Physiological Interaction Between α-Adducin and WNK1-NEDD4L Pathways on Sodium-Related Blood Pressure Regulation
The kidney plays an important role in salt and blood pressure (BP) homeostasis. In previous studies, variants in the genes for α-adducin (ADD1), WNK1, and NEDD4L, which all regulate renal sodium absorption, have been associated with increased BP. However, findings have been inconsistent. We tested whether this is because of physiological interactions between the effects of variants in these genes. We assessed the single and combined effects of the ADD1 (Gly460Trp), WNK1 (rs880054 A/G), and NEDD4L (rs4149601 G/A) polymorphisms on renal and BP response to an acute Na load (n=344 subjects), BP decrease after 1 month of treatment with 12.5 mg of hydrochlorothiazide (n=193), and ambulatory 24-hour BP (n=690). Individually, the variants showed modest effects on some of the studied phenotypes. We found the ADD1 Trp allele to be permissive for the effects of variants of the other genes. In combination, the same variants (ADD1 Trp/WNK1 GG/Nedd4L GA+AA) showed a consistent effect on renal Na handling (P=0.009) and acute BP response to a saline infusion (P=0.021), BP lowering after thiazide treatment (P=0.008), and nocturnal systolic BP (P=0.044). Physiological interaction between the ADD1 and WNK1-NEDD4L pathways influences the effects of variants in these genes on sodium-related BP regulation. Relatively common alleles in the ADD1, WNK1, and NEDD4L genes when present in combination may have significant effects on renal sodium handling, BP, and antihypertensive response to thiazides.
Regulation of body sodium and blood pressure (BP) is achieved through the interaction of several mechanisms, including sodium (Na) transport at the basolateral and luminal tubular cell membranes along specific nephron segments. Moreover, physical, nervous, and hormonal mechanisms modulate this constitutive capacity of tubular cells to transport Na according to the body’s needs.1 In fact, virtually all mendelian disorders leading to hypertension are caused by gene variants affecting salt reabsorption.2
A comprehensive approach to the genetics of the regulation of body Na and BP should take into account the interactions among the underlying variety of gene polymorphisms. At present, this approach is not feasible globally for the prohibitive size of the cohort required. However, a contribution along this line may be provided by focusing on genes affecting luminal (WNK1 and NEDD4L) and basolateral transport of Na (Adducin) in patients whose BP is changed by maneuvers affecting body Na.
These genes have been chosen for the following reasons: a missense mutation in the α-adducin (ADD1 Gly/Trp) gene has been shown to affect the function of the protein with the Trp allele resulting in greater renal Na-K pump activity in nephron segments proximal to the macula densa.3,4 Results of association studies of the Trp allele with BP are conflicting when this allele is considered alone,5 but they are much more consistent when dietary Na and appropriate gene modifiers are taken into account.4 A similar trend is also detectable in familial studies.4 Moreover, this allele is consistently associated with increased risk in hypertensive subjects,6–13 although, in normotensive subjects, this allele may be protective.9,14 In the distal tubule, WNK1 and WNK4, which encode proteins in the WNK (“with no lysine” [K]) family of serine-threonine kinases,15 affect Na homeostasis through their effect on the Na-Cl cotransporter and Na channel.16,17 Another protein that also plays a key role is NEDD4L, which encodes a ubiquitin ligase that controls cell surface expression of epithelial Na+ channels in the distal nephron.17,18 Recent studies have shown that, in addition to their role in mendelian forms of hypertension, common variants in both WNK1 (marked by the single nucleotide polymorphisms [SNPs] rs880054 and rs2301880) and NEDD4L (marked by rs4149601) contribute to BP variation in the general population.19,20 However, taken individually, the variants of these 2 genes have shown conflicting associations with BP, probably because the modifiers mentioned above were not considered.21–25
To avoid the confounding influence of previous therapy or the duration of hypertension on the genetic effect, we enrolled newly discovered (and consequently never treated) patients. Body Na was changed with 2 maneuvers: an acute saline infusion where the relation between the simultaneous changes in BP and renal Na excretion can be assessed and chronic diuretic treatment, which represents the most diffuse approach, to the reduction of BP through the increase in renal Na excretion. The genetic influence on the BP and renal Na excretion changes in these 2 studies were compared and used to evaluate their consistency with the genetic influence on 24-hour ambulatory BP (ABP) monitoring.
All of the studies were performed on newly discovered, unrelated, and never-treated patients referred for assessment of hypertension to the outpatient clinic at the San Raffaele Hospital between January 1997 and January 2007. The subjects in studies on acute Na load (study 2) and chronic diuretic treatment (study 3) are subsets of subjects enrolled in the ABP study (study 1).
Study 1: ABP
Inclusion criteria for the study were age 18 to 65 years; body mass index (BMI) <30 kg/m2; Na intake, evaluated as urinary Na excretion of <300 mEq/24 hours; and office systolic BP (SBP) >140 mm Hg and diastolic BP (DBP) >90 mm Hg in 3 consecutive visits to their family doctors. Exclusion criteria included history of myocardial infarction, stroke, congestive heart failure, liver disease, secondary cause of hypertension, diabetes, women on oral contraceptives, severe hypertension (>160/110 mm Hg), abuse of drugs or alcohol, and creatinine clearance <80 mL/m. Secondary forms of hypertension (eg, primary aldosteronism) were ruled out with specific investigations when deemed appropriate. A total of 84.7% consecutive patients approached agreed to participate, providing a sample of 690 patients.
As anticipated, some of the subjects were normotensive on ABP monitoring (ABPM). We included all of the subjects in the study and applied a case-only design, using BP as a continuous variable. ABPM was performed using Spacelabs 90207 devices (Space Labs). Subjects were monitored on a day chosen for typical weekly activity. Recordings were performed every 10 minutes during awake hours (daytime) and every 30 minutes during nighttime. The data were electronically processed for deletion of errors and “outliers” (SBP <70 or >240 mm Hg; DBP <40 or >150 mm Hg; and heart rate <20 or >200 bpm).
Study 2: Acute Na Load
The study protocol for acute Na load test was similar to that reported previously.25 Briefly the protocol began with a 2-hour equilibration period during which subjects were taken to a quiet room and a venous catheter was inserted into an antecubital vein. Subjects remained in the supine position until the end of the Na loading except for voiding. A steady state was considered to be achieved when the volume of urine collection and the values of the BP recordings varied by <1 mL/min and <3 mm Hg, respectively. The average equilibration period lasted 2 hours. After the equilibration period and achievement of a steady state, a constant-rate IV infusion of 2 L of 0.9% NaCl was carried out in 2 hours. BP (mean of 3 measurements taken 3 minutes apart) was measured every 30 minutes during the 2 hours of loading and 3 times at 3-minute intervals at the end of the infusion. These last 3 BP values were averaged and used in the analysis.
Fractional excretion of Na and potassium were calculated as Px*Ucrea/Pcrea*Ux, where Ux and Px are the urinary and plasma concentrations of the solute x, and Ucrea and Pcrea are the urinary and plasma concentrations of creatinine. The slope of the relationship between SBP and Na excretion (mm Hg/μEq per minute; pressure-natriuresis relationship) was calculated by plotting the Na excretion on the y axis as a function of SBP on the x axis observed both under basal conditions and after 2 hours of saline infusion, as described elsewhere.26
Study 3: Chronic Diuretic Protocol
This protocol is also similar to that reported previously.27 Briefly, newly diagnosed patients with essential hypertension (office BP >140/90 mm Hg but <160/110 mm Hg in ≥3 consecutive visits at weekly intervals) were enrolled after the exclusion of secondary hypertension. After the 1-month run-in phase, the study protocol consisted of 1 month of treatment with 12.5 mg of hydrochlorothiazide (HCTZ) once a day. The response to therapy was computed as the difference between the average of the last 3 BP values at the last pretreatment visit and the average of the last 3 BP values after 1 month of 12.5 mg of hydrochlorothiazide.
The protocols for all 3 of the studies were approved by the ethical committee of the San Raffaele Hospital. Informed consent was obtained from each individual enrolled.
DNA was extracted from venous whole blood by standard methods. All of the subjects were genotyped for ADD1 Gly460Trp (rs4961 at http://www.ncbi.nlm.nih.gov), NEDD4L rs4149601, and WNK1 rs880054, rs2107614, rs1159744, rs2301880, and rs2277869. SNPs were detected by Custom TaqMan SNP assay allelic discrimination (Applied Biosystems) using 5 to 15 ng of DNA amplified by the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Genotypes were determined by real-time fluorescent measurements.28 WNK1 genotypes were detected PCR using the ABI PRISM 7900HT, and genotypes were called using ABI PRISM SDS software version 2.1 (Applied Biosystems).
Data are expressed as means±SEMs. Several hypotheses were tested with ANOVA using a sequence of least squares models. The first analysis used a stepwise procedure to test for 2-way interactions between each of the genetic loci and age, BMI, and gender. The initial model included all of the possible 2-way interactions between the genotypes of each locus and these 3 anthropometric factors. In each successive step, the factor with the highest nonsignificant P value was removed from the model, and the analysis was repeated using a model including the remaining factors. This process was repeated until only statistically significant effects (P<0.05) were retained. These factors were then used in another set of analyses to test for the main effects of and interaction among the 3 genes. A similar stepwise procedure was followed. The initial model included all of the 2- and 3-way interactions among the adducin genes. Again, the single least significant factor was removed from the model in a series of steps until only significant effects remained. Statistical analysis was performed with SPSS (10.0.8 for Macintosh; SPSS Inc) and with SAS 8.2 statistical software (SAS Institute Inc). Our statistical methods also included single and multiple linear regressions. We searched for possible covariates of the arterial and renal phenotypes using stepwise multiple regression with the P value for independent variables to enter and stay in the model set at 0.15. Covariates considered for entry into the model were observer, age, BMI, and gender.
The clinical characteristics of the subjects investigated in the 3 studies are shown in Table 1. Please note that the subjects in the acute Na load study and the diuretic study are subsets of the ABPM study. The higher clinic BP values in the diuretic study subgroup reflect the selection criteria for this substudy (see Methods). Because the most informative SNP for the WNK1 gene was rs880054, we present the data on this SNP in the main article, together with those of ADD1 Gly460Trp and G/A NEDD4L polymorphisms. Data on the other WNK1 SNPs are reported, as appropriate, in Table S1 (available online at http://hyper.ahajournals.org).
Study 1: ABP
ABP was performed in 690 hypertensive patients (Table 1). In our study population, females had lower (P>0.01) BMI and both SBP and DBP.
The single variant analysis for ADD1, WNK1, and NEDD4L SNPs on ABP divided into daytime and nighttime periods is shown in Table S1. As reported previously,25 hypertensive subjects carrying ≥1 ADD1 Trp allele had both higher daytime and nighttime SBP. Among the other variants analyzed, only WNK1 rs2277869 showed an effect with the 10 subject homozygotes for the G allele having a significantly higher BP.
In 2-gene analyses, we observed a significant interaction on nighttime SBP between ADD1 Gly/Trp and WNK1 A/G (P=0.023; Table S2). Moreover, there was a trend toward a significant (P=0.06) interaction for daytime SBP and nighttime DBP for these variants. Evidence of interaction was also found between ADD1 Gly460Trp and NEDD4L on daytime DBP (P=0.041; Table S2).
When all 3 of the genes were analyzed together, there was a significant trend for differences for nighttime SBP in subjects carrying different genotype combinations (Figure 1). Subjects carrying the gene combination ADD1 Trp/WNK1 AA/NEDD4L AA had a nighttime SBP 13.3 mm Hg lower than those carrying the combination ADD1 Trp/WNK1 GG/Nedd4LG (P=0.044). No effect on the Gly background was found (data not shown). No such trends were discernible for daytime SBP or DBP in either period.
Study 2: Acute Na Load
We investigated the genotype dependence of the renal and BP responses to an acute Na load (310 mmol in 2 hours) in 344 patients (Table 2). At baseline (time 0), plasma renin activity was lower in ADD1 Trp carriers, as reported previously.3,6 Furthermore, fractional excretions of both Na and K were significantly lower in NEDD4L GG subjects. There were no significant differences in all of the other baseline (time 0) characteristics between subjects grouped by genotype (Table 2). The mean increase in SBP at 120 minutes was 5.2 mm Hg (Figure 2).
In single gene analysis, the ADD1 Trp allele was associated with a greater ΔDBP at 120 minutes, whereas the increase in SBP was not significant. There was no significant difference in BP response in the different WNK1 genotype groups. However, those carrying the WNK1 genotype showed a greater urinary Na and K excretion to the acute Na load than WNK1 G carriers (Table 2). NEDD4L AG and GG genotypes showed a greater SBP rise and a smaller urinary Na excretion than the other genotypes. Finally, the decrease in plasma aldosterone with Na loading was more pronounced in the NEDD4L GG patients.
In 2-gene analyses, we found an interaction between ADD1 and WNK1 polymorphisms in relation to absolute changes (Table 3) in SBP and Na excretion in response to Na loading. Indeed, no WNK1 GG effect was observed in the presence of ADD1 Gly. However, ADD1 Trp background amplified the WNK1 GG effect on SBP (10.7 mm Hg; P=0.041). Similarly, ADD1 Trp highlighted the NEDD4L AG effect on ΔDBP and change in mean BP. An additive effect (P=0.001) of WNK1×NEDD4L on urinary Na excretion was also observed (Table S3).
When we analyzed the combined effect of variants on all 3 of the genes on ΔSBP after acute Na load, treating the NEDD4L G allele as dominant for this analysis, we found a significant BP×genotype interaction (Figure 2; P=0.021). The 2 extremes resulted in a 15.3-mm Hg SBP difference, going from the “protective” combination ADD1 Trp/WNK1 AA/NEDD4L AA (−1.4 mm Hg) to the “salt-sensitive” one of ADD1 Trp/WNK1 GG/NEDD4L G (+13.9 mm Hg). On the ADD1 Gly background, no significant BP-genotype interaction was found, and the SBP differences between the 2 extremes of the same genotype combination were negligible (Figure S1).
After infusion of 2 L of saline, renal electrolyte handling can be affected by changes in kidney perfusion pressure. Thus, a more appropriate evaluation of the influence of genotypes on renal electrolyte handling should consider the simultaneous changes in BP by calculating the slope of the relationship between SBP and Na excretion both under basal conditions and at the end of the infusion period. As shown in Figure S2, the slopes of the pressure-natriuresis relationship were higher in patients who carried ADD1 Trp/WNK1 GG/ Nedd4L G genotypes (0.052±0.003 mm Hg/μEq per minute) than in those carrying the combination ADD1 Trp/WNK1 AA/Nedd4L AA (−0.0075±0.004 mm Hg/μEq per minute; Ptrend=0.009). This indicates that a larger increase of SBP was needed in the Trp/GG carriers to excrete the same amount of Na. In the ADD1 Gly background these differences were not present (ADD1 Gly/WNK1 AA/Nedd4L AA 0.012±0.01 mm Hg/μEq per minute; ADD1 Gly/WNK1 GG/Nedd4L G/A 0.032±0.008 mm Hg/μEq per minute; P value not significant).
Chronic Diuretic Protocol
The single variant analysis of BP values of 193 patients who carried out the diuretic protocol is reported in Table 4. The mean falls in SBP and DBP after 1 month were −8.5 mm Hg (Figure 3) and −5.1 mm Hg, respectively. The absolute SBP and DBP decrease (Δ) was significantly greater for ADD1 Trp carriers than ADD1 Gly subjects (Table 4). No consistent associations were seen with any of the other variants considered individually (data not shown). In the 2-gene analyses, ADD1 and WNK1 showed an additive effect similar to that found after saline infusion (Table S4).
When considered in combination, there was a significant trend (P=0.008) in different levels of fall in SBP at 1 month according to the combined genotypes ranging from −3.4 mm Hg for the combination of ADD1 Trp/WNK1 AA/Nedd4L AA to −23.2 mm Hg for the combination ADD1 Trp/WNK1 GG/ Nedd4L GG, respectively (Figure 3).
We report novel data supporting a role for variants in ADD1, WNK1, and NEDD4L on renal Na handling and consequently daily BP, BP response to saline infusion, and BP lowering after thiazide treatment. Most importantly, we demonstrate the importance of taking into account the physiological interactions between the systems in identifying and understanding genetic effects. Our studies show that, when individuals carry specific alleles of the 3 genes in combination, the effects on renal Na handling and BP homeostasis can be marked and of potential clinical relevance.
Several previous studies have investigated the association of ≥1 variant in ADD1, WNK1, and NEDD4L on BP. These findings have been inconsistent when these variants are considered alone. Indeed, we could not replicate here the previously reported single SNP associations of variants in WNK1 with regard to thiazide diuretics.25 We hypothesized that, in a highly integrated and spatially organized system, such as renal Na transport, the effect of a functional variant in 1 constitutive gene will invariably be modulated by the effect of functional alleles in other genes such that the final outcome will represent an overall balance of effects. This modulation may occur either between the luminal and basolateral membrane or between the more proximal and more distal segment of the nephron. In this situation, the effects of individual variants may be seen in some circumstances but not others. Our findings provide support for this hypothesis.
We found a remarkable degree of consistency and internal validity in the findings in the 3 studies. Thus, in combination, the same alleles of the 3 genes (ADD1 Trp, WNK1 G, and NEDD4L G), tracked with reduced Na excretion after a saline change, a greater acute rise in BP, an increased nocturnal SBP, and a larger BP reduction with thiazide diuretic treatment. These findings indicate that, in combination, the primary abnormality that these alleles are associated with is a tendency to greater Na retention. The effect on Na retention and, consequently, blood volume may explain why the association of the alleles is seen on SBP rather than DBP. Furthermore, in the ABP study, we found an association of the combined alleles on nocturnal rather than daytime SBP. This could reflect the fact that SBP during the daytime is much more variable and subject to other hemodynamic influences, whereas nocturnal SBP more accurately reflects the volume state.29
Our study suggests that the relative spatial location of the Na transport systems is also important. Thus, the effects of WNK1 G and NEDD4L G alleles were primarily visible on the ADD1 Trp rather than on the ADD1 Gly carriers. ADD1 Trp allele, therefore, appears to be permissive in this regard. In the first condition, mutated adducin (Trp) increases Na+-K+, ATPase activity in the proximal segments of the nephron, thus, the amount of Na delivered to the more distal ones, where WNK1 and NEDD4L are located, may determine an increased activity of these systems. However, in the presence of the wild-type ADD1, these effects are masked.
Our study has strengths and limitations, starting with the choice of the polymorphisms for study. There is evidence that the Gly460Trp change in α-adducin has direct functional effects.30 On the contrary, WNK1 rs880054 (which is located in intron 10), associated with ABP in a previous study,19 is probably a surrogate marker for functional variants still to be identified affecting the luminal Na transport. NEDD4L rs4149601 G→A substitution at the first nucleotide of exon 1 of the NEDD4L leads to an alternative splice site, which generates a transcript encoding a protein lacking the functionally crucial Ca++-dependent lipid-binding domain (C2 domain).31 This isoform of NEDD4L is abundantly expressed in the human kidney. NEDD4L, lacking the C2 domain, downregulates ENaC more potently than NEDD4L with an intact C2 domain,32 suggesting that carriers of the NEDD4L G allele have higher ENaC expression and higher renal Na reabsorption through ENaC than carriers of the A allele. Furthermore, although we observed a consistency of effects across the studies in the combined analysis and the trends were significant because putative “protective” alleles were progressively replaced by “deleterious” alleles, the number of individuals in the extreme genotype groups is small, and further studies in much larger groups are necessary to provide greater precision about the true size of the effect. In spite of these limitations, an interaction between genes affecting luminal and basolateral Na transport may product a synergic effect on transcellular Na transport.
BP has a strong genetic determination, with heritabilities for SBP and DBP estimated at between 40% and 60%. Yet, despite this, it has proved extremely difficult to define the major genetic regulators of BP. Our approach provides a paradigm that may need to be followed if the important determinants of BP are to be identified. It emphasizes that an integrative physiological interaction is crucial. Where such an approach can be reliably adopted, then significantly sized genetic effects of potential clinical relevance, from both a mechanistic and therapeutic perspective, may be discernible. Based on physiological knowledge, we show that relatively common alleles in the ADD1, WNK1, and NEDD4L genes, when present in combination, have significant effects on renal Na handling, BP, and antihypertensive response to thiazides.
We acknowledge Prof Jean-Marc Lalouel (University of Utah, Salt Lake City, Utah) for the scientific suggestions and Cinzia Scotti for the expert technical assistance.
Sources of Funding
Research included in this report was supported by the European Union (grants IC15-CT98-0329-EPOGH and LSMH-CT-2006-037093), InGenious HyperCare, Ministero Universitá e Ricerca Scientifica of Italy (progetti di ricerca di interesse nazionale [PRIN] grant 2006065339_01), the British Heart Foundation, and the Wellcome Trust Functional Genomics Programme. N.J.S. holds a British Heart Foundation Chair.
G.B. is a consultant of Prassis Sigma Tau Research Institute (Settimo Milanese, Milan, Italy). The remaining authors report no conflicts.
- Received March 27, 2008.
- Revision received April 14, 2008.
- Accepted June 7, 2008.
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