Kidney International, Vol. 63 (2003), pp. 1443–1449

Interactions between gender and the angiotensin type 1receptor gene polymorphism

HEATHER REICH, JOHN A. DUNCAN, JORDAN WEINSTEIN, DANIEL C. CATTRAN,JAMES W. SCHOLEY, and JUDITH A. MILLER

Department of Medicine, University of Toronto, Toronto, Ontario, Canada; and Department of Medicine, University of BritishColumbia, Vancouver, British Columbia, Canada

Interactions between gender and the angiotensin type 1 recep- The renin-angiotensin system (RAS) is an importanttor gene polymorphism. mediator of renal and cardiovascular physiology, and

Background. The cardiovascular effects of angiotensin II angiotensin II is the principal effector of the RAS [1, 2].are mediated by the angiotensin II type 1 receptor (AGT1R);Most of the cardiovascular effects of angiotensin II areone polymorphism of the AGT1R gene, A1166→C, has beenmediated by the angiotensin II type 1 receptor (AGT1R).associated with hypertension. The hemodynamic response to

angiotensin II is blunted in women compared to men, but interac- Although a number of polymorphisms of the AGT1R genetions between gender, blood pressure, and AGT1R gene poly- have been identified [3–5], one of the most widely studiedmorphisms are unclear. is an A→C substitution at position 1166 (A1166→C) [6–8].Methods. A total of 81 young healthy normotensive individ-

There have been several association studies of polymor-uals maintained regulated sodium and protein intake prior tophism of the AGT1R (A1166→C) gene polymorphismstudy. They were divided into four groups based on gender

and A1166→C genotype (AA versus AC/CC); serial supine and clinical end points such as myocardial infarction,blood pressures were obtained. A subset of 52 individuals re- hypertension [6–13], and the progression of renal diseaseceived graded infusions of angiotensin II. Inulin and paraamino-

[12, 14]. More recently, we have identified an associationhippurate clearance techniques were used to measure renal he-between renal hemodynamic function and the C allelemodynamic function at baseline and in response to the infusions.

Results. Men with the AC/CC genotype exhibited higher in normal healthy subjects [15] and an association be-blood pressures than men with the AA genotype; however, tween the systemic hemodynamic pressor response to highthis relationship was not found among women. Analysis of glucose and the C allele in subjects with type 1 diabetescovariance revealed a significant interaction between gender

mellitus [16].and AGT1R genotype in the determination of blood pressure.There is emerging evidence of a relationship betweenGlomerular filtration rate (GFR) declined variably in the study

subjects following infusion of angiotensin II, and a statistical gender and hypertension. Female gender has been shownmodel incorporating gender and genotype best predicted the to limit the development of hypertension in animal stud-fall in GFR. There was a trend for females of the AA genotype ies, in studies of ambulatory blood pressure monitoringto have a greater fall in GFR in response to angiotensin II

in age-matched individuals, and in population studiesinfusion, than any of the other groups.[17–20]. Estrogen regulates some components of the RAS,Conclusion. In young healthy subjects, there is an important

interaction between gender, the AGT1R A1166→C gene poly- there being an estrogen-responsive promoter in the an-morphism, and blood pressure. In addition, the renal hemody- giotensinogen gene [21], and RAS activity can be modu-namic response to angiotensin II infusion is a function of both lated by gender and estrogen status [22–24]. We havegender and the AGT1R genotype.

reported that the systemic and renal hemodynamic re-sponse to angiotensin II infusion is influenced by gender,suggesting that there may be an important interactionbetween gender and vascular responsiveness to angio-tensin II [25]. In this regard, there is also an associationbetween the AGT1R (A1166→C) gene polymorphism andblood pressure in females but not males, with essentialKey words: gender, angiotensin II, angiotensin type 1 receptor,

A1166→C polymorphism, blood pressure, glomerular filtration rate. hypertension [8].Our goal was to determine if there is an interactionReceived for publication July 2, 2002

between gender, the AGT1R (A1166→C) gene polymor-and in revised form September 19, 2002Accepted for publication November 14, 2002 phism, and blood pressure in young, healthy, normoten-

sive men and age-matched healthy premenopausal women. 2003 by the International Society of Nephrology

1443

Reich et al: Gender and angiotensin receptor gene polymorphism1444

Blood pressure values and the renal hemodynamic re- and PAH clearance methods. After collection of baselineblood samples of hematocrit (HCT), a priming infusionsponse to angiotensin II infusion were compared in four

groups based on gender and the AGT1R (A1166→C) containing 25% inulin (60 mg/kg) and 20% PAH (8mg/kg) was given. Inulin and PAH were then infusedgene polymorphism (AA versus AC/CC).continuously at a rate maintaining respective plasma con-centrations of 20 mg/dL and 1.5 mg/dL. After a 90-

METHODSminute equilibration period, three timed urine collec-

Subjects tions 20 minutes apart were obtained, and blood wascollected for inulin, PAH, and HCT levels.A total of 81 healthy females and males were enrolled

in this study, and were divided into two groups, according Angiotensin II infusion solution was prepared as pre-viously described [25], and was infused at one dose, 4to presence or absence of the C allele at position 1166

of the AGT1R gene (A versus AC/CC). All patients were ng/kg/minute for 45 minutes. Subjects remained supineexcept to void. Blood was collected once during theCaucasian, normotensive, and on no medications. Females

were of reproductive age, and no female subjects used angiotensin II infusion period, and urine was collectedat the end of the infusion period. MAP was measuredoral contraceptive medications. All subjects underwent

a detailed medical history and physical examination by throughout, and recorded at the midpoint.a qualified internist. In order to control confounding

Sample collection and analytical methodsvariables, all subjects were counseled by a dietitian toobserve a prescribed diet, maintaining their normal calo- Blood samples collected for inulin and PAH determi-

nations were immediately centrifuged at 3000 rpm forric intake, while taking 200 mmol/day sodium, and 1.5to 2.0 g/kg/day protein. Urine was collected to determine 10 minutes at 4�C. Plasma was separated, placed on ice,

and then stored at �70�C before the assay. Inulin concen-compliance with the diet, and data from subjects wereused only if the excretion rate of sodium was greater trations in plasma and urine were measured by a modi-

fied method of Walser, Davidson, and Orloff [26] andthan 150 mmol/day, and urea excretion rate was between3 to 6 mmol/kg/day. Data from four subjects were ex- PAH concentration was determined by a spectrophoto-

metric method according to Brun [27]. The mean of thecluded on this basis. Subjects reported to the renal physi-ology laboratory at 8:30 a.m. on the day of testing, after final two clearance periods represent glomerular filtra-

tion rate (GFR) and effective renal plasma flow (ERPF),an overnight fast. They lay supine in a warm, quiet roomduring the assessment. expressed per 1.73 m2. Filtration fraction (FF) repre-

sented the ratio of GFR to ERPF. Renal blood flowThe study was performed with the approval of theUniversity of Toronto Human Subjects Review Commit- (RBF) was calculated by dividing the ERPF by (1-HCT).

Renal vascular resistance (RVR) was derived by dividingtee. Documentation of informed consent was obtainedfrom each subject. MAP by the RBF.

Serum sodium concentration was measured by an ion-Study protocol selective electrode method, and urine sodium by a flame

photometry method. Blood for determination of angio-The study protocol is similar to that previously de-scribed [25]. Once resting in a quiet room, serial systolic tensin II was collected into prechilled tubes containing

ethylenediaminetetraacetic acid (EDTA) and angioten-blood pressue (SBP), diastolic blood pressure (DBP), andmean arterial blood pressure (MAP), as well as heart rate, sinase inhibitor (0.1 mL Bestatin Solution, Buhlmann

Laboratories, Basel, Switzerland). Samples were ana-were obtained by the use of an automated sphygmo-manometer (Dinamapp; Critikon Co., Tampa, FL, USA); lyzed using a competitive radioimmunoassay kit supplied

by Buhlmann Laboratories AG (Switzerland).readings were obtained each half hour for 3 hours. Finalblood pressure recordings reflect means of five consecu- Genomic DNA was extracted from peripheral blood

leukocytes as previously described [28]. The DNA wastive measurements.A subset of 52 subjects, 17 females and 35 males, were resuspended in 10 mmol/L Tris-HCl, 0.2 mmol/L sodium

EDTA, pH 7.5, and the concentration was determinedgiven angiotensin II infusions. An 18 gauge peripheralvenous cannula was inserted into an antecubital vein by spectrophotometry. To determine the AGT1R geno-

type of the subjects, 0.1 �g genomic DNA was subjectedfor infusions of inulin, paraaminohippurate (PAH), andangiotensin II (Clinalfa, Basel, Switzerland), and a sec- to polymerase chain reaction (PCR) amplification [28].

The reaction mixture (20 �L) contained 50 mmol/L KCl,ond catheter was inserted in the contralateral arm forblood testing. Subjects voided on arrival, and then drank 10mmol/L Tris, pH 8.3, 1.5 mmol/L MgCl2, 1 �mol of

each primer, 200 �mol of deoxy-adenosine triphosphatesufficient water to induce a diuresis. Adequate urineoutput (by spontaneous voiding) was ensured through- (ATP), guanosine triphospate (GTP), cytosine triphos-

phate (CTP), thymidine triphosphate (TTP), gelatin 200out the protocol by ingestion of 200 mL of water perhour. Renal hemodynamics were measured using inulin �g/mL, and 1 U of Taq polymerase. Thirty cycles of PCR

Reich et al: Gender and angiotensin receptor gene polymorphism 1445

Table 2. Mean blood pressure by genotype and genderTable 1. Baseline characteristics

Female Male Female Male P value(N � 34) (N � 47) P value

Genotype AASystolic blood pressure mm Hg 110 �3 114�2 NSAge years 26�1 29�1 NS

Body mass index kg/m2 22�0.6 25�0.5 �0.0001 Diastolic blood pressure mm Hg 65�2 66 �1 NSMean arterial pressure mm Hg 80�2 82 �1 NSUrine sodium mmol/kg/day 3�0.3 3�0.2 NS

Protein intake g/kg/day 1�0.04 1�0.05 NS Genotype AC/CCSystolic blood pressure mm Hg 109 �2 128�2 �0.0001Hematocrit 0.38�0.005 0.43�0.005 �0.0001

Systolic blood pressure Diastolic blood pressure mm Hg 68�2 78 �2 0.0002Mean arterial pressure mm Hg 81�2 95 �2 �0.0001mm Hg 110�2 121�2 �0.0001

Diastolic blood pressuremm Hg 67�2 72�2 0.01

Mean arterial pressuremm Hg 81�2 89�2 0.0002

RESULTS

Subject characteristics

A total of 34 healthy females and 47 healthy maleswere performed: 1 minute at 95�C, 1 minute at 55�C, andwere recruited to participate in this study. The baseline11⁄2 minutes at 72�C. The primers were 5�-GCACCATcharacteristics of the subjects are shown in Table 1. TheGTTTTGAGGTT-3� and 5�-CGACTACTGCTTAGCmean age was similar in both groups but the body massATA-3�. This PCR amplification yields a 546 bp product.index (BMI) was higher in the male subjects. ComplianceThe amplicon was subjected to overnight incubation withwith the prescribed sodium and protein diet was deter-Ddel, a restriction endonuclease, at 37�C, and the diges-mined by obtaining 24-hour urine collections for mea-tion products were separated by electrophoresis on 1.5%surement of the sodium excretion rate and urea excre-agarose gels containing ethidium bromide. The 1166Ction. Mean values for urinary sodium excretion andAGT1R allele contains a recognition site for the restric-protein intake corrected for body weight were similar intion endonuclease, Ddel, so that digestion of the PCRmale and female subjects. HCT was higher among maleproduct with Ddel yields 435 bp and iii bp fragments.subjects.The 1166A AGT1R allele does not contain a recognition

As expected, male subjects were found to have highersite for the restriction endonuclease, Ddel, so that themean values for SBP, DBP, and MAP than females (Ta-546 bp amplicon remains unaltered after incubation withble 1), although all values fell within the “normal range.”Ddel [29, 30].

Blood pressure by gender and genotypeStatistical analysisThe distribution of genotypes in the male and femaleComparisons between groups of baseline parameters

subjects was similar. In the women, 12 subjects were AA,were made using nonparametric methods (Wilcoxin rank20 were AC, and 2 were CC genotype. In the men, 21sum test). A P value �0.05 was designated to representmales were AA genotype, 22 were AC, and 4 were CC.statistical significance. Subjects were then segregatedGiven the small numbers of subjects homozygous forinto four subgroups on the basis of gender and the pres-the C allele, subjects were divided into four groups, ac-ence or absence of the C allele (AA versus AC/CC).cording to gender (male versus female) and genotypeData are presented as mean � SEM. We considered(AA versus AC/CC). The mean values for blood pres-gender and genotype as categorical variables, and bloodsure in each of the four groups are shown in Table 2.pressure as a continuous variable. Differences in bloodWhen SBP, DBP, and MAP were taken individuallypressure between the four groups based on gender andas continuous variables using the least square meansgenotype were then analyzed using analysis of covari-method, there was a statistically significant relationshipance. By constructing a model incorporating gender, ge-between blood pressure and gender genotype. Malesnotype, and an interaction term of the two variables (gen-in the AC/CC group exhibited significantly higher bloodder genotype), the relation of these factors to bloodpressure values than males in the AA group. Males inpressure was assessed. The differential effect of genderthe AC/CC group also had higher SBP, DBP, and MAPand genotype was analyzed using least square meansthan females in the AA and AC/CC group. SBP, DBP,procedures, using blood pressure as a continuous vari-and MAP were similar in women in the AA group andable. The differences in systemic and renal hemodynamicAC/CC group. In a statistical model that incorporatedresponses to angiotensin II were analyzed again usingboth gender and genotype as categorical independentanalysis of covariance to examine the relationship be-variables to predict blood pressure (gender genotype),tween gender genotype and hemodynamic responses.there was a significant interaction between genotype All statistical analyses were performed using the statisti-

cal package SAS (SAS Institute, Inc., Cary, NC, USA). gender, and blood pressure, with a P value � 0.03.

Reich et al: Gender and angiotensin receptor gene polymorphism1446

Table 4. Renal and peripheral hemodynamic responses toTable 3. Baseline renal hemodynamic parametersangiotensin II infusion

Female Male P valueFemale Male P value

Genotype AA N � 10 N � 12GFR mL/min/1.73 m2 120�11 101�4 0.06 Genotype AA N � 10 N � 12

MAP mm Hg 7�1 6�1 NSERPF mL/min/1.73 m2 608�60 606�23 NSRBF mL/min/1.73 m2 983�97 1050�42 NS GFR mL/min/1.73 m2 �29�12 �7�5 0.06

ERPF mL/min/1.73 m2 �244 �55 �185 �40 NSRVR mm Hg/min/L 100�15 87�3 NSFF 0.20�0.02 0.17�0.009 NS RBF mL/min/1.73 m2 �409 �87 �335 �43 NS

RVR mm Hg/min/L 10�2a 5�1 0.03Genotype AC/CC N � 13 N � 17GFR mL/min/1.73 m2 104�3a 98�3 NS FF 0.05 �0.01 0.06�0.009 NS

Genotype AC/CC N � 13 N � 17ERPF mL/min/1.73 m2 590�23 574�27 NSRBF mL/min/1.73 m2 954�40 1017�47 NS MAP mm Hg 7�1 5�1 NS

GFR mL/min/1.73 m2 �9�4 5�5b NSRVR mm Hg/min/L 88�4 96 �6 NSFF 0.18�0.005 0.18�0.008 NS ERPF mL/min/1.73 m2 �156 �33 �167 �32 NS

RBF mL/min/1.73 m2 �265 �43 �308 �44 NSAbbreviations are: GFR, glomerular filtration rate; ERPF, effective renalRVR mm Hg/min/L 5�1 5�1 NSplasma flow; RBF, renal blood flow; RVR, renal vascular resistance; FF, filtrationFF 0.05 �0.01 0.08�0.01fraction.

aP � 0.02 (AA females versus AC/CC males) Abbreviations are: MAP, change in mean arterial pressure in response toangiotensin II; GFR, change in glomerular filtration rate in response to angio-tensin II; ERPF, change in renal plasma flow in response to angiotensin II;RBF, change in renal blood flow in response to angiotensin II; RVR, changein renal vascular resistance in response to angiotensin II; FF, change in filtration

Hemodynamic response to angiotensin II infusion fraction in response to angiotensin II.aP � 0.05 (AA females versus AA males, AC/CC females, AC/CC males)At baseline and after angiotensin II infusion, 52 sub- bP � 0.01 (AA females versus AC males)

jects (23 female, 29 male) were studied. Baseline renalhemodynamic function data are shown in Table 3. Afterangiotensin II infusion (Table 4), male and female sub-

rationale for this study was based on reports of the associ-jects of both AGT1R genotypes exhibited similar risesation of the C allele (A1166→C) of the AGT1R genein SBP (6 � 2 versus 8 � 1 mm Hg for AA males versusand clinical parameters, including hypertension, cardio-females; 7 � 2 versus 7 � 2 mm Hg for AC/CC malesvascular outcomes, and our studies of systemic and renalversus females), DBP (6 � 2 versus 7 � 1 mm Hg forhemodynamic function [15, 16]. Finally, there is emerg-AA males versus females; 4 � 2 versus 7 � 2 mm Hging data that gender differences in RAS activity mayfor AC/CC groups), and MAP (Table 4). Analysis ofaccount, at least in part, for the difference in blood pres-variance confirmed that these differences were not statis-sure between men and women [18, 25, 31–33].tically significant.

The first major finding of this study was the demonstra-The renal hemodynamic response to angiotensin IItion of an association between the AGT1R (A1166→C)was also compared across the four groups (Table 4).gene polymorphism and blood pressure in male subjectsThere was no statistically significant difference in ERPF,but not in female subjects. Although blood pressure val-RBF, or FF across the four groups. Comparison of theues fell within the normal range in all of the study sub-four groups by least square means procedure revealedjects, male subjects in the AC/CC group had significantlydifferences in the GFR response to angiotensin II. Fe-higher blood pressure values than males of the AA geno-males demonstrated a greater fall in GFR in responsetype. This association, however, was not observed into angiotensin II compared with males (P � 0.02). Fur-female subjects; blood pressure values in the AC/CCthermore, females of the AA genotype demonstratedgroup were similar to the values in the AA group. Fur-the greatest fall in GFR compared with the other threethermore, a statistical regression model incorporatinggroups (P � 0.01 versus males of the AC/CC genotype).both gender and the AGT1R (A1166→C) gene polymor-Analysis of covariance revealed that a model incorporat-phism revealed a significant interaction between theseing gender genotype best describes the change in GFRtwo factors that correlated with blood pressure.in response to angiotensin II infusion (F � 4.1 for model,

As expected and previously reported, male subjects inP � 0.02). Comparing the RVR between the four groupsthis study had higher blood pressure values than femaleby least squares procedure also revealed that the greatestsubjects, even though all of the values fell within theincrease in RVR also occurred in females of the AAnormal “physiologic range.” Epidemiologic data fromgenotype (P � 0.05 versus all other groups).the Third National Health and Nutrition ExaminationSurvey showed that overall MAP was higher in both

DISCUSSION normotensive and hypertensive men than in women [17].This relationship has been shown to persist throughoutThe primary goal of this study was to determine if

there is an interaction between gender, blood pressure, adulthood and to be consistent across ethnic groups [34].The Framingham Heart Study also illustrates that femalerenal hemodynamics, and the AGT1R (A1166→C) gene

polymorphism in young healthy Caucasian subjects. The gender appears to have a protective effect on blood pres-

Reich et al: Gender and angiotensin receptor gene polymorphism 1447

lar wall stiffness [37–39] and perhaps increased RASactivity [15, 38], both of which may contribute to thepathogenesis of elevated blood pressure. In epidemio-logic studies, the genotype has also been observed tocorrelate with adverse clinical end points such as hyper-tension, renal, and cardiovascular disease, but results ofthese association studies have been inconsistent [5, 7, 8,40–43]. The finding of elevated blood pressure in subjectswith the C allele is consistent with the data mentionedabove; however, the absence of a relationship betweenthe C allele and elevated blood pressure in females sug-gests that there is a critical link between gender, theAGT1R (A1166→C) gene polymorphism, and bloodpressure.Fig. 1. Mean arterial pressure (MAP) in females (�) and males ( )

Our finding of a highly significant interaction betweenwith the AA and AC polymorphisms. *P � 0.05 (AC/CC males versusAA males); †P � 0.05 (AC/CC males versus AC/CC females). gender, the AGT1R (A1166→C) gene polymorphism,

and blood pressure is in accord with other recent studiesof gender and gene polymorphisms of components ofthe RAS. An analysis of a large population from thesure; mean SBP and DBP of women did not exceed thatFramingham Heart Study subjects has revealed a rela-of men at all ages [19]. Our study reveals an associationtionship between the angiotensin-converting enzymein young healthy normotensive individuals studied in a(ACE) deletion-insertion polymorphism and blood pres-salt-replete state with monitored protein intake. It is insure in men but not in women [44]. Another populationaccord with observations of large epidemiologic studiesstudy, the Copenhagen City Heart Study, assessed poly-and suggests that males maintain consistently highermorphisms of the angiotensinogen gene and found anmean blood pressure, even within the “normal range”association between double homozygosity of the Thr235of readings.and Thr174 gene variants and elevated blood pressureThe mechanisms responsible for gender-associatedin women but not in men [42]. Our study extends thesedifferences in blood pressure remain unclear. The focusobservations because, unlike the aforementioned stud-of trials designed to investigate the processes underlyingies, we focused on young, healthy, normotensive individ-gender differences in hemodynamic function has cen-uals maintained on prescribed sodium and protein diets

tered around the role of sex hormones on blood pressure to reduce the confounding effect of salt and protein in-and their influence upon the RAS. Estrogen has been take on blood pressure and RAS activity.demonstrated to have a favorable effect on blood pres- The second major finding of our study was the differ-sure, as shown in a murine model examining the develop- ence in renal hemodynamic response to angiotensin IIment of diet-induced hypertension in ovariectomized infusion by gender and genotype. There was a greaterrats [35]. Androgens may also play a role in gender fall in GFR in response to angiotensin II in females,differences in blood pressure, as suggested by studies of particularly of the AA genotype. This was accompaniedambulatory blood pressures in children at the onset of by a greater increase in RVR. Further statistical analysispuberty. Higher blood pressures are observed in boys revealed that gender and AGT1R genotype were thecompared with girls, and this phenomenon becomes ac- most important factors determining the change in glo-centuated postpuberty [36]. Significant gender differ- merular filtration in response to angiotensin II.ences in the RAS have also been implicated. For in- Our finding of a decline in GFR in women after angio-stance, plasma renin activity in normotensive men has tensin II infusion is in keeping with previous work bybeen shown to be consistently higher than that in women, our group [25], where we demonstrated that the renalregardless of age [32]; this finding has also been con- hemodynamic response to angiotensin II infusion is in-firmed in animal models [18, 31]. In addition, differing fluenced by gender. In the present study, however, wesensitivity to angiotensin II infusion according to gender demonstrate that this renal hemodynamic change is alsohas been demonstrated, further implicating gender-spe- influenced by the AGT1R genotype, with females of thecific differences in the RAS [25, 33]. AA genotype appearing to exhibit an augmented GFR

The functional importance of genetic polymorphisms of decline in response to angiotensin II, which was possiblycomponents of the RAS, including the AGT1R (A1166→C) due to exaggerated afferent arteriolar vasoconstrictiongene polymorphism, remains to be elucidated. Data from or mesangial contraction. This finding is surprising, inphysiologic trials including both male and female sub- that van Geel et al [45], using isolated human arteries,jects suggest that the A1166→C polymorphism is associ- noted that the C allele predicted an increased vasocon-

strictive response to angiotensin II. The possibility existsated with hemodynamic changes such as increased vascu-

Reich et al: Gender and angiotensin receptor gene polymorphism1448

Reprint requests to Judith A. Miller, M.D., F.R.C.P.(C), M.Sc., To-that the decline in GFR we observed in the women withronto General Hospital, 11EN-221, 200 Elizabeth Street, Toronto, On-

the AA genotype was actually due to reduced constriction tario, Canada, M5G 2C4.of the efferent arteriole rather than exaggerated afferent E-mail: judith.miller@utoronto.ca

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19. Vokonas PS, Kannel WB, Cupples LA: Epidemiology and risktion, Inc. (Drs. Miller and Cattran), and operating grants from theof hypertension in the elderly: the Framingham Study. J HypertensKidney Foundation of Canada and the Canadian Institutes of Health(Suppl 6):S3–S9, 1988Research (Dr. J.W. Scholey). The authors would like to thank Clinalfa

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