Essential hypertension (EH) is a major global health concern that contributes to millions of disability and premature death worldwide every year due to cardio-cerebrovascular events, congestive heart failure, and endstage renal disease (Mills et al., 2016). The recent survey published on Circulation showed that approximately 23.2% (≈244.5 million) of the Chinese adult population ≥18 years of age had hypertension During 2012 and 2015 (Wang et al., 2018). Hypertension in Northern China is especially a serious problem, with high prevalence and a lack of control (Xu et al., 2021). The etiology and pathogenesis of EH are regarded as a highly heterogeneous disorder resulted from multiple genetic and environmental factors, as well as their complex interactions. Evidences from pedigree study have demonstrated that genetic factors are a determinant of hypertension to a large extent (Hong et al., 1994). Approximately 20%–60% of the blood pressure (BP) variation among the general population is genetically determined (Levy et al., 2000). Therefore, identification for the potential genes susceptible to hypertension is of crucial importance, which would help to realize the early detection of individuals, treatment and prevention of hypertension.

WNK1 (lysine deficient protein kinase 1) is a member of the WNKs subfamily, the gene of which was identified a candidate gene for BP regulation and EH (Lifton et al., 2001; Wilson et al., 2001; Zambrowicz et al., 2003). Human WNK1 maps to chromosome 12p13.33, is encoded by 31 exons, and spans over 160 kb of genomic DNA. There are two major isoforms of WNK1, a kinase-active full long isoform (L-WNK1) with complete kinase domain and a kinase-deficient kidney specific short isoform (KS-WNK1). L-WNK1 is ubiquitously expressed in various tissues, particularly in heart and muscle, whereas KS-WNK1 is produced mainly in kidney (Delaloy et al., 2003). It is thought that the elevation of BP in PHA2 patients may partially due to increasing reabsorption of water and sodium via L-WNK1/KS-WNK1 mediated upregulation of Na-Cl cotransporter (NCC), resulting volume expansion-induced hypertension (Vidal-Petiot et al., 2013; Brown et al., 2021; Meor Azlan et al., 2021). Hence, WNK1 is an obvious candidate gene, and the possibility has been proposed that genetic variants in WNK1 alter susceptibility to EH.

Highly polymorphic was observed in WNK1, with >100 validated single nucleotide variant (SNVs). The SNV rs1468326, located 3 kb from the WNK1 promoter, was first found to be associated with severity of hypertension in families from the British Genetics of Hypertension (BRIGHT) Study in 2005 (Newhouse et al., 2005). Subsequently, a case-control study in the Chinese Han population suggested allele A carriers of rs1468326 in WNK1 had higher risk for EH by Han et al. (2011). In accordance with the Chinese study, Persu et al. (2016) reported the rs1468326 was associated with hypertension in multicenter study of white European populations. Additionally, consistent result was found that the SNV rs1468326 associated with EH for Tibetan individuals in China, which supports the role of WNK1 as potential hypertension susceptibility genes (Shi et al., 2018). However, among the coding regions of WNK1, conflicting results were reported regarding the association of common variants and EH. Kokubo et al. (2004) first tested for the association among 7 WNK1 SNVs (rs956868, rs3858703, and rs2286007 et al.) and EH in Japanese population of 771 hypertensives and 1047 controls selected from within the Suita cohort, but no association was found. However, Han and Hui (2008) reported the rs956868, located in the WNK1 exon 13, was significantly associated with EH in the Han Chinese population (dominant model, AA + AC vs. CC: p = 0.007, OR = 1.39, 95%CI [1.10–1.77]) after adjusting the covariates by logistic regression analysis. Moreover, significant association was observed between the WNK1 exon 1 variant (rs34880640) and EH in the Hani minority of China, but no positive result was found in the Yi minority (Cun et al., 2011). In aspect of WNK1 introns, Newhouse et al. (2009) reported the major allele (A) of rs765250, located in intron 1, demonstrated the evidence for association with hypertension (p = 0.01, OR = 1.3, 95%CI [1.0–1.7]), whereas others failed to replicate this finding (Persu et al., 2016; Shi et al., 2018). Another SNV, rs880054 is in the 10th intron of WNK1 gene. Tobin et al. (2005) found statistically significant association of rs880054 in WNK1 with BP variation. Nevertheless, another study in the Tibetan population of China showed no significant association between rs880054 variant with hypertension (Shi et al., 2018). Therefore, the relationship of WNK1 SNVs with EH was still a worthy research problem, and further studies are in needed. In the present study, we investigated the association between the SNV loci of WNK1 gene and EH risk in Northern Han Chinese in the Beijing area.

The present study was intended to investigate the relationship between the WNK1 gene and EH. A total of 12 tagging SNVs of the WNK1 gene were identified by Haploview software and genotyping was further performed. Binominal logistic regression analyses were also performed to exclude the influences of the confounding factors. Data obtained from this study have shown that the rs7305099 variant in the WNK1 gene was significantly related to EH risk in the Northern Han Chinese population after Bonferroni correction for multiple testing, and T allele was a protective factor for hypertension. In the two haplotype blocks, the rs4980973-rs4980974-rs880054-rs956868-rs12828016-rs2051852-rs7972490 A-A-A-C-G-G-G haplotype might be a risk factor for EH. It is worth noting that hypertensive subjects taking antihypertensive medication were included in the current study, accounting for 78.57% of hypertensive patients. Accordingly, the mean SBP value of all patients was quite low. However, 21.43% of individuals did not take medication or take antihypertensive drugs irregularly, this also reminded us to further exhort patients to take medicine as directed.

EH is a multifactorial disease, involving the interaction of multiple genes and environmental factors (Wang et al., 2020). Ample evidence supports a crucial role for the kidney in BP regulation and pathogenesis of EH (Rossier et al., 2013; Franceschini et al., 2014). In particular, several rare Mendelian forms of hypertension are proved to be due to genetic mutations involved in sodium and water reabsorption (Lifton et al., 2001; Delles et al., 2010). WNK1 is a serine-threonine kinase, it can regulate various ion channels, which participate in the sodium and chloride transport in the kidney (Náray-F et al., 2004; Cope et al., 2006). NCC is a critical sodium reabsorption channel located in the DCT, and it is a key regulator resulting in volume expansion and hypertension when it is hyperactive (Simon et al., 1996; Subramanya et al., 2014). The reabsorption of NCC must be switched on with direct phosphorylation by Ste20-like proline-alanine rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1), phosphorylated and activated by a family of proteins called WNKs (Richardson et al., 2008). WNK1 mutations cause increased expression of L-WNK1 in the distal tubule (Brown et al., 2021). Therefore, the WNK-SPAK/OSR1-NCC pathway is overactivated, and NaCl reabsorption is increased, leading to hypertension (Brown et al., 2021).

A number of potential candidate genes for BP regulation and EH have been identified through studies of rare Mendelian forms of hypertension. Previous studies also found the possible relationship between WNK1 SNVs and EH risk, and several SNVs have been included in the current study. In Kokubo et al. (2004) selected seven WNK1 SNVs (rs956868, rs3858703, and rs2286007 et al.) and investigated the association between these SNVs and EH risk in the Japanese general population. The results suggest no association of WNK1 with hypertension was observed (Kokubo et al., 2004). Similarly, a family-based association study by Newhouse et al. (2005) demonstrated no relationship with hypertension was found for the WNK1 rs956868 variant in white European populations. However, Han and Hui (2008) reported the increased risk of EH was found to be associated with rs956868 of WNK1 in the Han Chinese population after adjustment for conventional risk factors by logistic regression analysis (dominant model, AA + AC vs. CC: p = 0.007, OR = 1.39, 95%CI [1.10–1.77]). Unfortunately, our present study failed to find a positive connection between WNK1 rs956868 variant and HT risk in the Northern Han Chinese. Rs956868 is located on the exon 13 of the WNK1 gene, and its association with the genetic susceptibility to hypertension has been reported in various ethnic groups. The following factors may explain the observed negative result for rs956868 of WNK1 and EH. First, lack of association may result from ethnic differences. Different races may have different genetic backgrounds and heterogeneity. In the above studies, the frequency of A allele for rs956868 was 0.134 in the Japanese population, and 0.177 in the British white population. In addition, the data reported by Han et al. was 0.234 in the Chinese Han population, whereas the frequency of A allele in our current study was 0.174 which is similar to that reported in the British white population. Second, we cannot rule out the possibility that the results have limitations due to sampling bias. Furthermore, it is worth mentioning that another possibility is a “true” mutation, as yet unidentified, exists in the linkage disequilibrium with rs956868. It is expected that further studies with large samples will be conducted in the future.

Rs880054 is another SNV of WNK1, and it is located in intron 10. Tobin et al. (2005) reported that the rs880054 contributes to BP variation in a population-based sample of 996 subjects from 250 white European families. However, a Chinese study on Tibetan population has shown that both rs880054 and rs4980973 mutations were not associated with EH susceptibility (Shi et al., 2018). Consistently, no significant connections between WNK1 genetic variants (rs880054 and rs4980973) and EH were found in our study. As reported, the minimum allele frequencies of rs880054 and rs4980973 were 0.323 and 0.418 respectively in the current study population. In the Tibetan ethnic group of China, the data were 0.33 and 0.42 respectively (Shi et al., 2018), which were in accord with the Northern Han Chinese. However, the minimum allele frequency of rs880054 in white European subjects was 43.8%, according to Tobin’s Report (Tobin et al., 2005). As we all know, EH is the result of the joint influence of both genes and environmental factors (Zhang et al., 2022). Genetic background, growth environment and dietary habits et al. are all the root causes of ethnic heterogeneity, the influence of confounding factors should be more rigorously controlled to increase the accuracy of the results (Shi et al., 2021). In view of this, the findings need to be validated in other populations.

Notably, rs7305099, located in the eighth intron of the WNK1 gene, was found to be significantly correlated with EH in our study. It is not surprising that most tag SNVs detected by Haplotype software locate in the introns of gene (Sakharkar et al., 2004). Although introns can not be translated into amino acids directly, their key role in gene expression regulation cannot be ignored (Le Hir et al., 2003; Jo and Choi, 2015). Mutation of the rs7305099 locus could elevate the expression of WNK1, and may therefore be involved in the pathogenesis of EH. Alternatively, tag SNV located in intron maybe just a site which was in close linkage disequilibrium (LD) with the other functional genetic variants (Dong et al., 2017; Grossi et al., 2018). With the consideration of these findings, the potential function of the rs7305099 variant was further predicted by using HaploReg (version 4.0) (http://pubs.broadinstitute.org/mammals/haploreg/haploreg_v4.php). It was found that there were 9 SNVs in LD with rs7305099. These SNVs are reported to be associated with enhancer histone marks, DNAse, proteins bound, motifs changed and selected expression Quantitative Trait Loci (eQTL) hits, many of which are involved in the regulation of blood pressure (Manunta et al., 2008; Liu et al., 2013). Of course, the functional role of rs7305099 in EH should be verified in future studies.

Since the interaction of multiple SNVs on the biological phenotype, haplotype analysis was further performed in the present study, which may be more powerful to identify genetic variants of complex diseases, such as EH. The present haplotype analysis results showed that the haplotype A-A-A-C-G-G-G (rs4980973-rs4980974-rs880054-rs956868-rs12828016-rs2051852-rs7972490) was significantly associated with increased risk for EH.

Our research team has been committed to the study of SNVs for EH. According to a previous study by Li et al. (2019) the prediction models to recognize individuals with high risk for EH were presented, and rs7305099 was identified to be a genetic factor for EH prediction, which was consistent with our present findings. Although it is undeniable that some hypertensive subjects were from the Anzhen community in our study, which overlapped with Li et al.’s, they were two distinct studies with different designs. Firstly, our research was designed in a community based trial, and subjects were from the community of Anzhen, Liuliqiao and Guozhuang in Beijing. While the subjects in Li et al.’s study were hospital based patients, who were screened in clinic and physical examination center at Anzhen hospital. In general, community or population-based sampling strategy is more representative and brings less bias compared to the hospital-based study. Secondly, the purposes and research perspectives of the two studies were different. Li et al.’s study aimed to develop prediction models to recognize individuals with high risk for EH or BP. Thus many variables for genes, SNVs, and other factors were included. By contrast, our present study focused on genetic patterns of WNK1 and feature of haplotypes between hypertensives and controls. To some extent, the present research can be seen as a validation of Li et al.’s study in community-based design.

Although both inner authenticity and the representativeness of the sampling population were considered in the current study, several limitations still existed. First, it is worth noting that EH is regarded as a complex polygenic disease responsive to environmental factors. A SNV or gene likely has weak effects on the individual’s phenotype, because both genetic and environmental factors have been implicated in the occurrence and development of EH, as well as their interactions. This was the main limitation of the study. Second, the present study covered most of the common WNK1 variants, but, unavoidably, some regulatory regions and functional domains might be missed, especially other functional SNVs with lower frequency that are still worthy of study. Third, although the subjects were adequately matched for age and gender between groups, some participants in control group might inevitably develop EH later in the life. Fourth, the study was performed in only one region (Beijing), which may cause some geographic bias. Fifth, considering low statistical power for several SNVs in our study, caution should be exercised when extrapolating the insignificant association between the SNV for WNK1 and hypertension. Therefore, in future studies, evaluation of potential interactions such as gene-gene, gene-environment is required in countrywide large samples to the pathological mechanism behind the correlation. Results produced can only demonstrate correlations rather than causality.

In conclusion, the rs7305099 T allele of WNK1 gene conferred to a significantly decreased risk for EH in the Northern Han Chinese population in Beijing, and individuals with haplotype A-A-A-C-G-G-G (rs4980973-rs4980974-rs880054-rs956868-rs12828016-rs2051852-rs7972490) are more susceptible to developing EH. These could provide evidences to the risk assessment, early prevention and individualized therapy of EH to some extent. Further functional studies are needed to confirm above discovery, and should be testified in different populations.