(Hypertension. 2001;37:882.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
G-Protein ß3 Subunit Gene (GNB3) 825T Allele Is Associated With Enhanced Renal Perfusion in Early Hypertension
From the Department of Medicine/Nephrology (R.Z., C.D., M.S., R.E.S.), University of Erlangen-Nürnberg (Germany); and the Department of Pharmacology (W.S.), University of Essen (Germany).
Correspondence to Prof Dr med Roland E. Schmieder, Department of Medicine IV/4, University of Erlangen-Nürnberg, Breslauer Str 201, D-90471 Nürnberg, FRG. E-mail roland.schmieder{at}rzmail.uni-erlangen.de
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
The C825T polymorphism of the gene encoding the G-protein ß3 subunit (GNB3) is associated with increased intracellular signal transduction and arterial hypertension. The aim of the study was to investigate the impact of this polymorphism on early adaptive processes of the left ventricle and renal hemodynamic changes in young normotensive to mildly hypertensive subjects. Ninety-five white male students with normal or mildly elevated blood pressure were genotyped for the GNB3 C825T polymorphism. In each participant, 24-hour ambulatory blood pressure, left ventricular structure and function (2D-guided M-mode echocardiography), renal plasma flow (para-aminohippurate clearance), glomerular filtration rate (inulin clearance), and 24-hour urinary sodium excretion were determined. The GNB3 825T allele was not associated with casual or ambulatory blood pressure, parameters of left ventricular structure or function, glomerular filtration, or 24-hour urinary sodium excretion. However, in T-allele carriers (CT+TT), renal plasma flow was higher than in CC subjects (CT/TT: 659±96 versus CC: 614±91 mL/min, P=0.019). ANOVA disclosed that renal plasma flow was independently influenced by both genotype and blood pressure, with hypertensives having a higher renal plasma flow than normotensive subjects. This was the fact irrespective of the criteria used for the definition of hypertension (World Health Organization or 24-hour ambulatory blood pressure criteria). The GNB3 825T variant is associated with increased renal perfusion in this study. Because early renal hemodynamic changes play a pivotal role in the pathogenesis of essential hypertension, our data suggest a relevance of increased G-protein activation in the pathogenesis of hypertension.
Key Words: G proteins • polymorphism • genes • hypertension, essential • hemodynamics
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The early course of essential hypertension is characterized by structural and functional changes in the systemic and renal circulation. In addition, early structural changes of the left ventricle (LV) have been reported. Increased concentric remodeling of the LV and impaired diastolic function have been recognized to reflect early changes of the myocardium in early essential hypertension.1 Renal hemodynamics were shown to be abnormally regulated in a prehypertensive stage, and increased renal perfusion was reported in early hypertension.2 3 4 It has been proposed that renal vascular changes may not simply be a complication but the cause for blood pressure elevation and thus may play a pivotal role in the pathogenesis of essential hypertension.5
In the search for mechanisms by which genetic markers
contribute to the development of hypertension, recent interest focused
on a novel gene polymorphism closely associated with a
well-established intermediate phenotype, that is, the enhanced
Na+/H+ exchanger
activity in hypertensive
subjects.6 Immortalized
lymphoblasts of these patients have been found to respond with enhanced
G-protein activation on
stimulation.7 Subsequently, a
single nucleotide polymorphism
(C/T at position 825) in the
gene encoding the ß3 subunit of heterotrimeric
G proteins (GNB3) has been
identified and was demonstrated to be significantly associated with
essential hypertension.8 The
825T allele was also
associated with the expression of a novel splice variant (Gß3-s)
displaying a 123-bp in-frame deletion within exon 9, resulting in the
loss of 41 amino acids and 1 WD repeat domain of the Gß subunit.
Increased binding of [35S]GTP
S in Sf9
insect cells expressing Gß3-s suggests that this splice variant
results in the enhanced activation of pertussis toxin–sensitive G
proteins.
The aim of this study was to determine in early hypertension the impact of the C825T polymorphism on early adaptive processes of the LV and hemodynamic changes in the renal circulation. Therefore, we measured LV mass as well as renal hemodynamics and glomerular filtration rate (GFR) in young normotensive and mildly hypertensive subjects.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Study Population
Young white male students were elicited by announcement for participation in the study at the campus of the University of Erlangen-Nürnberg. Study participants were screened for normal to mildly elevated blood pressure, and written informed consent was obtained from each participant before study inclusion. A total of 95 subjects were subsequently enrolled in the study. The study protocol was approved by our clinical investigation committee.
Inclusion criteria were age between 20 and 40 years, male gender, no current or previous treatment for arterial hypertension, no cardiovascular disease, and no secondary hypertension or World Health Organization (WHO) stage III of hypertensive disease. Therefore, exclusion criteria were advanced hypertensive fundoscopic changes, myocardial infarction, or any other evidence of coronary artery disease, congestive heart failure (New York Heart Association classes II to IV), previous cerebrovascular event, or hepatic or renal insufficiency.
Each participant was examined for the presence of secondary hypertension and target-organ damage by 12-lead ECG at rest, a fundoscopic evaluation, sonography of the kidneys and adrenal glands, Doppler sonography of the renal arteries, and routine laboratory tests. Detailed evaluation of hormones and endocrine metabolites was conducted if indicated.
Blood Pressure Measurements
Casual blood pressure was measured 4 times on 2
different occasions in our outpatient clinic (at least 2 weeks apart).
The cuff size of the sphygmomanometer was adjusted according to the
persons’ arm circumference, and blood pressure readings were taken
with the participant seated after 5 minutes of rest. Subjects were said
to be mildly hypertensive if the mean blood pressure was 
140
mm Hg systolic or 90 mm Hg diastolic,
according to WHO recommendations. To exclude white-coat hypertension,
ambulatory 24-hour blood pressure was additionally recorded with a
portable device (SpaceLabs 90207). Measurement intervals were every 15
minutes during the daytime (6
AM to 10
PM) and every 30 minutes
during the nighttime (10 PM
to 6 AM). Blood pressure
was said to be hypertensive if the mean daytime blood pressure values
were 135 mm Hg systolic and/or 85 mm Hg
diastolic.
Echocardiography
Two-dimensionally guided M-mode
echocardiography at rest (Picker-Hitachi CS 192,
2.5-MHz probe) was performed in the third to fourth intercostal space
lateral to the left sternal border, with the patient lying in the
partial left decubitus position. LV structure was assessed by
measurement of septal wall thickness, posterior wall thickness, and LV
end-diastolic diameter. Midwall fractional fiber shortening
was taken as a parameter of systolic
function.9 Concentric LV
hypertrophy was assessed by calculation of the relative
wall thickness as 2xposterior wall thickness divided by
end-diastolic
diameter.10 LV mass was
calculated according to the American Society of
Echocardiography (ASE)
recommendations11 but was
subsequently corrected by the regression LV=0.8 (ASE cube LV mass)+0.6
g, following the suggestions of Devereux et
al.12 LV
diastolic filling was determined by pulsed-wave Doppler
sonography of the LV inflow, as previously described in
detail.13
Measurement of Renal
Hemodynamics and Urinary Sodium Excretion
GFR and renal plasma flow (RPF) were determined after
1 hour of rest in a supine position. We applied the constant infusion
technique to measure RPF
(para-aminohippurate clearance)
and GFR (inulin clearance) without urinary sampling, as previously
described in
detail.14 15
Briefly, the excreted amount of
para-aminohippuric acid (PAH)
and inulin is equal to the infused dose of the compounds under
steady-state conditions. A bolus injection was applied followed by a
constant infusion for a total of 150 minutes to achieve steady state.
The doses of bolus and constant infusion of PAH and inulin were
adjusted to body weight. PAH and inulin were measured as outlined in
detail
elsewhere.16 17
This method overestimates RPF by 10% to 20%, but differences between
genotypes are not affected by this potential bias. The 24-hour
urinary sodium excretion was measured with participants on their usual
diet. To ensure complete collection of urine, all samples containing
<0.6 L and/or the expected creatinine per kilogram of body
weight were excluded.
Genotyping
Genomic DNA was extracted from whole blood according
to standard procedures with a QIAamp Blood Midi Kit (QIAGEN GmbH).
Genotyping for the GNB3 C825T
polymorphism was performed at the Department of Pharmacology at the
University of Essen, as recently described in
detail.8 Briefly, polymerase
chain reaction (PCR) with
GNB3-specific primers resulted
in a 368-bp fragment. PCR products were subsequently
restriction-digested with the enzyme
BseD1, leading to the
generation of a 116-bp and a 152-bp fragment with the C allele. The
T allele is not cleaved by
BseD1. Hence,
CT heterozygotes exhibit all 3
fragments (368, 152, and 116 bp).
Statistics
For statistical analysis, subjects either
heterozygous or homozygous for the GNB3
825T allele were taken together into one group because
it has been reported that 1 T
allele is sufficient for the expression of the Gß3 splice variant
and because the number of homozygous
T-allele carriers was too
small for separate analysis. All statistical calculations were
carried out with the use of SPSS
software.18 A
t test and 2-way ANOVA were
performed to compare CC and
TC/TT genotypes. Unless
otherwise specified, values are expressed as mean±SD. A 2-tailed
probability value of <0.05 was considered
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The clinical characteristics of the study group are given in Table 1. In our homogeneous study cohort of young white male students, the frequencies of the GNB3 825C and T alleles were 70% and 30%, respectively. Forty-four subjects were genotyped as homozygous for the C allele, 45 subjects were CT heterozygotes, and 6 individuals were homozygous for the T allele. There were no significant differences according to age, body mass index, body surface area, and casual and 24-hour ambulatory blood pressure between the CC and the TC/TT genotypes (Table 1).
|
In the whole study group, renal hemodynamic parameters were associated with the G-protein ß3 subunit C825T polymorphism (Table 2). Subjects with the 825T allele (TC and TT genotypes) had a greater RPF than those homozygous for the C allele (CT/TT: 659±96 versus CC: 614±91 mL/min; P=0.019). Furthermore, T-allele carriers had a lower renal vascular resistance than subjects with the CC genotype (CT/TT: 83±16 versus CC: 91±14 mm Hg/[mL/min], P=0.021).
|
Two-way ANOVA disclosed that genotype and blood pressure independently influenced RPF (Table 3). Thus, first the group of hypertensive subjects had an increased RPF compared with the group of normotensive subjects, irrespective of their genotypes. This was found irrespective of whether the examined subjects were stratified for hypertension according to the WHO (hypertensives: 661±99 versus normotensives: 611±84 mL/min, P=0.003) or ambulatory (hypertensives: 665±96 versus normotensives: 618±90 mL/min, P=0.011) blood pressure criteria. And, second most important, T-allele carriers had a greater RPF than individuals with the CC genotype (659±96 versus 614±91 mL/min, P=0.005). Hypertensive T-allele carriers had the greatest RPF, followed by normotensive individuals with the CT/TT genotype or hypertensive individuals homozygous for the C allele with intermediate values, and finally normotensive homozygous C allele carriers having the lowest values for RPF (Table 3).
|
GFR, 24-hour sodium excretion, and echocardiographic parameters for LV structural adaptation, LV systolic function, and LV diastolic filling were similar between groups of subjects stratified according to their G-protein genotype (Table 2). Accordingly, 2-way ANOVA did not disclose any significant differences for GFR or LV mass between the genotypes.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In this study, we show that the 825T allele of the G-protein ß3 subunit is associated with increased RPF in young male normotensive to mildly hypertensive subjects. Previous studies have reported normal to enhanced renal blood and/or plasma flow in the prehypertensive stage and in borderline hypertension,2 4 19 20 21 22 whereas in sustained hypertension, the consistently observed pattern of renal hemodynamics is characterized by a decreased renal blood and/or plasma flow and a normal to slightly decreased GFR, resulting in an elevated renal filtration fraction.23 24 25 Renal hemodynamic changes have been supposed to play an important role in the development of hypertension. Evidence for this hypothesis comes from the observation of an abnormal control of renal circulation in subjects with a positive family history of hypertension in response to mental stress, and Bianchi et al4 found that normotensive offspring of hypertensive parents were characterized by high renal blood flow (RBF) despite normal cardiac output, suggesting a specific renal vasodilation in the prehypertensive stage.3 4 Because RBF and cardiac output (CO) are strongly correlated, the ratio of both is a reliable parameter for the assessment of renal perfusion.24 25 In this study, CO was not measured. However, potential confounding factors such as blood pressure or the size and structure of the heart, which reflect profound alterations in systemic hemodynamics, were similar between the different genotypes in our study population. Hence, renal hyperperfusion observed in our study is most likely due to a selective renal vasodilation rather than to increased CO. Consequently, the GNB3 polymorphism per se might account for the observed variance in renal perfusion and therefore might be of significance in the early steps of the development of arterial hypertension.
The pathogenetic relevance of the C825T polymorphism relies on the fact that the 825T allele of GNB3 is related to enhanced stimulated G-protein activation in cell lines from hypertensive patients.26 There is substantial evidence that the enhanced Na+/H+-exchange activity observed in 30% to 50% of patients with essential hypertension is mediated by this genetically fixed G-protein activation.6 Na+/H+ exchange is involved in the regulation of pHi and contributes to sodium reabsorption in the kidney.7 Thus, a faster tubular sodium reabsorption may lead to an increase in RPF by means of the macula densa feedback mechanism and inhibition of renin secretion in subjects with the GNB3 825T allele. However, the fact that we have not found any change in GFR is in conflict with this hypothesis. Alternatively, one may speculate that a faster Na+/H+ exchange could be involved in selective renal vasodilation by a direct effect at the vascular smooth muscle or endothelial cell levels.
An additional finding of our study is that RPF was higher in hypertensive than in normotensive subjects regardless of the definition used for arterial hypertension. This observation is in line with the above-mentioned studies that found enhanced renal perfusion in the prehypertensive and in very early stages of hypertension.2 4 19 20 21 22 Because we examined a young homogeneous study cohort, including only mildly hypertensive subjects without signs of advanced target organ damage, it can be presumed that our hypertensive individuals had early essential hypertension.
Besides renal perfusion, we also determined GFR and cardiac structural adaptation to assess abnormalities occurring with early essential hypertension. Thus, an increase in LV mass and impaired LV systolic function has been demonstrated in young patients with borderline to mild hypertension.27 Furthermore, glomerular hyperfiltration has been related to incipient LV hypertrophy in mild hypertension, indicating early target organ damage.28 In this study, GFR and functional or structural parameters of the heart were not linked to the G-protein ß3 subunit gene polymorphism. Poch et al29 recently reported an association between the GNB3 825T allele and LV hypertrophy in hypertensive patients. In a study including patients with mild to moderate hypertension, we found that the CT/TT genotype was associated with impaired LV diastolic filling, which is an early sign of hypertensive heart disease.30 Because we have investigated young volunteers with normal or mildly elevated blood pressure, it is not surprising that we did not find such associations in the present study.
Study Limitations
There are several limitations of our study that must be
emphasized. We studied young white subjects and therefore we do not
know whether or not our results can be extended to older individuals,
to subjects with more advanced stages of essential hypertension, or to
other populations. Furthermore, because we only included men in our
study, we cannot extrapolate the results to women. However, we believe
that the use of a homogeneous study population is also
advantageous because none of the participants has ever received any
antihypertensive or cardiovascular medication, thereby
ruling out any potential effect of such previous or current
antihypertensive therapy. Moreover, the results are extremely unlikely
to be due to an unexpected admixture of populations because of the
ethnic homogeneity of our study population.
Conclusions
We have demonstrated that the
825T allele of the
ß3 subunit of heterotrimeric G proteins is
associated with increased renal perfusion in early hypertension and
thus may be of relevance in the pathogenesis of essential hypertension.
However, we currently can only speculate on how the
GNB3 825T allele leads to
increased RPF. We anticipate that this is due to functional changes
because we have not found any association of the G-protein
ß3 subunit gene variant with structural
alterations. Subsequent studies are needed to further clarify the role
of the GNB3 825T allele in
the regulation of renal
hemodynamics.
Received March 29, 2000; first decision May 3, 2000; accepted August 30, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Frohlich ED. State of the art lecture: risk mechanisms in hypertensive heart disease. Hypertension. 1999;34:782–789.
2. Hollenberg NK, Borucki LJ, Adams DF. The renal vasculature in early essential hypertension: evidence for a pathogenetic role. Medicine. 1978;57:167–178.[Medline] [Order article via Infotrieve]
3.
Hollenberg NK,
Williams GH, Adams DF. Essential hypertension: abnormal renal vascular
and endocrine responses to a mild psychological stimulus.
Hypertension. 1981;3:11–17.
4. Bianchi G, Cusi D, Gatti M, Lupi GP, Ferrari P, Barlassina C, Picotti GB, Bracchi G, Colombo G, Gori D, Velis O, Mazzei D. A renal abnormality as a possible cause of "essential" hypertension. Lancet. 1979;1:173–175.[Medline] [Order article via Infotrieve]
5. Rosenberg WL. The glomerular origin of essential hypertension. Med Hypotheses. 1983;10:167–171.[Medline] [Order article via Infotrieve]
6. Siffert W. G proteins, hypertension, and coronary heart disease: novel findings and hypotheses. Kidney Blood Press Res. 1996;19:71–80.[Medline] [Order article via Infotrieve]
7.
Siffert W, Düsing
R. Sodium-proton exchange and primary hypertension: an update.
Hypertension. 1995;26:649–655.
8. Siffert W, Rosskopf D, Siffert G, Busch S, Moritz A, Erbel R, Sharma AM, Ritz E, Wichmann HE, Jakobs KH, Horsthemke B. Association of a human G protein beta3 subunit variant with hypertension. Nat Genet. 1998;18:45–49.[Medline] [Order article via Infotrieve]
9. De Simone G, Devereux RB, Roman MJ, Ganau A, Saba PS, Alderman MH, Laragh JH. Assessment of left ventricular function by the midwall fractional shortening/end-systolic stress relation in human hypertension. J Am Coll Cardiol. 1994;23:1444–1451.[Abstract]
10. Devereux RB, Savage DD, Sachs I, Laragh JH. Relation of hemodynamic load to left ventricular hypertrophy and performance in hypertension. Am J Cardiol. 1983;51:171–176.[Medline] [Order article via Infotrieve]
11. Devereux RB, Alonso DR, Lutas EM, Gottlieb GJ, Campo E, Sachs I, Reichek N. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings. Am J Cardiol. 1986;57:450–458.[Medline] [Order article via Infotrieve]
12. Devereux RB, Lutas EM, Casale PN, Kligfield P, Eisenberg RR, Hammond IW, Miller DH, Reis G, Alderman MH, Laragh JH. Standardization of M-mode echocardiographic left ventricular anatomic measurements. J Am Coll Cardiol. 1984;4:1222–1230.[Abstract]
13. Schobel HP, Langenfeld M, Gatzka C, Schmieder RE. Treatment and post-treatment effects of alpha- versus beta-receptor blockers on left ventricular structure and function in essential hypertension. Am Heart J. 1996;132:1004–1009.[Medline] [Order article via Infotrieve]
14. Cole RB, Giangiacomo J, Ingelfinger JR, Robson AM. Measurement of renal function without urine collection: a critical evaluation of the constant-infusion technique for determination of inulin and para-aminohippurate. N Engl J Med. 1972;287:1109–1114.
15. Schmieder RE, Veelken R, Schobel H, Dominiak P, Mann JF, Luft FC. Glomerular hyperfiltration during sympathetic nervous system activation in early essential hypertension. J Am Soc Nephrol. 1997;8:893–900.[Abstract]
16. Smith HW, Finkelstern N, Aliminosa L, Crawford B, Grabner M. The renal clearance of substituted hippuric acid derivatives and other aromatic acids in dogs and man. J Clin Invest. 1945;24:388–398.
17. Rosenbaum JL, Kramer MS, Raja RM, Manchanda R, Lazaro N. Determination of inulin and p-aminohippurate clearance without urine collection. Nephron. 1973;10:347–359.
18. Norusis MJI. SPSS in SPSS for Windows. Professional Statistics, Release 6.0. Chicago, Ill: 1993.
19. London GM, Safar ME, Weiss YA, Laurent S, London AM. Renal and systemic hemodynamics in borderline hypertension. Am J Hypertens. 1988;1:127S-130S.[Medline] [Order article via Infotrieve]
20. Hollenberg NK, Merrill JP. Intrarenal perfusion in the young "essential" hypertensive: a subpopulation resistant to sodium restriction. Trans Assoc Am Physicians. 1970;83:93–101.[Medline] [Order article via Infotrieve]
21.
Messerli FH, De
Carvalho JG, Christie B, Frohlich ED. Systemic and regional
hemodynamics in low, normal and high cardiac output
borderline hypertension.
Circulation. 1978;58:441–448.
22. Temmar MM, Safar ME, Levenson JA, Totomoukouo JM, Simon AC. Regional blood flow in borderline and sustained essential hypertension. Clin Sci. 1981;60:653–658.[Medline] [Order article via Infotrieve]
23.
Lowenstein J,
Steinmetz PR, Effros RM, Demeester M, Chasis H, Baldwin DS, Gomez DM.
The distribution of intrarenal blood flow in normal and hypertensive
man. Circulation. 1967;35:250–259.
24. Hollenberg NK, Adams DF. The renal circulation in hypertensive disease. Am J Med. 1976;60:773–784.[Medline] [Order article via Infotrieve]
25.
London GM, Safar
ME, Sassard JE, Levenson JA, Simon ACH. Renal and systemic
hemodynamics in sustained essential hypertension.
Hypertension. 1984;6:743–749.
26. Siffert W, Rosskopf D, Moritz A, Wieland T, Kaldenberg-Stasch S, Kettler N, Nartung K, Beckmann S, Jakobs KH. Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension. J Clin Invest. 1995;96:759–766.
27.
Palatini P,
Mormino P, Santonastaso M, Mos L, Dal Follo M, Zanata G, Pessina AC.
Target-organ damage in stage 1 hypertensive subjects with white coat
and sustained hypertension: results from the HARVEST study.
Hypertension. 1998;31:57–63.
28.
Schmieder RE,
Messerli FH, Garavaglia G, Nunez B. Glomerular
hyperfiltration indicates early target organ damage in essential
hypertension. JAMA. 1990;264:2775–2780.
29.
Poch E, Gonzalez
D, Gomez-Angelats E, Enjuto M, Pare JC, Rivera F, de la Sierra A.
G-protein ß3 subunit gene variant and left ventricular
hypertrophy in essential hypertension.
Hypertension. 2000;35:214–218.
30. Jacobi J, Hilgers KF, Schlaich MP, Siffert W, Schmieder RE. 825T allele of the G protein beta3 subunit gene (GNB3) is associated with impaired left ventricular diastolic filling in essential hypertension. J Hypertens. 1999;17:1457–1462. [Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  M. Sartori, A. Semplicini, W. Siffert, P. Mormino, A. Mazzer, F. Pegoraro, L. Mos, M. Winnicki, and P. Palatini G-Protein {beta}3-Subunit Gene 825T Allele and Hypertension: A Longitudinal Study in Young Grade I Hypertensives Hypertension, November 1, 2003; 42(5): 909 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Ruiz-Velasco and S. R. Ikeda A splice variant of the G protein {beta}3-subunit implicated in disease states does not modulate ion channels Physiol Genomics, April 16, 2003; 13(2): 85 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Rankinen, T. Rice, A. S. Leon, J. S. Skinner, J. H. Wilmore, D. C. Rao, and C. Bouchard G protein {beta}3 polymorphism and hemodynamic and body composition phenotypes in the HERITAGE Family Study Physiol Genomics, February 28, 2002; 8(2): 151 - 157. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||