Angiotensin I–Converting Enzyme Gene Polymorphism and Salt Sensitivity in Essential Hypertension
Abstract We undertook the present study in 66 Japanese patients with essential hypertension to identify genetic factors associated with salt sensitivity. Patients were classified into salt-sensitive or salt-resistant groups on the basis of changes in their mean blood pressures from a week of a low salt diet (50 mmol/d) to a week of a high salt diet (340 mmol/d). Salt sensitivity and resistance were studied in relation to a 287-bp insertion/deletion (I/D) polymorphism of the angiotensin I–converting enzyme gene detected by a polymerase chain reaction method and the haptoglobin phenotype determined by polyacrylamide gel electrophoresis. Patients with the angiotensin I–converting enzyme gene genotype II were more apt to be salt sensitive than patients with the ID and DD genotypes, although plasma renin activity was similar in each group. The frequency of the I allele in the salt-sensitive group was significantly higher than that in the salt-resistant group (χ2=7.4, odds ratio=2.78). However, there was no significant relationship between haptoglobin phenotype and salt sensitivity. These data suggest that an I/D polymorphism of the angiotensin I–converting enzyme gene is a genetic factor associated with salt sensitivity of blood pressure independently of plasma renin activity in Japanese patients with essential hypertension.
High BP may be an expression of the combined and interactive effects of genetic and environmental factors. Among environmental factors, dietary sodium has been generally accepted as an important contributor to hypertension.1 However, the BP response to a high salt intake, called salt sensitivity, varies among individuals. Although mechanisms by which salt intake increases BP are still not completely understood, inappropriate suppression of the renin-angiotensin-aldosterone system,2 3 4 greater volume expansion,5 and the intracellular accumulation of sodium and calcium6 have been proposed factors. We have previously revealed that essential hypertensive patients with an apparent hereditary component of hypertension could be characterized as a salt-sensitive subgroup with the accumulation of intracellular sodium.7 However, there is limited information about actual genetic markers for salt sensitivity in patients with essential hypertension. Therefore, in this study, we focused on the candidates of genetic markers associated with salt sensitivity of BP.
Genetic studies in stroke-prone spontaneously hypertensive rats have demonstrated that a locus linked to the ACE gene is strongly associated with BP.8 Rigat et al9 and Tiret et al10 have identified an I/D polymorphism in intron 16 of the ACE gene in humans and demonstrated that this polymorphism is related to serum ACE levels. Recently, the DD genotype of the ACE gene has been reported to be associated with an increased risk of cardiovascular diseases, such as myocardial infarction.11 A single study in humans established a small but significant association between the ACE gene polymorphism and hypertension by demonstrating that the I allele was associated with an increased incidence of hypertension12 ; however, other studies have failed to document such an association.10 13 Thus, the results are still controversial, and a conclusive association has not yet been established in patients with essential hypertension.
In addition, Weinberger et al14 have reported a relationship between haptoglobin phenotype and salt sensitivity in black and white populations in the United States although the significance of this polymorphism in other races such as Japanese is unclear.
In the present study, we determined whether polymorphism of the ACE gene and haptoglobin phenotype may be associated with the salt sensitivity of BP in patients with essential hypertension.
Sixty-six Japanese inpatients (36 men, 30 women; mean age, 51.4±8.9 years; age range, 33 to 65 years) with mild to moderate essential hypertension were studied. Patients older than 65 years were excluded because most of them have significant salt sensitivity.15 Hypertension was defined as a systolic BP greater than 160 mm Hg and/or diastolic BP greater than 95 mm Hg with patients in the sitting position on at least three different occasions recorded in the outpatient clinic. Patients with secondary forms of hypertension and/or extensive target-organ damage were excluded after appropriate clinical and laboratory examinations. No medication was permitted for at least 4 weeks before the study. Informed consent was obtained from all patients.
Patients were initially maintained on a “regular” diet of 170 mmol/d NaCl to allow stabilization of the systemic sodium balance and BP. Subjects were then given a low salt diet (50 mmol/d) for 1 week followed by a high salt diet (340 mmol/d) for an additional week. Throughout the study, the patients ingested a constant amount of K+ (2000 mg/d), Ca2+ (500 mg/d), and calories (40 kcal/kg). A daily 24-hour urine collection was performed for determination of urinary Na+ excretion.
On the last morning of each diet period, BP was determined with a mercury sphygmomanometer every minute for 10 minutes by the same physician throughout the study after patients were kept in a supine position for 30 minutes in a quiet, dark room. The mean of consecutive BP readings was used for analysis. Mean BP was calculated as diastolic BP plus one third pulse pressure. Salt sensitivity was decided on the basis of the percentage change in mean BP from a low to a high salt diet. Patients were classified as salt sensitive when the mean BP measured during the high salt period exceeded that in the low salt period by 10%. Patients whose change in mean BP after salt loading was less than 10% were classified as salt resistant. Blood glucose, blood urea nitrogen, and serum and urinary concentrations of creatinine and electrolytes were determined by routine chemical methods. PRA and plasma aldosterone concentration were assayed by radioimmunoassay.
DNA Analysis for ACE Genotype and Haptoglobin Phenotype
Samples for DNA analysis were obtained from peripheral leukocytes in 66 patients. An I/D polymorphism of the ACE gene was identified by detecting an alu repetition sequence of 287 bp in intron 16 with the polymerase chain reaction technique of Rigat et al.16 The sense oligonucleotide primer was 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′, and the antisense primer was 5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′. These primers allowed detection of a genomic DNA segment of 490 bp corresponding to the I allele as well as a segment of 190 bp corresponding to the D allele. The ACE genotype was classified as II, ID, or DD depending on whether each allele had this sequence. Reactions were performed in a final volume of 50 μL containing 25 pmol of each primer, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.4), 0.1 mg/mL gelatin, 0.2 mmol/L of each dNTP, and 1.25 U Taq DNA polymerase (Wako Pure Chemicals). The amplification profile included an initial denaturation at 95°C for 1 minute and 35 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 2 minutes, and extension at 72°C for 3 minutes. The PCR products were electrophoresed on 2% agarose gels, and DNA was visualized with ethidium bromide staining. In 56 patients, the haptoglobin phenotype was identified by examining their serum by the polyacrylamide gel electrophoresis method and classifying the haptoglobin phenotype as 1-1, 2-1, or 2-2.14 17
Values are expressed as mean±SD. The distribution of salt-sensitive and salt-resistant patients among groups with different polymorphic markers and alleles was compared with the χ2 test. Differences between groups were analyzed by one-way ANOVA. A value of P<.05 was considered statistically significant.
There was no significant difference between the salt-sensitive and salt-resistant groups in age, sex, body mass index, mean BP on the regular salt diet, degree of hypertensive retinopathy, duration of hypertension, or routine biochemical indexes such as blood glucose and serum concentrations of creatinine and electrolytes (Table 1⇓). However, mean BP and urinary excretion of sodium and calcium increased significantly, and serum total calcium concentration and PRA decreased significantly in each subgroup with the change from a low to high salt diet (Table 2⇓). Mean BP during the low salt diet was significantly lower, and that during the high salt diet was significantly higher in the salt-sensitive than salt-resistant group. Pulse rate during each salt diet period was similar in the two groups. PRA values during both the regular and low salt diet were lower in the salt-sensitive than salt-resistant group (Tables 1⇓ and 2⇓).
The ACE genotype distribution and derived allele frequency are shown in Table 3⇓. The number of patients with ACE genotypes II, ID, and DD were 32, 24, and 10, respectively. There were no significant differences in age (II, 53±7; ID, 52±11; DD, 50±10 years), sex, or mean BP with the regular salt diet as well as PRA for each salt diet period (PRA with regular salt: II, 0.49±0.66; ID, 0.49±0.06; DD, 0.41±0.25 ng angiotensin I/mL per hour) among the three groups. The prevalence of salt sensitivity was significantly higher in patients with genotype II than in those with the two other genotypes. Also, the frequency of the I allele in the salt-sensitive group was significantly higher than that in the salt-resistant group.
The number of patients in groups with haptoglobin phenotypes 1-1, 2-1, and 2-2 were 6, 25, and 25, respectively (Table 4⇓). There were no significant differences among these three groups in the prevalence of salt sensitivity and parameters such as age, sex, and mean BP with the regular salt diet as well as PRA for each salt diet period. Comparison of allele frequencies between the salt-sensitive and salt-resistant groups also failed to show a significant difference.
We have previously reported that the elevation in mean BP with a high salt diet is greater in patients with essential hypertension whose parents are both hypertensive than in hypertensive patients without a family history of hypertension.8 Thus, we hypothesized that the genetic backgrounds of patients with essential hypertension may determine at least in part the heterogeneity in the pressor response to salt loading. In the present study, we focused on genetic markers for specific subtypes of essential hypertension to assess the role of the candidate gene in the determination of salt sensitivity of BP. This is the first report to document a significant association between the pressor response to salt loading and polymorphism of the ACE gene. This finding supports our previous proposal regarding the genetic contribution to salt sensitivity of a gene-environment interaction in Japanese patients with essential hypertension. This method for discriminating salt-sensitive patients according to ACE gene polymorphism may have significant pathogenic and clinical implications. The marginal statistical significance of the ACE gene polymorphism may reflect the fact that multiple factors are involved in the genesis of salt sensitivity.
As the renin-angiotensin system is a key factor in BP regulation, one could hypothesize that genes related to the renin-angiotensin system might contribute to the development of primary hypertension. Studies of stroke-prone spontaneously hypertensive rats have shown a close linkage between their elevated BPs and the chromosome region containing the ACE gene.8 18 These studies also have indicated that the phenotypic expression of a hypertensive locus linked to the ACE gene could be augmented by a high salt diet.8 18
Recently, the DD genotype of the ACE gene in humans has been reported to be associated with an increased risk of cardiovascular diseases such as myocardial infarction11 and ischemic or idiopathic dilated cardiomyopathy,19 especially in patients without other coronary risk factors. However, the results of analysis of the association between this polymorphism and essential hypertension are controversial, although this polymorphism is thought to account for half of the variance in serum ACE levels.9 Only one report by Zee et al12 has documented a small but significant association between ACE gene polymorphism and essential hypertension, but others have failed to find such an association.10 13 These discrepancies in results may be due to heterogeneity in the backgrounds of the patients with essential hypertension studied. Since factors regulating BP are complex and essential hypertension should not be regarded as a single disease entity, many investigators have divided this disease into distinct subgroups with their own characteristics. Patients with essential hypertension with a strong family history of hypertension may represent one such subgroup, as we have previously reported.7 Zee et al have reported a higher frequency of the I allele in essential hypertensive patients with hypertensive parents than in normotensive control subjects with normotensive parents. Notably, both the presence of the I allele and a positive family history have been demonstrated to be involved in salt sensitivity by our laboratory.
The purpose of the present study was to detect a genetic factor associated with salt sensitivity in patients with essential hypertension. Because most elderly patients are salt sensitive, the salt sensitivity in patients older than 65 years may be acquired rather than genetically determined. From the viewpoint of the genetic factor, the patients older than 65 years should be excluded in this study. Although we decided salt sensitivity of BP and assessed the polymorphism of the ACE gene and haptoglobin phenotype in five patients older than 65 years, when these patients are included in the present study, our results do not change; the significance of the ACE gene still exists.
It is well recognized that salt sensitivity is greater in individuals with lower PRA levels.4 In the present study, we found no significant difference among the three ACE genotype subgroups in background factors and PRA during each salt diet period. Therefore, the I polymorphism of the ACE gene may represent a genetic marker for salt sensitivity of BP that is independent of PRA in patients with essential hypertension.
The I/D polymorphism in the ACE gene lies within an intron, and there is no evidence for differential splicing that might incorporate this sequence into a primary transcript. Although several studies have documented that the level of serum ACE activity was in the order DD>ID>II for individuals with different genotypes,9 10 the mechanisms of how polymorphisms of the ACE gene contribute to salt sensitivity of BP remain unknown. Our study merely illustrates that the I/D polymorphism in the ACE gene may be used as a genetic marker for salt sensitivity of BP in patients with essential hypertension; the cause of the relationship between the gene and salt sensitivity of BP awaits further investigation.
In the present study, we failed to find a significant contribution of the haptoglobin phenotype to salt sensitivity of BP. However, Weinberger et al14 have reported that those individuals with the haptoglobin 1-1 phenotype are more likely to be salt sensitive, whereas those with the 2-2 phenotype are more likely to be salt resistant, although they assessed salt sensitivity by a rapid protocol (intravenous saline followed by furosemide). Kojima et al20 have also shown a significant association between the haptoglobin 1-allele and salt sensitivity. The discrepant results regarding haptoglobin phenotypes may have been caused by differences in study populations, the protocol of dietary salt manipulation (such as duration or the amount of salt), or the criteria of salt sensitivity of BP.
In conclusion, we found that an I polymorphism of the ACE gene was associated with salt sensitivity of BP in patients with essential hypertension. Therefore, salt sensitivity is partially determined by a genetic predisposition. Discrimination of salt-sensitive patients according to this I/D polymorphism of the ACE gene may have clinical implications and provide further insight into the pathogenesis of essential hypertension and the mechanism underlying salt sensitivity of BP.
Selected Abbreviations and Acronyms
|PRA||=||plasma renin activity|
This work was supported by a Grant-in-Aid for Scientific Research (06672293) from the Ministry of Education, Science, and Culture, Japan. We thank Yasuaki Nakasaki, MD, for invaluable advice and Yuko Omura for secretarial assistance.
MacGregor GA. Sodium is more important than calcium in essential hypertension. Hypertension. 1985;7:628-637.
Weinberger MH, Miller JZ, Luft FC, Grim CE, Fineberg NS. Definitions and characteristics of sodium sensitivity and blood pressure resistance. Hypertension. 1986;8(suppl II):II-127-II-134.
Luft FC, Weinberger MH, Grim CE, Fineberg NS, Miller JZ. Sodium sensitivity in normotensive human subjects. Ann Intern Med. 1983;98(part 2):758-762.
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990;86:1343-1346.
Cambien F, Poirier O, Lecerf L, Evans A, Cambou J-P, Arveiler D, Luc G, Bard J-M, Bara L, Ricard S, Tiret L, Amouyel P, Alhenc-Gelas F, Soubrier F. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992;359:641-644.
Weinberger MH, Miller JZ, Fineberg NS, Luft FC, Grim CE, Christian JC. Association of haptoglobin with sodium sensitivity and resistance of blood pressure. Hypertension. 1987;10:443-446.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1). Nucleic Acids Res. 1992;20:1433.
Smithies O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochem J. 1955;61:629-641.
Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T, Soubrier F, Dubay C, Cartwright P, De Gouyon B, Julier C, Takahashi S, Vincent M, Ganten D, Georges M, Lathrop GM. Chromosomal mapping of two genetic loci associated with blood-pressure regulation in hereditary hypertensive rats. Nature. 1991;353:521-529.