Journal of Human Hypertension (2003) 17, 187–191
& 2003 Nature Publishing Group All rights reserved 0950-9240/03 $25.00
ORIGINAL ARTICLE
Renin-angiotensin-aldosterone system
and G-protein beta-3 subunit gene
polymorphisms in salt-sensitive essential
hypertension
E Pamies-Andreu, R Ramirez-Lorca, P Stiefel Garcı́a-Junco, O Muñiz-Grijalbo,
I Vallejo-Maroto, S Garcia Morillo, ML Miranda-Guisado, JV Ortı́z and
J Carneado de la Fuente
Unidad de Hipertensión Arterial y Lı´pı´dos, Departamento de Medicina Interna, Hospitales Universitarios
Virgen del Rocı´o, Sevilla, Spain
Approximately 50% of hypertensive patients are salt
sensitive (they increase their Blood Pressure in re-
sponse to sodium intake or volume expansion). Mechan-
isms underlying salt sensitivity are not completely
elucidated although there is evidence that they may be
genetically determined. The aim of this study is to
establish the relation among some genetic polymorph-
isms of the renin–angiotensin system (RAAS) and the
beta-3 subunit of the protein G and salt sensitivity. We
studied 102 essential hypertensive patients, stage 1–2
and without target organ damage. Salt sensitivity was
assessed by the rapid protocol of Weinberger. We
determined by polymerase Chain reaction techniques
the following polymorphisms: insertion/deletion (I/D) of
the angiotensin-converting enzyme (ACE), A1166C of
the angiotensin II type 1 receptor (AT1R),
344C/T and
Keywords: salt sensitivity; genetic polymorphisms; renin–angiotensin system; aldosterone synthase; protein G
Introduction
The blood pressure (BP) response to changes in
sodium and extracellular volume balance is hetero-
geneous among hypertensive patients. Patients
who increase their BP with a high sodium diet or
volume expansion or in whom BP is reduced
following volume depletion are called salt sensitive.
The rest of patients are salt resistant.1–3 Approxi-
mately 50% of hypertensive patients are found
to be salt sensitive.4 Although the mechanisms
responsible for salt sensitivity are not completely
Correspondence: Dr J Carneado de la Fuente, Unidad de
Hipertensión Arterial y Lı́pı́dos, Departamento de Medicina
Interna, Hospitales Universitarios Virgen del Rocı́o, Avda, Manuel
Siurot s/n, 41013 Sevilla, spain,
E-mail: jcarnead@hvr.sas.cica.es
Received 5 August 2002; revised 1 December 2002; accepted 9
December 2002
www.nature.com/jhh
intron 2 conversion (IC) of the aldosterone synthase
(CYP11B2), and C825T of the beta-3 subunit of the
protein G (GNB3). 41 patients (40.19%) were salt
sensitive. The distribution of the different polymorph-
isms was similar in both groups of patients, but subjects
carriers of the W allele of the CYP11B2 IC polymorphism
had a greater risk for salt sensitivity as compared with
no carriers (37 of 41, 90.2% vs 4 of 41, 9.8%, OR 3.02,
Po0.05). Although there is no association between salt
sensitivity and the different studied genotypes of the
RAAS and of the GNB3, our data show a greater risk for
salt sensitivity among carriers of the W allele of the
CYP11B2 1C polymorphism.
Journal of Human Hypertension (2003) 17, 187–191.
doi:10.1038/sj.jhh.1001534
elucidated, there is evidence that they may be
genetically determined.5,6 The relation between
salt sensitivity and some genetic polymorphisms
of the RAAS has been studied in the last years
with contradictory results.7–11 It has also been
recently reported that there is no association
between a polymorphism in the GNB3, implicated
with enhanced G-protein activation and increased
activity of the sodium–proton exchanger, and
dietary salt response in normotensive men.12 In
order to further clarify the role of these polymorph-
isms in salt sensitivity, the aim of this study is to
examine the relationship between salt sensitivity
and the following polymorphisms: the angiotensin-
Converting enzyme (ACE) insertion/deletion (I/D),
the AT1R A1166C, the CYP11B2 -344 C/T
and CY11B2IC and the GNB3 C825T gene poly-
morphism in a group of patients with essential
hypertension.
 Genetics and salt sensitivity
EP Andreu et al
188
Materials and methods
Patients
A total of 102 outpatients with essential hyper-
tension classified as stage 1 or 2, according to the VI
Report of the Joint National Committee,13 were
consecutively recruited from the Hypertension and
Lipids Unit of the Hospitales Universitarios Virgen
del Rocı́o, Sevilla (Spain). Patients were under 60
years of age and the diagnosis of hypertension was
established after at least three office BP measure-
ments 4140/90 mmHg. Secondary causes of hyper-
tension were ruled out after complete clinical,
biochemical and radiological examinations. None
of the patients had renal impairment, papilledema,
cardiac, coronary or cerebrovascular diseases, dia-
betes or pregnancy. We included patients who were
not previously treated or in which antihypertensive
treatment was discontinued for at least 3 weeks
before testing. Written informed consent was
obtained from all the participants and the protocol
was approved by our Hospital Ethics Committee.
Protocol
Patients were initially maintained in a ‘regular’ diet
of 150 mEq/day of sodium for 15 days, to allow
equilibrium of the systemic sodium balance and BP.
Subjects then came to the Clinic after overnight
fasting and without smoking, and they were
assessed for salt sensitivity according to the rapid
protocol of Weinberger et al.14 Briefly, rapid volume
expansion was achieved by the intravenous admin-
istration of 2l of normal (0.9%) saline over a 4 h
period between 9 am and 1 pm. BP was measured
three times, at 5 min intervals the last 15 min of the
infusion, and the mean of these three measures was
taken as the BP representative of this period. On the
following day, sodium and volume depletion was
accomplished by administration of a 10 mEq sodium
diet and three 40 mg doses of furosemide given
orally at 10 am, 2 pm and 6 pm. On the third day,
patients came again to the Hospital and their BP
was measured in the way previously described. We
classified those individuals as salt sensitive in
whom mean BP (calculated as diastolic Blood
Pressure plus 1/3 of the pulse pressure) decreased
X10 mmHg when comparing the measurement
made at the end of the saline infusion with the
one following volume depletion. The rest of patients
were classified as salt resistant. Blood samples and
24 h urine collection (to confirm volume and sodium
expansion and depletion) were also obtained in each
period. Blood glucose, blood urea, serum creatinine
and serum and urinary concentrations of electro-
lytes were determined by routine chemical methods.
Plasma renin activity (PRA) and plasma aldosterone
(PA) concentrations were assayed by radioimmuno-
assay.
Journal of Human Hypertension
Genomic DNA was prepared from whole blood
according to standard procedures. Polymerase chain
reaction (PCR) was conducted in a 50 ml volume
reaction containing 20 mM Tris–HCl, pH 7.5,
100 mM KCI, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween
20, 0.5% Nonidet P40, 50% glycerol, 1.5 mM MgCl2,
0.1 mM dNTPs, 0.4 mmol/l each primer, 2 U Taq
polymerase (Roche) and 250 ng genomic DNA.
Detection of ACE I/D polymorphism
The I/D polymorphism of the ACE gene was
assessed by detecting the presence (allele I, inser-
tion) or absence (allele D, deletion) of a 287 bp in
intron 16 of the ACE gene in chromosome 17 with
the PCR technique and agar electrophoresis as
described by Rigat et al.15
Since the D allele is preferentially amplified in
heterozygous subjects,16 each sample found to be
DD was reanalysed by a second independent PCR
amplification with a primer that specifically recog-
nises an insertion-specific sequence.17
Genotype determination for AT1R A1166C
polymorphism
The A1166C polymorphism of the AT1 receptor was
genotyped using the PCR-restriction fragment length
polymorphism (RFLP) analysis.18
Genotyping of CYP11B2 polymorphisms
The subjects were also genotyped by PCR for two
recently discovered polymorphisms in the CYP11B2
gene, chromosome 8q22 as previously described.19
Genotype determination for GNB3 C825T
polymorphism
TT, CC and CT genotypes of the GNB3 gene
polymorphisms were determined according to the
previous reports.20,21
Statistical analysis
The association of salt sensitivity with genotype
distribution of patients was tested by w2 test. For
each genotype, we also studied the association of
salt sensitivity with distribution of patients in two
groups according to the condition of being or no
carriers for the different alleles. One-way ANOVA
was used to compare means among different groups.
Hardy–Weinberg equilibrium was tested by w2,
values are expressed as means 7 s.d.: Po0.05 was
considered to be statistically significant.
 Genetics and salt sensitivity
EP Andreu et al

189
Results Table 2 Distribution of insertion/deletion (I/D) angiotensin-
converting enzyme (ACE), A1116C angiotensin III type 1 receptor
Following the mentioned criteria, we found that (AT1R), -344C/T aldosterone synthase (CYP11B2) and C825T beta-
41 patients were salt sensitive (40.19%). General 3 subunit of the protein G (GNB3) gene polymorphisms in salt-
sensitive (SS) and salt-resistant (SR) patients
characteristics, blood chemistry, basal PRA and PA
of salt-sensitive and salt-resistant patients are Genotypes SS (n=41) SR (n=61)
expressed in Table 1. The distribution of different
genotypes followed that expected by the Hardy– ACE DD 22 (53.6%) 34 (55.7%)
Weinberg equilibrium. I/D ID 14 (34.1%) 22 (36%)
Table 2 shows the distribution of ACE I/D, AT1R II 5 (12.2%) 5 (8.2%)
A1166C, GNB3 C825T and CYP11B2 -344C/T geno- AT1R
A1116C AA 18 (43.9%) 31 (51%)
types in salt-sensitive and salt-resistant patients. AC 18 (43.9%) 23 (37.7%)
There were no differences between both groups of CC 5 (12.2%) 7 (11.4%)
patients with regard to these genotypes. If we GNB3
consider the distribution according to the condition C825T CC 11 (26.8%) 23 (37.7%)
CT 25 (60.9%) 31 (51%)
of being or no carriers for the different alleles, the TT 5 (12.2%) 7 (11.4%)
results do not change (data not shown). The CYP11B2
decrease of BP from volume expansion to volume -344C/T CC 15 (36.5%) 12 (19.6%)
depletion was similar in the different genotypes CT 16 (39%) 28 (46%)
(Table 3). TT 10 (24.4%) 21 (34.4%)
In Table 4, we report the distribution of CYP11B2
P=NS for all comparisons.
IC gene polymorphism and W vs no W allele
carriers in salt-sensitive and salt-resistant patients.
There were no statistically significant differences
for the distribution of the three different geno- Table 3 Decrease of BP from volume expansion to volume
types between the groups but because the depletion according to the different genotypes
values were almost significant (w2 ¼ 4.74, P ¼ 0.09)
and the very low frequency of patients with CC Genotypes Decrease of blood
genotype in the group of SS patients could be pressure (mmHg)
the factor determining the lack of significance,
ACE DD 9.07 7 8.44
to avoid this schew we performed the w2 for I/D ID 8.24 7 6.19
the trend with the following results: odds ratio for II 9.02 7 8.88
salt sensitivity for the group with CC genotype 1; AT1R
for the CW genotype 2.46 and for the WW geno- A1116C AA 8.88 7 8.13
type 4.58 (w2 ¼ 4.58, P ¼ 0.03). When comparing AC 10.42 7 7.40
CC 7.55 7 8.41
patients carriers of the W allele (WW plus WC GNB3
genotypes) vs no carriers (CC genotype), the C825T CC 7.09 7 6.96
former group had a greater risk for salt sensitivity CT 9.79 7 8.02
(Po 0.05; OR 3.02). TT 7.20 7 9.77
CYP11B2
-344C/T CC 10.31 7 6.49
CT 7.90 7 7.35
TT 9.04 7 10.76
CYP11B2
Table 1 General characteristics, blood chemistry, plasma renin Intron 2 conversion CC 6.32 7 9.88
activity (PRA) and plasma aldosterone (PA) in salt-sensitive (SS) WC 9.29 7 9.10
and salt-resistant (SR) patients WW 9.68 7 6.21

SS (n=41) SR (n=61) ACE (I/D): Insertion/deletion of angiotensin-converting enzyme,

A1166C of angiotensin II 1 receptor (AT1R),C825 T of the beta-3
Age (years) 39.38 7 8.77 35.86 710.41* subunit of protein G (GNB3), -344C/T and intron 2 conversion (W:
Sex (male/female) 21/20 37/24 wild allele, C: conversion allele) of aldosterone synthase (CYP 11B2)
BMI (kg/m2) 27.56 7 3.65 27.45 7 3.61 gene polymorphisms. P=NS for all comparisons.
MBP (mmHg) 107.34 7 1.03 107.1 7 10.45
Blood glucose (mg/dl) 91.40 7 9.85 90.59 7 9.81
Plasma creatinine (mg/dl) 0.86 7 0.13 0.87 7 0.1 2
Na+ (mEq/l) 140.33 7 2.22 140.6 7 1.93
K+ (mEq/l) 4.07 7 0.27 3.97 7 0.36 Discussion
Basal PRA (mgr/ml/h-1) 1.17 7 1.21 1.45 7 1.04
Basal PA (nmol/l) 0.481 7 0.45 0.408 7 0.26 The present study shows that there is no relation
24-h UNa+ (mEq) 126.91 7 57.71 142.62 7 65.36 among salt sensitivity and ACE I/D, AT1R A1166C,
24-h UK+ (mEq) 72.26 7 25.39 70.86 7 27.43
CYP11B2 -344C/T, nor GNB3 C 825T polymorph-
isms in essential hypertensive patients. Previous
BMI: body mass index; MBP: mean blood pressure; 24-h UNa+:
urinary excretion of sodium in 24 h; 24-h UK+: urinary excretion of studies addressing the relation between ACE I/D
potassium in 24 h; *Po0.05. genotype and salt sensitivity had contradictory

Journal of Human Hypertension

 Genetics and salt sensitivity
EP Andreu et al
190
Table 4 Distribution of the intron 2 conversion of aldosterone
synthase (CYP11B2) gene polymorphism and W allele carriers vs
no W carriers in salt-sensitive (SS) and salt-resistant (SR)
hypertensive patients
SS (n=41)
Genotype*
CC 4 (9.7%)
WC 19 (46.3%)
WW 18 (43.9%)
W allele carriers vs no carriers**
W carriers (WW plus WC) 37 (90.2%)
No W carriers (CC) 4 (9.8%)
W=wild allele C=conversion allele.
*Po0.1 (NS),
**Po0.05.
results: association with either II or DD genotypes
or no association.7–11 One possible explanation for
these discrepancies is the different protocols used to
assess salt sensitivity and probably the different age
and ethnicity of the studied patients. We classified
patients as salt sensitive or salt resistant according
to the rapid protocol of Weinberger et al,14 this
method has been validated and has a good correla-
tion with dietary techniques to assess salt sensitiv-
ity,14,22 and the frequency of salt sensitivity in our
patients (40.19%) was the expected according to the
literature.4 In addition, we only included subjects
below the age of 60 years to increase the genetic
component of the studied phenotype.23
With regard to the association between salt
sensitivity and the AT1R A1166C polymorphism,
our negative results are in agreement with that
previously described by Giner et al9 and Poch et al.10
To our knowledge there are no further studies
addressing this subject.
The GNB3 C825T polymorphism has been studied
in relation with hypertension with contradictory
results.24–27 We did not find any association between
this polymorphism and salt sensitivity in hyper-
tensive patients. Similar results were found in a
previous study made in normotensive men and with
a dietary protocol.12
Tamaki et al28 and Komiya et al29 described an
association between the C-344 allele of the CYP11B2
and low renin hypertension with inappropiated
elevation of aldosterone in Japanese hypertensive
patients. On the contrary, a study made in Italian
people found an increased frequency of the allele T
and a relative excess of TT over CC homozygosity in
patients with low-renin hypertension in comparison
with both normal-to high- renin hypertension and
normotensive controls.30 Both studies suggested that
this polymorphism might be related with salt
sensitivity. However, this relation was not con-
firmed in a further study in Spanish population.10
We did not find any relation between the CYP11B2
-344C/T polymorphism and salt sensitivity in
Journal of Human Hypertension
agreement with the Spanish report 1010 and other
study in Caucasian population.31
In our study, there was no association between the
IC of the CYP11B2 genotypes and salt sensitivity as
SR (n=61) has been previously reported.10 This polymorphism
has also been studied in relation with hyper-
tension32 with negative results. We did not find
15 (24.5%) any difference between salt-sensitive and salt-
29 (47.5%) resistant patients for the distribution of the different
17 (27.8%)
genotypes of this polymorphism, although patients
carriers of the W allele had a greater risk for salt
46 (75.4%) sensitivity than no carriers. To our knowledge, this
15 (24.6%) is the first report about the possible implication of
this allele in salt sensitivity. Our hypothesis is that
the W allele could be associated with increased
activity of the enzyme determining the condition
of salt sensitivity. This is plausible in view of the
importance of aldosterone in sodium and water
homeostasis. The similar levels of aldosterone in
both groups of patients in our study are not against
this possibility.
In conclusion, although we did not find any
relationship among the different genotypes of the
RAAS, beta-3 subunit of the G-protein and CYP11B2
and salt sensitivity in accordance with previous
reports, our data show a greater risk for salt
sensitivity among W allele carriers of the CYP11B2
IC polymorphism. Further studies are needed to
clarify this subject.
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