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Original Studies |
Centre d’Investigations Cliniques 9201 (M.A., M.-C.H., T.T.G., J.M.) and Laboratoire de Biologie Moléculaire (X.J.), Assistance Publique des Hôpitaux de Paris (AP-HP) et INSERM, Hôpital Européen Georges Pompidou, 75908 Paris, France
Address all correspondence and requests for reprints to: Michel Azizi, Centre d’Investigations Cliniques 9201, Hôpital Européen Georges Pompidou, 20–40 rue Leblanc, 75908, Paris Cedex 15, France. E-mail: michel.azizi{at}egp.ap-hop-paris.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The discovery that the human AGT gene is involved in essential hypertension revived interest in AGT (10). One of several molecular variants, the M235T polymorphism, was associated with a 10–30% increase in plasma AGT concentration (11). Its association with plasma AGT is thought to result from almost complete linkage disequilibrium with a nucleotide substitution close to the initial transcription start site (G-6A), which affects the basal transcription rate of the AGT gene in vitro (12) and in vivo (13, 14, 15). Chronic increases in plasma AGT concentration may slightly increase blood pressure and facilitate hypertension, in conditions in which AGT synthesis is stimulated.
Whether the M235T polymorphism may influence the plasma concentration of the protein in plasma in response to estrogens has not been tested yet in humans. Therefore, the aim of this study was to analyze the influence of the M235T polymorphism on the ethinylestradiol (EE)-induced increase in plasma AGT concentration and on the resulting generation of Ang I and Ang II in plasma. Two different protocols were used, involving short-term and sustained oral administration of EE in healthy male subjects selected on the basis of their AGT genotype. To investigate more in depth the influence of the M235T polymorphism on the physiology of renin secretion, acute renin release was induced by mild sodium depletion followed by a single oral administration of captopril.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This experiment was designed to observe the effect of the M235T genotype on early changes in renin-angiotensin system (RAS) activity after a small increase in plasma AGT concentration.
Twenty-four normotensive Caucasian male subjects homozygous for the M235T genotype (MM = 12 and TT = 12), 18–35 yr old, were included in this short-term stimulation study. They were given two oral doses of 50 µg EE on the first (day 1) and third (day 3) days at 0900 h. Throughout the study, volunteers were instructed to follow their usual sodium diet.
Blood samples were taken before the study and on days 2, 3, 4, 5, and 6 (at 0900 h) for plasma AGT determination. Plasma active renin and plasma renin activity (PRA) were determined before EE intake (day 1) and on day 4.
Exp 2: 7 days’ oral administration of 50 µg EE
From the results of Exp 1, we calculated that seven subjects per
group were necessary for Exp 2 to achieve a difference in stimulated
plasma AGT concentrations of 700 ng Ang I/mL between the TT and the MM
subjects with an SD of 300 ng Ang I/mL and an 
-risk of
5% and a ß-risk of 20%.
Fifteen normotensive Caucasian male homozygous subjects, 18–35 yr old (MM = 8 and TT = 7), were instructed to arrive at the center at 0800 h each day, from the first (day 1) to the seventh (day 7) day of the study. They were given 50 µg EE orally with tap water every day. In addition, eight heterozygous Caucasian subjects (MT) were given a matched placebo in a single-blind fashion during 7 days.
During this first phase, volunteers were ambulatory and followed their usual sodium diet. Blood samples were taken on days 1 and 7, at 0900 h, for PRA, plasma active renin, AGT, Ang I, and Ang II determinations.
To investigate, in more detail, the consequences of the increase in AGT concentration on the reactivity of the RAS, subjects underwent sodium depletion, followed by acute inhibition of the RAS, as follows: On day 7, subjects were instructed to return to the center at 1800 h. They were given a single oral dose of 40 mg furosemide at 2100 h, followed by a sodium-restricted diet (30 mmol NaCl) to induce mild sodium depletion.
On day 8, after a light caffeine- and fat-free breakfast at 0700 h, subjects were comfortably installed in a semirecumbent position on their beds. An indwelling cannula was inserted into a brachial vein for blood sampling. At 0900 h, after a 1-h rest in the semirecumbent position (to allow equilibration of hormones), each subject was given a single oral dose of 50 mg captopril with 50 mL water and remained in the same position for 6 h. Fluid intake was unrestricted, and subjects were given a light meal, 6 h and 12 h post dose. After the first meal, subjects were allowed to move from their beds. For hormone determinations performed 10 and 24 h post dose, subjects were again placed in the semirecumbent position, 1 h before sampling.
Blood samples were taken before captopril administration and 1, 2, 4, 6, 10, and 24 h after captopril administration for PRA, plasma active renin, AGT, Ang I, and Ang II determinations.
Volunteers gave their written informed consent for participation in the protocol, which was approved by the Comité Consultatif de Protection des Personnes se prêtant à des Recherches Biomédicales (Paris-Cochin, France). The procedures followed were in accordance with guidelines proposed in The Declaration of Helsinki.
Laboratory methods
For each hormone determination, subjects were placed in a semirecumbent position, 1 h before blood sampling. Heparinized tubes were used to collect blood for plasma AGT, active renin, and PRA determinations. For plasma angiotensin RIAs, blood samples (10 mL) were collected rapidly (within 10 sec) into prechilled EDTA-K3 vacutainers; and 0.5 mL of an inhibitor mixture containing 62.5 mmol/L EDTA, 100 µM of the renin inhibitor, remikiren, and 100 µmol/L enalaprilat was immediately added to prevent the generation and degradation of angiotensins in vitro (16). Blood samples were immediately centrifuged at 3500 rpm at 4 C and were stored at -80 C until assay.
Plasma AGT was determined by measuring the generation of Ang I after addition of an excess human renin to obtain complete cleavage to Ang I. Plasma (0.25 µL) was incubated at 37 C for 1 h with 0.062 pmol/L pure human recombinant renin (0.025 Goldblatt Units; final concentration of 0.125 nmol/L; a gift from Dr. Walter Fischli, Hoffman-LaRoche Inc.) in 0.5 mL of 0.15 mol/L citrate/phosphate buffer (pH 5.7) containing 50 mmol/L EDTA[Na2], 1.4 mmol/L phenylmethylsulfonylfluoride, and 0.2% BSA. The reaction was stopped by placing the tubes in an iced-water bath at 4 C. The amount of Ang I generated was determined using a polyclonal antibody, as previously described (17). The standard Ang I used in these experiments was purchased from the National Biological Standards Board (Holly Hill, Hampstead, London, UK). For each sample, the mean of two Ang I concentrations was calculated with the Multicalc software of the CliniGamma 1272 counter (LKB, Finland). The limit for detection of Ang I was 40 pg/mL incubated. AGT concentrations were expressed as ng Ang I/mL of plasma. To check intraassay precision, 3 samples were tested 8 times in the same assay. The following coefficients of variation were obtained: 5.5% for 619 ng Ang I/mL, 2.2% for 1053 ng Ang I/mL, and 5.5% for 5600 ng Ang I/mL. For interassay precision, the same 3 samples were measured in 12 assays, giving coefficients of variation of 14%, 7%, and 7%, respectively. Plasma AGT was determined in triplicate, and a mean of the 3 values was then calculated.
Plasma active renin was determined by immunoradiometric assay (18) using a commercially available kit (ERIA, Diagnostics Pasteur, Marnes-la Coquette, France). PRA was determined at pH 7.4, as previously described (19). The anti-Ang I polyclonal antibody used was the same as that used for the AGT enzyme assay. The PRA/active renin ratio was used to estimate the amount of Ang I (pg/h) generated per picogram renin.
Plasma angiotensins were determined after extraction on a single Bondelut column, as previously described (20).
All hormone determinations were performed blind to the genotype and treatment.
M235T genotyping was performed using a mutagenetically separated amplification method, as previously described (21).
Statistical methods
Exp 1. The area under the plasma-time curves (AUC) up to the last measured time-point (0–120 h) was calculated according to the trapezoidal rule. The maximum value, at peak for plasma AGT concentration, was determined from the concentration-time curves for each subject and then averaged. The initial slope for the increase in plasma AGT concentration with time (up to 72 h) was calculated for each subject by determining the slope of the regression line best fitting the plot of plasma AGT concentration vs. time. The values were then averaged. Between-genotype differences were analyzed using unpaired t tests.
For plasma active renin, PRA, and AGT, a one-way ANOVA with one repeated factor (time) and one grouping factor (genotype) was used to test the effect of EE administration.
Exp 2. We first assessed the effects of EE vs. placebo independently of M235T genotype. Data from the stimulation phase (from day 1 to day 7) were analyzed by a one-way ANOVA with one repeated factor (time) and one grouping factor (treatment). For the captopril study on day 8, baseline values on day 8, the AUCs (between day 8 and day 9), and the values at peak were compared using unpaired t tests.
We then assessed the effect of the M235T genotype on the response to EE administration. From day 1 to day 7 (stimulation phase), data were analyzed using a one-way ANOVA with one repeated factor (time) and one grouping factor (genotype). For the second phase (captopril study), baseline values on day 8, the AUCs (between day 8 and day 9), and the values at peak were compared between genotypes using unpaired t-tests.
The homogeneity of the variance was checked for each variable, and logarithmic transformation was used where appropriate.
Calculations were done with Statview 5.1 statistical software (Abacus Concepts Inc., Berkeley, CA). Data are expressed as means ± 1 SD in the tables, means ± 1 SEM in the figures, and are otherwise specified. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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At baseline, plasma AGT, active renin, and PRA levels did not
significantly differ between the TT and the MM subjects (Table 1
).
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).
There was a high level of interindividual variability in plasma AGT
concentration at baseline (range, 652-1651 ng Ang I/mL) and after EE
stimulation (range, 950-2564 ng Ang I/mL). The difference in peak
plasma AGT levels between the MM and TT subjects was not significant
(1448 ± 284 vs. 1577 ± 390 ng Ang I/mL,
respectively). There was also no significant difference for the slope
of plasma AGT concentration vs. time and for the AUC
0–120 h of plasma AGT concentration
vs. time (not shown).
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Exp 2
Effects of repeated administration of EE vs.
placebo. At baseline (day 1), none of the hormonal variables
differed significantly between the EE and placebo groups (Table 2 and Fig. 2). EE administration resulted in
significantly larger increases in plasma AGT levels on day 7, by a
median of 77% (range, 26–293%), than for the controls [12% (range,
-14% to +39%), P < 0.001]. The in vitro
Ang I production rate (PRA/active renin ratio) increased significantly
in the EE group, whereas it remained stable in the placebo group
(F1,22 = 14, P < 0.01, Table 2).
Plasma active renin, Ang I and Ang II concentrations, and PRA were not
significantly affected by EE administration, as shown by comparison
with placebo and baseline levels (Table 2).
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). Thus, the PRA/active renin ratio was significantly higher in
the EE group than in the placebo group (34 ± 16 vs.
22 ± 6 pg Ang I/h·pg, P < 0.05,
respectively).
After captopril administration, the increase in plasma active renin
concentration was significantly smaller for the EE group than for the
placebo group (AUC of plasma active renin vs. time,
1747 ± 901 vs. 2996 ± 1923 pg·h/mL;
P < 0.05, respectively; Fig. 2), whereas PRA
concentrations at peak were similar in both groups (Fig. 2). The
in vitro Ang I production rate (PRA/active renin ratio) was
significantly higher in the EE group than in the placebo group, but
similar plasma Ang I and Ang II concentrations were observed in both
groups after captopril administration, because of a decrease in renin
release (Fig. 2).
Effect of the M235T genotype on the response to repeated EE
administration. At baseline (day 1), none of the hormonal
variables differed significantly between genotypes (Table 3).
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and Fig. 3]. The slope of the increase
in plasma AGT vs. time was significantly steeper in TT
subjects (261 ± 131 ng Ang I/mL·day) than in MM subjects
(146 ± 75 ng Ang I/mL·day, P = 0.05).
Interindividual variability in plasma AGT levels was greater in TT
subjects than in MM subjects on day 1 and was even more so on day 7
(Table 3).
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and Fig. 3). On day 8, 12 h after
furosemide intake, plasma Ang I and Ang II concentrations did not
significantly differ between genotypes. Plasma active renin
concentration was lower in the TT subjects than in the MM subjects, but
the difference between groups was not statistically significant
(27 ± 11 vs. 40 ± 18 pg/mL, P =
0.10, respectively). In contrast, PRA was similar in the two groups.
Thus, the in vitro Ang I production rate was significantly
higher in TT than in MM subjects (41 ± 13 vs. 29
± 16 pg Ang I/h·pg, P < 0.05, respectively; Fig. 3).
After captopril intake, plasma active renin levels remained
significantly lower in the TT group than in the MM group (AUC of plasma
active renin vs. time, 1237 ± 691 vs.
2194 ± 853 pg·h/mL, P < 0.05; and peak plasma
active renin, 201 ± 145 vs. 350 ± 157 pg/mL,
P < 0.05, respectively; Fig. 3). PRA did not differ
significantly between the TT and MM subjects (6651 ± 3054
vs. 9587 ± 3416 pg Ang I/mL·h; P =
0.13) and PRA/active renin ratio remained significantly higher in TT
than in MM subjects after captopril intake (46 ± 15
vs. 31 ± 13 pg Ang I/h·pg, P <
0.05, respectively). Plasma Ang I levels did not differ significantly
between the TT and MM subjects (AUC of plasma Ang I vs.
time, 844 ± 331 vs. 1512 ± 936 pg·h/mL;
P = 0.13, respectively).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We first analyzed the effects of EE independently of genotype. Both
short-term and repeated oral administration of EE significantly
increased plasma AGT levels. The increase in renin substrate was
accompanied by a full readjustment of the circulating RAS, which was
more complete after the seventh dose than after the second dose of EE.
Despite a 77% (range, 26–293%) increase in plasma AGT concentration
with EE treatment, the plasma Ang I and Ang II concentrations, achieved
by stimulation of renin release by administration of furosemide and
captopril, were similar for the EE and placebo groups (see Fig. 2). The
similarity in plasma Ang I and Ang II concentrations, observed in both
groups, was attributable to a decrease in renin release after sodium
depletion and angiotensin-converting enzyme (ACE) inhibition in the EE
group, even though the in vitro Ang I production rate
(PRA/active renin ratio) was significantly higher in the EE group than
in the placebo group. The decrease in renin release after furosemide
and ACE inhibition after EE administration was probably caused by the
inhibition of renin release by high local Ang II concentrations at the
level of the juxtaglomerular cells in the presence of an increase in
AGT (23). Actually, EE administration reduced, although
not significantly, plasma active renin concentrations from day 1 to day
7. Partial suppression of renin release and synthesis, as a consequence
of high concentrations of renin substrate during the administration of
synthetic estrogens, has been previously reported in rat models
(8, 23) and in humans (3). It has also been
reported during the oral administration of natural estrogens in
postmenopausal women (24).
The physiological response to an estrogen-induced increase in plasma AGT concentration involves complete readjustment of the RAS via the effects of the Ang II negative feedback loop on renin release. This confirms that hypertension caused by estrogen-containing oral contraceptive pills cannot be solely caused by the increase in renin substrate concentration (9).
The second step of our study was to look for possible effects of the M235T polymorphism on EE-induced AGT stimulation and on the subsequent readjustment of the RAS. Before EE administration, no difference was observed between the plasma AGT concentrations of MM and TT subjects, likely explained by the slight basal effect of the genotype (10, 25) and the small size of our sample. The short-term administration of EE (50 µg, twice) did not induce a differential genotype-dependent increase in AGT concentration, although TT subjects had slightly (but not significantly) higher plasma AGT concentrations than MM subjects. In the 7-day study, TT subjects did achieve higher plasma AGT concentrations than MM subjects despite both groups having similar baseline plasma AGT levels. There was, however, considerable interindividual variability in baseline and, particularly, in stimulated plasma AGT concentrations. The observed increase in the variability of plasma AGT levels, after 7 days of EE administration, is probably attributable to the high level of variability in EE pharmacokinetics (26) and metabolism (27). In addition, the reentry of the drug into circulation via the enterohepatic cycle (28) and its secondary release from adipose tissue pools (29) may also be involved in whole-plasma AGT level variability. Even greater variability was observed in TT subjects and could be attributed to a greater genetic diversity in AGT genes carrying T235, compared with their M235 counterparts (21). A careful control salt diet may have also limited the interindividual variability and facilitate the detection of genotypic differences.
The influence of the M235T polymorphism on plasma AGT concentration and its consequences on the readjustment of the RAS after EE administration provides strong indirect support for these results. After 7 days of EE administration, the more pronounced increase in AGT concentration in TT subjects resulted in similar PRA levels, with lower plasma active renin concentration and, therefore, higher PRA/active renin ratios. This effect of the concentration of the renin substrate on Ang I generation is consistent with the kinetics of its reaction with renin (5) and is operative in vitro and in vivo (3). The difference in renin stimulation according to genotype was even more pronounced after furosemide and captopril intake. The genotype difference in renin secretion led to a readjustment of Ang I generation in vivo, with even a trend toward lower PRA and plasma Ang I levels in TT subjects. These results are consistent with those obtained by Danser et al. (30) for the German population of the MONICA survey. Analysis of 228 men and 168 women in the German cohort showed that the T235 allele was significantly associated with lower concentrations of plasma active renin and prorenin, thus indicating a decrease in both renin release and synthesis. This epidemiological study and our physiological investigation provide altogether strong evidence that the T235 allele promotes increased Ang I generation compensated at the plasma level by the negative feedback exerted by Ang II on renin release.
It is difficult to extrapolate these results to estrogen- induced hypertension or pregnancy-induced hypertension because: 1) our study was restricted to male subjects who were given EE during a short period of time; and 2) the consequences of EE administration were only assessed on the circulating RAS. Campbell et al. (23) found that the administration of synthetic estrogens to rats induces an increase in the concentration of angiotensin peptides in the kidney, whereas a decrease is observed in plasma. It is therefore possible to observe no change in the equilibrium of the circulating RAS despite an increase in angiotensin production in the kidney. Because the T235 allele has been associated with the nonmodulating phenotype in hypertensive patients, a phenotypic trait characterized by a failure to modulate renal blood flow to Ang II during high sodium intake (31), the TT genotype may therefore be one of several susceptibility factors for the development of hypertension under EE administration, especially by increasing the local formation of Ang II, independently of the circulating system. Subtle changes in renal blood flow have indeed been observed in women whose AGT levels increased because of oral administration of synthetic estrogens (32).
In conclusion, this study demonstrates that the T235 allele of the AGT gene is associated with an enhanced stimulation of AGT secretion after the administration of EE in normotensive male subjects. Over a short period of time, a full readjustment of the circulating RAS occurs, blunting the effects of the renin substrate increase. Whether these changes may favor EE-induced hypertension during oral contraception or pregnancy-induced hypertension remains a plausible hypothesis.
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Acknowledgments |
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Footnotes |
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Received May 22, 2000.
Revised July 20, 2000.
Accepted July 25, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) and
12 TT subjects (•) after 2 oral doses of 50 µg EE given 48 h
apart.
) or
placebo (
), from day 1 to day 7, followed by a stimulation of renin
release by furosemide and captopril on day 8, in healthy male subjects.