Association of Genetic Variation in the Epithelial Sodium Channel Gene with Urinary Sodium Excretion and Blood Pressure

This study was performed to investigate whether genetic variation in the epithelial sodium channel (ENaC) is associated with 24-h urinary sodium excretion and blood pressure. A total of 3345 participants of the KoGES_Ansan and Ansung study were eligible for this study. Genomic DNA samples were isolated from peripheral blood and genotyped on the Affymetrix Genome-Wide Human SNP Array 5.0. Thirty-four single nucleotide polymorphisms (SNPs) were extracted for gene regions (SCNN1A, SCNN1B, and SCNN1G) as additive components by using Plink. Twenty-four-hour sodium excretions were estimated from spot urine samples using the Tanaka formula. The general linear model (GLM) was applied to assess the association between SNPs and urinary sodium excretion or blood pressure. In the SCNN1G gene, six SNPs (rs4073291, rs12934362, rs7404408, rs4494543, rs5735, and rs6497657) were significantly different in 24-h urinary sodium excretion according to gene variants. However, no difference was found in blood pressure among participants with gene variants of ENaC. Our finding indicated that 24-h urinary sodium excretions were different according to variants of the SCNN1G gene in large samples. Further studies to replicate these findings are warranted.

ENaC is located in the distal nephron and plays a crucial role in controlling sodium balance. ENaC is mostly expressed in the luminal membrane of connecting tubule cells and plays an important role in renal sodium reabsorption and excretion [3,4]. Excessive sodium reabsorption by the kidney has been known to increase the risk of hypertension. In the kidney, the final control of sodium reabsorption takes place in the distal nephron through ENaC [4]. Liddle's syndrome, a hereditary form of hypertension due to gain-of-function mutations in the genes coding for ENaC subunits, has shown the key role of ENaC in the sodium balance [8].
ENaC is located within taste cell membranes and is the primary mediator of salt taste. ENaC seems to be responsible for the appetitive behavioral responses caused by salt taste [1,2]. Taste perception plays Genotyping Algorithm (DNA Link, Seoul, Korea) for 500,568 SNPs, and 352,228 SNPs remained after quality control. SNP imputation was conducted using the IMPUTE program [15,16]. Based on NCBI build 36 and dbSNP build 126, the JPT and CHB in HAPMAP were used as a reference panel comprising 3.99 million SNPs (HapMap release 22). All genetic variants were examined for Hardy-Weinberg equilibrium. Markers with Hardy-Weinberg equilibrium p value < 10 −6 were discarded. More details on the genotype calling, quality-control, and imputation processes are described in a previous study [13].

Statistical Analysis
By using Plink (http://pngu.mgh.harvard.edu/~purcell/plink2/), we extracted SNP genotype data for gene regions (SCNN1A, SCNN1B, and SCNN1G) as additive (0, 1, and 2) components. A total of 34 SNPs were identified. Association analyses between SNPs and urinary sodium excretion or blood pressure were conducted using SAS software (version 9.3; SAS Institute, Cary, NC, USA). The general linear model (GLM) was used to assess the association between SNPs and urinary sodium excretion or blood pressure according to the number of minor alleles after being adjusted for age, sex, BMI, and smoking status (non-smoker and current smoker). p-Values < 0.05 were considered significant. Tukey's multiple comparison test was applied to search for specific differences between pairs of groups at p < 0.05. The linear trend test was performed by GLM in an additive genetic model with 1 degree of freedom. Table 1 shows the general characteristics of the study subjects. The mean ages of male and female subjects were 51.9 years and 51.0 years, respectively. The means of BMI for male and female subjects were 24.0 kg/m 2 and 24.5 kg/m 2 . The proportions of the subjects with a family history of hypertension were 14.3% for male subjects and 15.6% for female subjects. Table 2 demonstrates allele distributions of SNPs in putative salt taste receptors within the study population. Major allele homozygous, heterozygous, and minor allele homozygous are presented as MM, Mm, and mm, respectively. The associations of genetic variation in the ENaC gene with estimated 24-h urinary sodium excretion obtained with the Tanaka formula are presented in Table 3. In the SCNN1G gene, individuals homozygous for the C allele of rs4073291 (A>C) showed lower 24-h urinary sodium excretion than those with either the AA or AC genotype. Individuals with homozygous for the C allele of rs12934362 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. Individuals homozygous for the T allele of rs7404408 (C>T) showed lower 24-h urinary sodium excretion than those with either the CC or CT genotype. Individuals homozygous for the G allele of rs4494543 (A>G) showed lower 24-h urinary sodium excretion than those with either the AA or AG genotype. Individuals homozygous for the C allele of rs5735 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. Individuals homozygous for the C allele of rs6497657 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. In addition, as using the general linear model with an additive model after adjusting for age, sex, BMI, and smoking status, 24-h urinary sodium excretion showed significant decreasing trends in the SCNN1G gene (rs4073291, rs12934362, rs7404408, rs4494543, rs5735, rs6497657, rs4260062, rs5740, rs4470152, rs4309398, and rs4341748). The associations of genetic variation in the ENaC gene with systolic blood pressure and diastolic blood pressure are shown in Tables 4 and 5, respectively. No differences were found in systolic blood pressure and diastolic blood pressure among the genotype groups.

Discussion
The present study was performed to determine the association of genetic variation in ENaC (SCNN1A, SCNN1B, and SCNN1G) with urinary sodium excretion and blood pressure in Korean adults. Polymorphisms of six SNPs in the genes that code for the ENaC γ subunit (SCNN1G) may modify urinary sodium excretion. However, no difference was found in blood pressure among the gene variants of ENaC.
ENaC is located in the distal nephron. Aldosterone and vasopressin regulate ENaC activity. A low-sodium diet stimulates the renin-angiotensin-aldosterone system (RAAS), and aldosterone increases sodium reabsorption by activating ENaC in the distal nephron. Vasopressin is secreted in response to increases in plasma osmolality, which means a water deficit. Vasopressin stimulates ENaC activity and increases water conservation by increasing sodium reabsorption [4]. In addition, proteolytic ENaC activation by serine proteases may contribute to sodium reabsorption [17][18][19]. Proteases cleave specific sites in the extracellular domains of the αand γ-subunits [17][18][19].
Low 24-h urinary sodium excretion can be associated with high sodium reabsorption in the kidney or low sodium intake. Thus, the persons who are homozygous for the minor alleles of six SNPs (rs4073291, rs12934362, rs7404408, rs4494543, rs5735, and rs6497657) of the SCNN1G gene may reabsorb more sodium in the kidney or consume less sodium than the persons having carriers of major alleles. Excessive sodium reabsorption by the kidney has been known to increase the risk of hypertension. In a South African study, there were no differences in urinary sodium excretion between the wild type and carriers of the R563Q variant of the ENaC [20]. However, the G442V polymorphism in SCNN1B showed greater Na retention in normotensive young people (both black and white) [21]. In addition, Vormfelde SV et al. reported that carriers of the variant G-allele of rs5723 in SCNN1G tended to excrete less sodium in healthy white adults [22]. Therefore, variants of ENaC, particularly SCNN1B and SCNN1G, may be associated with reabsorption of more sodium, but there may be racial differences.
Twenty-four-hour urinary sodium excretion can reflect dietary sodium intake; thus, the people who are homozygous for the minor alleles of SNPs in the SCNN1G may consume less sodium than those who are carriers of major alleles. These differences seem to be related to taste perception. Salt taste perception has been the focus of studies on salt taste receptors such as the ENaC and transient receptor potential cation channel subfamily V member 1 (TRPV1) [23]. ENaC is located within taste cell membranes and is the key mediator of salt taste. In the study by Dias et al. [24], two SNPs (rs239345 (A>T) and rs3785368 (C>T)) in intronic regions within SCNN1B modified suprathreshold taste sensitivity. In the TRPV1 gene, one SNP (rs8065080 (C>T)) polymorphism modified salt taste perception. Carriers of the T allele perceived salt solutions stronger than those homozygous for the C allele. Even though the types of taste receptors were not the same as in the present study, genetic variation in salt taste receptors seems to modify taste perception, which results in the differences in salt intake. However, according to a twin study, salt taste perception appears to be determined more by environmental influences [25]. Further clinical trials on salt taste perception are needed.
In the present study, the SCNN1G gene was associated with 24-h urinary sodium excretion but was not related to blood pressure. The SCNN1A and SCNN1B genes were not associated with blood pressure either. The present study was performed to focus on urinary sodium excretion reflecting salt intake. Hypertensive patients were excluded from the study since they tried to modify their sodium intake. Previous studies reported conflicting results about the associations of ENaC with blood pressure [11,[26][27][28][29]. The Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study [26] conducted in the Han Chinese population reported that rs13306613 in the SCNN1A gene was associated with diastolic blood pressure (DBP) and that rs12447134 in the SCNN1B gene was associated with systolic blood pressure (SBP) under a codominant model. In addition, 5 SNPs in SCNN1G and 4 SNPs in SCNN1B were associated with SBP, DBP, or mean arterial pressure (MAP) under the additive model. In addition, in the other GenSalt study, SCNN1A SNP rs11064153 and SCNN1G SNP rs4401050 were related to longitudinal changes in SBP [11]. However, the T594M variant of the ENaC gene was not associated with hypertension among individuals of African ancestry [27] and Indo-Aryan ancestry [28]. The γ649 ENaC polymorphism was not related to hypertension or salt sensitivity either [29].
There are some limitations to consider when interpreting the results of this study. First, 24-h urinary sodium excretion was estimated using the Tanaka formula with spot urine samples. The 24-h urine collection is hardly realistic in large-sample epidemiologic studies. Second, sodium reabsorption in the kidney is affected by hormones, such as aldosterone and vasopressin, but they were not measured in the present study. Third, sodium intake and salt perception were not measured; thus, it is not known whether the difference in urinary sodium excretion according to ENaC gene variants is due to sodium intake (salt taste perception) or sodium reabsorption in the kidney. Fourth, persons with hypertension were excluded in the present study because hypertension patients tended to try a low sodium diet and we were more interested in sodium intake according to gene variants.
Despite these limitations, this study confirmed that there was a difference in 24-urinary sodium excretion according to variants of the SCNN1G gene in large samples, and this difference did not appear for blood pressure. Further studies are needed to determine whether the difference in 24-h urinary sodium excretion according to the ENaC gene variant is due to salt taste perception differences or sodium reabsorption differences in the kidney. In addition, since ENaC activity is also related to urinary potassium excretion, urinary Na/K ratio should be compared by genetic variation in the ENaC gene.