variations in blood pressure response to dietary sodium and potassium intake.

Table 6 Interactions between WNK1 haplotypes and sodium to potassium (Na/K) intake ratio
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Discussion

Although epidemiological and genetic studies have shown a high heritability of blood pressure variation, identification of the genes responsible for essential hypertension has been very difficult owing to the complexity and heterogeneity of the trait. The regulation of net renal sodium reabsorption and potassium excretion in the kidney is a major determinant of blood pressure.3, 25 Furthermore, the most widely studied environmental aspect of hypertension is the relationship between sodium and potassium intake.4 However, the blood pressure response to dietary sodium and potassium intake is believed to vary considerably among individuals. Genetic factors may also have an important role not only in determining blood pressure levels but also in determining the blood pressure responses to dietary sodium and potassium intake.6

The WNK kinases and several ion transport systems in the kidney work together for maintaining sodium and potassium homeostasis, and consequently blood pressure regulation.8, 9, 12, 26 WNK1 is expressed in two splicing variants in the kidney, namely KS-WNK1 and L-WNK1.17, 18 They have remarkably different effects; KS-WNK1 antagonizes the ability of L-WNK1 to inhibit renal outer medullary K+ (ROMK) and to activate the epithelial Na channel (ENaC) and Na+Cl− cotransporter (NCC). Therefore, a decrease in the ratio of L-WNK1 to KS-WNK1 enhances potassium secretion through ROMK and decreases sodium reabsorption through ENaC and NCC.8, 12 Therefore, the ratio of L-WNK1 to KS-WNK1 is very important for the regulation of renal potassium secretion and sodium reabsorption. Recent studies have indicated that the expression of the mRNA and protein of KS-WNK1 is significantly increased by a high potassium diet compared with a low potassium diet in rats.19, 20 These findings indicate the possibility that variations in dietary potassium intake cause reciprocal changes in the ratio of L-WNK1 to KS-WNK1 in human kidneys, and consequently in the regulation of renal potassium secretion and sodium reabsorption.

In this study, we have detected that the haplotypes of the WNK1 gene that are associated with individual variations of blood pressure in the general Japanese population, and that the individual blood pressure response to dietary sodium and potassium intake differs among the WNK1 haplotypes. We speculate that the L-WNK1 and KS-WNK1 expression ratio in the kidney is affected not only by dietary potassium intake but also by variations in the WNK1 gene. The possibility exists that KS-WNK1 expression is not increased in carriers of haplotype 3 when their potassium intake is increased. On the other hand, for the carriers of haplotype 1 or haplotype 2, the expression ratio of L-WNK1 to KS-WNK1 in the kidney may be modulated by the amount of dietary sodium and potassium intake.

The possibility exists that some of the individual differences in blood pressure response to dietary sodium and potassium intake are represented by variations in the WNK1 gene. However, at present, we have no direct evidence that such variations in the WNK1 gene cause alterations in the expression ratio of L-WNK1 and KS-WNK1 and/or the in the activity of L-WNK1 in vivo. Further in vivo and in vitro studies are required to determine the functional changes in WNK1 and the ion transport systems in the kidney.

We found that an appropriate Na/K intake ratio is effective in decreasing blood pressure levels for carriers of haplotype 1 or haplotype 2 in the WNK1 gene; however, this is not effective for carriers of the haplotype 3. It will be important to gather scientific evidence of any interactions or otherwise between genetic and modifiable environmental factors such as dietary nutrient intake to establish a preventive method for common diseases, such as hypertension. If such interactions are detected, then modification of such dietary exposure may be suitable for certain population subgroups with important health consequences.