α-Adducin Gly460Trp Polymorphism and Renal Hemodynamics in Essential Hypertension
Previous studies have shown an association between the α-adducin Gly460Trp polymorphism and salt-sensitive hypertension. Not much is known about the effects of the variants of this polymorphism on renal hemodynamics and function. Therefore, we performed the present study to investigate the effect of the 460Trp allele of the α-adducin gene on renal hemodynamics in one hundred and seventeen essential hypertensive patients who were put on a low and high sodium diet (randomized order). On the last day of each one-week dietary period, blood pressure, effective renal plasma flow (ERPF), glomerular filtration rate (GFR), and neurohormones were measured. Effective renal blood flow (ERBF), renal vascular resistance, and filtration fraction were calculated. ERPF, ERBF, and GFR were lower in patients homozygous for the 460Trp allele compared with patients with the Gly460Gly genotype on low sodium diet but no differences were found at the higher sodium intake. On the other hand, levels of atrial natriuretic peptide were significantly higher in patients with the Trp460Trp genotype as compared with patients with the Gly460Gly genotype on both diets. In multivariate analysis, Trp460Trp genotype, age, and mean arterial pressure were predictors of ERPF, whereas Trp460Trp genotype and age were predictors of GFR during the phase of low sodium diet. The present study shows that the Trp460Trp genotype is significantly associated with reduced renal plasma flow and glomerular filtration rate as compared with the wild-type variant.
Essential hypertension is a complex disease, involving a variety of genetic, physiological, and environmental factors. However, according to modern physiology, no form of hypertension can persist without the presence of a renal defect.1 Predictably, a rise in blood pressure by whatever cause is followed by enhanced renal sodium output (pressure natriuresis) until the original situation is restored. Only when the sodium excretory capacity of the kidney is impaired, hypertension may develop. Not surprisingly, therefore, virtually all forms of monogenic hypertension are characterized by disturbances in sodium metabolism.2
In Milan hypertensive rats (MHS), a genetically determined cellular defect caused by a mutation in the α-adducin gene involving faster Na transport across the renal cell membranes is considered to be a probable cause of the elevated blood pressure in these animals. Considering the many similarities in renal and cellular dysfunctions between MHS rats and patients with essential hypertension and given the very high degree of sequence homology between rat and human α-adducin, it is possible that this molecular mechanism is also relevant to humans.3 Indeed, the α-adducin Gly460Trp polymorphism has been associated with enhanced renal tubular sodium reabsorption, which could account for a sodium-sensitive rise in blood pressure.4,5 Several studies have been performed on the association between this mutation in the α-adducin gene and sodium-sensitive hypertension. By and large, the results of these studies confirm that the 460Trp variant is associated with a greater degree of sodium sensitivity.4–7 In contrast to the many studies investigating the association between the α-adducin variants and the reaction of blood pressure to sodium, far less is known about the effect of these variants on renal hemodynamics and renal function. This is surprising considering the central role of the kidney and its blood supply on the regulation of sodium homeostasis. This lack of information prompted us to study the impact of the 460Trp allele of the α-adducin gene on renal hemodynamics in hypertensive patients during different sodium diets.
Patients and Protocol
One hundred and seventeen patients with hypertension attending the outpatient clinic of the University Hospital Maastricht were enrolled in this study. Hypertension was defined as a systolic blood pressure ≥140 mm Hg and/or a diastolic blood pressure ≥90 mm Hg without treatment. Inclusion criteria were an age between 30 and 75 years, no secondary causes of hypertension, and no other significant medical illness, including diabetes mellitus and renal insufficiency. Antihypertensive treatment, if any, was withdrawn 3 weeks before the study. Patients were investigated after 1 week of low sodium diet (55 mmol Na per day) and 1 week of high sodium diet (220 mmol Na per day), given in randomized order. Compliance with the diet was checked by measuring sodium, potassium, and creatinine output in 24-hour urine collections obtained on the last 2 days of each dietary period. Patients had to refrain from smoking, alcohol, and caffeine from 10 pm the evening before the measurements and they remained supine during the entire session. Effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) were measured by infusion of para-aminohippurate (PAH) and inulin. Before infusion, baseline blood samples were taken. After a 2-hour equilibration period, which was necessary to reach steady state plasma concentrations of PAH and inulin, blood samples were drawn at 120, 130, 140, and 150 minutes. ERBF, filtration fraction (FF), and renal vascular resistance (RVR) were calculated.8 Blood samples were taken for measurement of active plasma renin concentration (APRC), aldosterone and atrial natriuretic peptide (ANP). To ensure diuresis, patients consumed 200 mL of water every hour until the last blood samples had been drawn. The study was approved by the Medical Ethical Committee of the University Hospital Maastricht. Informed consent was obtained from each individual recruited.
DNA was extracted from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Leiden, the Netherlands). The genotypes of the α-adducin Gly460Trp polymorphism (Gly460Gly, Gly460Trp, and Trp460Trp) were detected by mutagenically separated PCR,9,10 as described previously.11
Hemodynamic and Assay Methods
Methods for the measurements of blood pressure and renal hemodynamics (ERPF and GFR) as well as the assays for the determination of APRC, aldosterone, ANP, PAH, and inulin levels have been described earlier.8
We estimated sample size by a power calculation based on the assessment of ERPF, the variable with the highest variation (6.4% variability for repeated measurements). To detect a difference in ERPF between groups of more than 10% with a type 1 error of 0.05, a statistical power of 80%, and a standard deviation of 60 mL/(min · 1.73 m2), we calculated that there should be at least 9 subjects in the Trp460Trp group and 80 subjects in the 460Gly group, using a power calculation program with sample size technique for unequal group sizes.12 The calculation of allele frequencies to test for Hardy-Weinberg equilibrium was performed using χ2 analysis comparing expected against observed frequencies. Because of the small number of patients in the Trp460Trp group (n<20), nonparametric tests were used for analysis of the data. Kruskal Wallis test was used to determine the association between the 3 genotype groups of the α-adducin Gly460Trp polymorphism and clinical characteristics and for univariate testing of differences in renal hemodynamics and hormonal levels between the genotype groups. When the Kruskal Wallis test revealed a significant effect of genotype, Mann-Whitney U test with Hochberg correction was used as post-hoc analysis.13 Linear regression analysis was done to determine the effect of the α-adducin genotype on ERPF and GFR, taking potential confounders (age, gender and MAP) into account. Analysis of covariance was used to detect differences between the 3 genotype groups with respect to the relationship between ERPF and age. Wilcoxon signed ranks test was performed to test for differences in neurohormones between the 2 sodium diets. Because the α-adducin Gly460Trp polymorphism was the only genetic polymorphism studied in this article, no adjustments were made regarding multiple testing.
Because many patients could not fully comply with the initially high sodium diet of 220 mmol Na per day, we redefined the 2 dietary periods as low and normal-to-high rather than low and high. The clinical characteristics of the 3 genotype groups on both diets are summarized in Table 1. No differences with respect to the use of antihypertensive medication before the washout period were found among the groups. The genotype distribution of the α-adducin polymorphism did not deviate significantly from Hardy-Weinberg’s expectation (χ2=1.187, P=0.552). However, the distribution of the α-adducin Gly460Trp polymorphism was significantly different compared with another white population (χ2=12.1, P=0.002),4 the frequency of the Trp460Trp genotype being higher in our population (10% versus 4%). No significant differences between the 3 genotype groups were found with the exception of ANP, which was significantly higher in patients homozygous for the 460Trp allele on both diets (Kruskal Wallis test, P=0.026 and P=0.009, respectively). Post-hoc analyses revealed a significant difference in ANP on the low and the high sodium diet between patients with the Trp460Trp and Gly460Gly genotype (P=0.015 and P=0.009, respectively). Aldosterone levels tended to be lower in the Trp460Trp group, especially during the low sodium diet, but the differences were not statistically significant.
Low Sodium Diet
In univariate analysis, differences in renal hemodynamics between the 3 genotype groups were statistically significant (Kruskal Wallis test, P=0.041 and P=0.036, respectively, Table 2; Figure 1). Post-hoc analyses revealed a significant difference in ERPF and ERBF between patients with the Trp460Trp and Gly460Gly genotypes (P=0.039 and P=0.030, respectively). GFR was also different between the 3 genotype groups (Kruskal Wallis test, P=0.035, Table 2). Patients homozygous for the 460Trp allele had a lower GFR compared with patients homozygous for the 460Gly allele (post-hoc analysis, P=0.018; Figure 2). FF did not differ between the 3 genotype groups. RVR was higher in the Trp460Trp genotype group compared with the other groups but the difference was only of borderline significance (0.05<P<0.10). Taking possible confounders into account, we performed a linear regression analysis to determine predictors for renal hemodynamics. As expected, age and mean arterial blood pressure (MAP) proved to be important predictors of ERPF and GFR. Sex, on the other hand, did not predict renal plasma flow or glomerular filtration rate. In Table 3 the results of the linear regression models are given. The Trp460Trp genotype, age, and MAP were predictors of ERPF. Regarding GFR, the Trp460Trp genotype and age were predictors of GFR. When ERPF was plotted against age, no differences in slopes for the 3 genotype groups were found (Analysis of Covariance, P=0.968).
Normal-to-High Sodium Diet
Univariate analyses failed to show significant differences in ERPF, ERBF, RVR, GFR, and FF between the 3 genotype groups (Table 2). As on the low sodium diet, no differences in slopes for the 3 genotype groups were found when ERPF was plotted against age (ANCOVA, P=0.955).
Changes During the Dietary Switch
Because there was no time-treatment effect concerning the order of the sodium diets, the data from both periods were pooled for analysis. When changing from the lower to the higher sodium diet, no significant changes in blood pressure, ERPF, ERBF, RVR, and GFR were found in either genotype group. Nevertheless, FF was higher during the normal-to-high sodium diet in the Gly460Gly genotype group [0.31 (0.27, 0.35) versus 0.29 (0.25, 0.31) Wilcoxon Signed Ranks test, P=0.004]. In patients carrying at least one 460Gly allele, both APRC and aldosterone fell significantly after the switch from the lower to the higher sodium intake, whereas ANP significantly increased. In the Trp460Trp genotype group, only a decrease in aldosterone was seen. These changes in hormonal levels differed significantly among the genotype groups.
This study shows that the α-adducin Gly460Trp polymorphism has a demonstrable influence on renal hemodynamics in hypertensive patients. With low sodium intake, the Trp460Trp genotype was significantly associated with decreased renal plasma flow and glomerular filtration rate. On the normal-to-high sodium diet, no differences between the 3 genotype groups were seen regarding renal hemodynamics. In addition, we found that Trp460Trp patients were characterized by markedly elevated ANP levels on both diets and reduced suppression of renin with an increase in sodium intake.
Earlier studies in MHS rats indicated that the mutated human adducin variant displayed an increased affinity for Na-K-ATPase over that of the wild-type variant.14 In MHS rats, during the development of hypertension, GFR is higher and plasma renin lower compared with Milan normotensive (MNS) rats. This is in contrast to our finding that patients with the Trp460Trp genotype exhibit a significantly lower GFR. This discrepancy may be related to the duration of hypertension. Indeed, in the MHS rats GFR is higher only during the development of hypertension and not in the established phase. Because hypertension has been present for a long period of time in our patients, we suppose that secondary renal changes account for the lower GFR. A transient phase of renal sodium retention that could lead to an increase in body fluid volumes has also been observed in MHS rats. The maintenance of a normal sodium balance, despite a higher GFR, could be explained by assuming an increased renal tubular reabsorption.15 Although the 460Trp allele has been associated with an increase in renal (proximal) tubular reabsorption,16 it is not certain whether body fluid volumes are elevated in the Trp460Trp genotype group. However, levels of ANP were markedly elevated in this genotype group, which could point to a mechanism counteracting the tendency to retain more sodium. In this regard, the increase in ANP could be viewed as a compensatory mechanism to increase postglomerular resistance and hydrostatic pressure in the glomerular capillaries.17 The same mechanism may also be responsible for a reduced ERBF.
Despite the small number of patients in the Trp460Trp group, we clearly found a reduction in ERPF and GFR in this group. Because patients homozygous for the 460Trp allele are rare in most white populations, the Gly460Trp and Trp460Trp genotypes are usually combined to find an effect of this polymorphism on outcome parameters like blood pressure. This implicitly suggests that the 460Trp allele is dominant. However, the present data indicate that patients homozygous for the 460Trp allele are actually different from patients carrying the Gly460Trp genotype. The 460Gly allele seems to be the dominant one regarding renal hemodynamics. This is an important issue in studies on genetic polymorphisms.
In our study, the relationship between ERPF and age was not different between the 3 genotype groups. This is in contrast with the findings of Castellano et al,18 who have suggested that the pathogenic effect of the α-adducin Gly460Trp polymorphism is more evident at older age, possibly because of a reduced efficiency of compensatory mechanisms. Although our results do not substantiate this finding, we cannot exclude the possibility that we would have found age-related effects in the presence of other disturbances in kidney function. On the other hand, it should also be noticed that the population of Castellano et al concerns a sample of the general population, which is basically different from our sample of hypertensive patients.
Based on the hypothesis that the Trp460Trp genotype predisposes to a sodium-sensitive form of hypertension, one might have expected more marked alterations in renal hemodynamics during the normal-to-high salt phase in patients with this genotype. However, renal hemodynamics are not the only determinant of sodium sensitivity in hypertension. In fact, we found the expected suppression of renin and aldosterone and a rise in ANP in patients with at least one 460Gly allele but very little change in these hormones in the Trp460Trp patients. This could mean that, in the latter group, the renin system was more fixed, which is a recognized feature of sodium sensitivity. A fixed renin system, in turn, may again be associated with reduced renal blood flow.
The present study shows that the Trp460Trp genotype is significantly associated with a reduction in renal plasma flow and a reduction in glomerular filtration rate. Future studies will have to concentrate on subgroups of hypertensive patients with additional risk factors such as diabetes mellitus or renal insufficiency to assess whether such risk factors enhance the effect of the gene on renal hemodynamics. Moreover, it would be interesting to find out if patients with the Trp460Trp genotype group respond differently to interruption of the renin system compared with patients with 1 or 2 Gly460 alleles. In this respect, it is worthwhile also to evaluate whether the degree of sodium-dependency of blood pressure is related to the altered renal hemodynamics or the renin-angiotensin system or both.
This study was supported by grant NHF2002B205 from the Dutch Heart Foundation.
- Received February 24, 2004.
- Revision received March 12, 2004.
- Accepted July 12, 2004.
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