2550 Volume 7 - Number 12 - 1996

The Deletion Polymorphism of the Angiotensin I-ConvertingEnzyme Gene Is Associated with Target Organ Damage inEssential Hypertension1Roberto Pontremoli, Antonella Sofia, Angelito Tirotta, Maura Ravera, Clizia Nicolella,

Francesca Viazzi, Gian Paolo Bezante, Luigi Borgia,

Giorgio Sacchi, and Giacomo Deferrari2

R. Pontremoli. A. Sofia. A. Tirotta. M. Ravera. C.Nicolella. F. Viazzi. G.P. Bezante, G. Deferrari, Depart-ment of Internal Medicine, University of Genoa.Genoa, Italy

L. Borgia. Institute of Ophthalmology. University ofGenoa, Genoa, Italy

N. Bobola, P. Pavazzolo, Institute of Biology and Ge-netics. University of Genoa. Genoa, Italy

G. Sacchi. Institute of Methodology, Economics andStatistics, University of Urbino, Urbino, Italy

(J. Am. Soc. Nephrol. 199#{243};7:2550-2558)

ABSTRACTThe activity of the renin-angiotensin-aldosterone sys-tem is thought to play a significant role in the devel-opment of target organ damage in essential hyper-tension. An insertion/deletion (I/O) polymorphism of

the angiotensin I-converting enzyme (ACE) gene hasrecently been associated with increased risk for leftventricular hypertrophy and coronary heart diseasein the general population. The D allele is associatedwith higher levels of circulating ACE and thereforemay predispose to cardiovascular damage. Thestudy presented here was performed to investigatethe association between the ACE genotype, mi-croalbuminunia, retinopathy, and left ventricular hy-pertrophy in 106 patients with essential hypertension.ACE gene polymorphism was determined by poly-merase chain reaction technique. Microalbuminuniawas evaluated as albumin-to-creatinine ratio (A/C) inthree nonconsecutive first morning urine samples(negative urine culture) after a 4-wk washout period.Microalbuminunia was defined as A/C between 2.38to 19 (men) and 2.96 to 20 (women). Hypertensiveretinopathy was evaluated by direct funduscopicexamination (Keith-Wagener-Barker classification)and left ventricular hypertrophy by M-B mode echo-cardiography. The distribution of the DD, ID, and I!

1 Received April 19, 1996. Accepted July 9. 1996.

2 Correspondence to Dr. G. Deferrari, Department of Internal Medicine, University

of Genoa, Viale Benedetfo XV 6, 16132 Genoa, Italy.

1046.6673/0712-2550103.00/0Journal of the American Society of NephrologyCopyright © 1996 by the American Society of Nephrology

Nicoletta Bobola, Roberto Ravazzolo,

genotypes was 27, 50, and 23%, respectively. Theprevalence of microalbuminunia, retinopathy, andleft ventricular hypertrophy was 19, 74, and 72% re-

spectively. There were no differences among thethree genotypes for age, known duration of disease,

body mass index, blood pressure, serum glucose, uricacid, and lipid profile. DO and ID genotypes weresignificantly associated with the presence of mi-croalbuminunia (odds ratio, 8.51 ; 95% confidenceinterval, 1.07 to 67.85; P = 0.019), retinopathy (oddsratio, 5.19; 95% confidence interval, 1.71 to 15.75; P =0.005) and left ventricular hypertrophy (odds ratio,5.22; 95% confidence interval, 1.52to 17.94; P= 0.016).

Furthermore, patients with DO and ID genotypes

showed higher levels of A/C (3.6 ± 0.9, DO: 2.6 ± 0.7,ID; 0.9 ± 0.2 mg/mmol, II; P = 0.0015 by analysis ofvariance) and increased left ventricular mass index(152 ± 4.7, DD+!D versus 133 ± 5.7 g/m2, II; P = 0.01)compared with I! patients. The 0 allele was signifi-

cantly more frequent in patients with microalbumin-

unia (odds ratio, 2.59; 95% confidence interval, 1.24 to5.41 ; P = 0.013) and in those with retinopathy (oddsratio, 2.44; 95% confidence interval, 1.21 to 4.90; P =0.015). Multiple regression analyses performed

among the entire cohort of patients demonstratedthat ACE genotype significantly and independentlyinfluences the presence of retinopathy, left ventricularhypertrophy, and microalbuminunia. In conclusion,the D allele of the ACE gene is associated with mi-croalbuminunia as well as with retinopathy and left

ventricular hypertrophy, and seems to be an inde-

pendent risk factor for target organ damage in essen-

tial hypertension.

Key Words: ACE gene polymorphism. renin-angiotensin-aldo-

sterone system. left ventricular hypertrophy. retinopathy, mi-croalbuminuria

P atients with essential hypertension are heteroge-

neous in their clinical characteristics and prog-

nosis. Several factors besides the bevel of blood pres-

sure values may contribute to the development of

target organ damage and cardiovascular complica-

tions. Among these factors. an unambiguous role isplayed by the renin-angiotensmn-aldosterone system

( 1 ).

The genes encoding components of the renin-angio-

Pontremoli et al

Journal of the American Society of Nephrology 2551

tensin system are appealing candidates for a signifi-

cant role in the development of cardiovascular damage

in patients with hypertension. The renin-anglotensln-aldosterone system is present in circulating and tis-

sue-based forms and is Involved In sodium homeosta-

515. cardiovascular remodeling, and maintenance of

vascular tone. Angiotensin I-converting enzyme (ACE)

is a key component within the renin-anglotensin-

system. where It hydrolyzes anglotensin I to generate

the powerful vasoconstnictor angiotensin II, and

within the kalllkrein-kinin system, where It macti-

yates the vasodilator bradykinmn. In humans, the

plasma level of ACE is genetically determined. The

ACE gene. which has been mapped to human chro-

mosome 1 7q23. has an insertion/deletion (lID) poby-

morphism In Intron 16. The mean plasma ACE level inDD subjects Is about twice that of II subjects with ID

subjects having intermediate bevels (2.3). Although the

lID polymorphism of the ACE gene is considered to

play a small robe in the pathogenesis of hypertension,

the higher bevels of ACE associated with the D allele

may lead to greater angiotensmn II formation In cardiac

and vascular tissues, predisposing a subject to car-

diovascular damage.

In fact, the DD genotype has recently been reported

to be a risk factor for left ventricular hypertrophy (4,5)

and lschemic heart disease (6,7), both in the general

population and in patients with diabetes mellitus (8),

even though other studies could not confirm this

association (9).

The study presented here was performed to explore

the possibility that ACE gene polymorphism may be

related to the development of target organ damage In

essential hypertension.

METHODS

Study Population

Since January 1994, 106 consecutive patients (all Cauca-sian Europeans) with essential hypertension who were at-tending the outpatient clinic of our institution were asked toparticipate in this study. which was part of a larger clinicaltrial (MAGIC. : Microalbuminuria: A Genoa Investigation

on Complications) approved by the Ethical Committee of ourInstitution. Exclusion criteria were the presence of neoplas-tic. hepatic and/or renal disease, chronic heart failure (NewYork Heart Association Classes III and IV). diabetes mellitus,severe obesity (defined as body weight > 1 50% of ideal bodyweight), disabling diseases (such as dementia), or the inabil-Ity of the patients to cooperate. Diagnosis of essential hyper-

tension was made by the attending physician after complete

medical history, physical examination. and routine blochem-Ical analyses of blood and urine were obtained from everypatient. Further investigation was carried out only whenabnormalities were found In these analyses, or when othersymptoms or signs suggesting secondary hypertension were

present. Hypertension was defined according to Joint Na-tional Committee V criteria as an average blood pressure� 140/90 mm Hg on at least three different occasions. or bythe presence of antlhypertensive treatment. None of thepatients was on drug treatment at the time of the study. Theyhad either never been treated for hypertension or had been

taken off therapy at least 4 wk before the study. Twenty-four

urinary collection was obtained In each patient for the mea-

surement of creatinine and electrolytes On the study day,height and weight were measured. then venous blood was

drawn after an overnight fast in order to measure hemato-chemical parameters. Blood pressure was measured by a

trained nurse on the right arm, with the patient In a sitting

position after a 5-mm rest. with a mercury sphygmomanom-eter (cuff size 1 2.5 x 40 cm). The systolic and diastolic blood

pressures were read to the nearest 2 mm Hg. Disappearance

of Korotkoffs sounds (Phase V) was the criterion for diastolic

blood pressure. The lowest of three consecutive readingswere recorded. Body mass index (BMI) was calculated withthe formula: BMI weight (Kg)/height (m)2. Standard elec-trocardiogram ( 1 2 leads) and funduscopic examination wereobtained for each patient. Creatinine, BUN. electrolytes. uricacid, triglycerides. total and high-density lipoprotein chobes-terol, and other standard chemistry evaluations were per-

formed on serum and urine according to routine methods.Low-density bipoprotein cholesterol was calculated using

Friedewald’s formula ( 10). FamIly history and lifestyle habitswere assessed by means of a standardized questionnaire.

Assessment of ACE Genotype

DNA was isolated from frozen whole blood containingEDTA as anticoagulant by a standard salting-out procedure.and was resuspended in a it ( 10 mM Tris/ 1 mM EDTA, pH7.6) buffer. Genomic DNA (approximately 0.25 �.tg) was am-plified by polymerase chain reaction in a 50-g.tL reactionmixture containing 10 mM Tris-HC1. pH 8.8. 50 mM KCI. 1.5mM MgCb2. 0.1% TrIton X-100 (Sigma Chemical Co., St.Louis, MO). 200 �.tM each dATP. dCTP. dGTP. dTTP, 0. 1 2 �tMeach primer, and 0.5 U thermostable DNA polymerase (Dy-nazyme. Espoo, Finland). The mixture was overlaid withmineral oil. In any given set of reactions. a negative control

containing no genomic DNA and a positive control of known

genotype were always included. The amplification was ob-

tamed after the following steps: 5 mm of denaturation at

95#{176}C.followed by 30 cycles of 1 mm at 94#{176}C.1 mm at 58#{176}C.and 2 mm of DNA synthesis at 72#{176}C.First, all of the sampleswere analyzed using a pair of primers that amplilv the entireintron 16. as already reported ( 1 1). To avoid ID/DD mistyp-ing. a pair of primers that amplify a region inside intron 16were also used to analyze all samples showing DD genotypeas described (7). The samples were visualized after electro-phoresis on a 2% agarose gel and ethidium bromide staining.

Investigators were blinded as to type when genotyping was

undertaken.

Microalbuminuria

The presence of microalbuminurla was evaluated. at theend of the wash-out period (if any) as the albumin-to-creatinine ratio (A/C) on three nonconsecutive. first morning

urine samples. Only samples from patients with a negative

urine culture were collected. Whenever a positive urine cul-

ture was found. urine samples were discarded, appropriateantibacterial treatment was instituted, and urine collectionsfor albuminuria were repeated only after a second culturetested negative. The A/C was calculated as follows: urinaryalbumin concentration (milligram per liter)/urinary creati-nine concentration (mmol per liter). Serum and urine creat-

mine levels were determined by the routine Jaffe’s reaction.Urine albumin concentration was measured by a commer-cially available RIA kit (Sclavo. Ciniselbo Balsamo. Italy). An

A/C between 2.38 and 19 (men) and between 2.96 and 20

ACE Gene and Organ Damage in Hypertension

2552 Volume 7 - Number 12 - 1996

(women) was used to define microalbuminuria. These criteriaproved to have good sensitivity and specificity in the detec-

tion of albumin excretion rate between 20 and 200 ug/min(12). The average of each patient’s three A/C was used toIndicate the level of albumin excretion.

Retinopathy

The presence, type. and extent of hypertensive retinopathywere Investigated in a darkened room and under pupil

dilatation. Direct ophthalmoscopy was carried out with a

halogen ophthalmoscope: the first arteriovenous crossing at

least one disc diameter from the disc in each quadrant wasselected and assessed. Each quadrant of the fundus wasphotographed by means of a Zeiss fundus camera(Oberkochen. Germany) with a 30#{176}field. Photographic slideswere projected and evaluated by the same ophthalmologist.who was unaware of the patients’ clinical data. Retinalfeatures sought were venule nipping. venube deviation, andlight reflex change at all clearly visualized crossings with

comparably sized arterioles at least one disc diameter from

the disc. The mean ratio of diameters of equivalent-orderarterioles and venules in each quadrant was measured andthe presence of focal arterlolar narrowing. hemorrhages.exudates. and papilledema was sought. Retinal lesions wereclassified according to Keith-Wagener-Barker classification

(13).

Echocardiography

All echocardiographic studies were performed using an

Acuson XP-128 ultrasound machine (Mountain View. CA).

Echocardiograms were obtained at rest with patients supinein the left lateral position. by using standard parasternal andapical views. The overall monodimensionab left ventricularmeasurements and the bidimensional (apical four and twochamber) views were obtained according to the recommen-dations ofthe American Society ofEchocardiography (14.15).

All tracings were obtained and read by a single observer who

was blinded to the clinical characteristics of the patients

under observation. Left ventricular mass was derived fromthe formula described by Devereux and associates (16):

LV mass (g) = 0.80 x 1 .04 [(VSTd + LVIDd + PWTd)3-(LVIDd)3j + 0.6

where VSTd is ventricular septal thickness at end diastole,LVIDd Is LV internal dimension at end diastole. and PWI’d isLV posterior wall thickness at end diastole. Left ventricularmass was corrected for body surface area (LVMI), and ex-pressed in units of grams/meter squared (g/m2). The pres-ence of left ventricular hypertrophy (LVH) was defined forLVMI �l34 g/m2 (men) and �110 g/m2 (women) (17). Nopatients showed dyssynergic areas that would invalidate thetheoretical assumptions behind the cardiac mass calcula-

tions.

Statistical Analysis

All data are expressed as mean � SE. Differences betweenvariables were assessed using the appropriate statistical testbased on the underlying distribution of the variables. One-way analysis of variance (ANOVA) with multiple comparisonpost-test were used to analyze data from patients with thethree ACE genotypes. Either a nonparametric (Welch’s I test)or a parametric (Student’s I test) test was used to assess

differences between patients with DD+ID and II genotypes.

Differences between prevalences were assessed by chi-

squared test or Fisher’s exact test as appropriate. The calcu-bation of odds ratios was performed to provide an estimate ofthe relative risk of microalbuminuria, retinopathy, and leftventricular hypertrophy in groups of patients with different

genotypes. Piecewise linear regression analysis was per-formed to assess the contribution of ACE genotype and othervariables on albumin excretion. This analysis may provide a

better assessment of data when the nature of the relation-

ship between one or more independent variables and adependent variable changes over the range of the dependentvariable. It also permits the identification of the point where

discontinuity in the regression line occurs (i.e. , the break-

point of the regression) ( 1 8). MultIple regression analysis wasperformed to assess the independent contribution of ACEgenotype (II = 0, ID+DD = 1 ) and other variables on thepresence of retinopathy and left ventricular hypertrophy. AK-means cluster analysis was performed on the entire cohort

of patients to identify different subgroups of patients accord-ing to the presence / absence of target organ damage and ACE

D genotype (II = 0, DD+ID = 1 ). The appropriateness of theclassification has been assessed by performing a standardbetween-group analysis of variance for each variable ( 1 9). Allstatistical analyses were performed using SAS (SAS Institute,Cary. NC) and Statistica (Statsoft Inc. , Tulsa, OK) software.

RESULTS

The observed overall genotype distribution (DD,

27%; ID. 50%; and II 23%) was consistent with Hardy-

Weinberg equilibrium and similar to previous reports

in caucasian populations (20). There was an overall

frequency of 54% for the D allele and 46% for the I

allele, which compares closely with other white popu-

latlons studied (2 1 ). The overall prevalence of ml-

croabbummnunia. retinopathy. and left ventricular hy-

pertrophy were 19, 74, and 72%, respectively. Therewere no differences in age, prevalence offamiby history

for hypertension. current smokers, known duration of

disease. BMI. blood pressure. serum glucose, uric

acid, and lipid profile when patients were divided

according to their genotype (Table 1).

Patients with DD and ID genotypes were signifi-

cantly more likely to be microalbuminunic (DD, 37%;

ID, 26%; II, 6%; chi-squared. 6.74, P 0.034; chi-

squared for trend, 6.50, P = 0.0 1 ; Table 1 ) and showedhigher levels of A/C (DD. 3.6 ± 0.9; ID, 2.6 ± 0.7; II,

0.9 ± 0.2 mg/mmob; P = 0.027 by ANOVA; Table 1)

compared with II patients. The odds ratio for the

presence of microabbuminunia was 8.51 (95% confi-

dence interval. 1.07 to 67�85, P = 0.019; Figure 1) in

patIents with DD and ID genotypes. The frequency of D

allele was significantly higher in patients with mi-

croabbuminunia (0.69 versus 0.46; odds ratio, 2.59;

95% confidence interval, 1.24 to 5.41; P 0.013;

Table 2) compared with subjects who were nor-

moabbuminuric. A piecewise linear regression anaby-

515 was performed among the entire cohort of patients

to identify factors that independently influence A/C. A

two-slope linear model was identified with a break-

point value of 2.4, almost identical to the one pre-

defined to indicate the passage from normo- to mi-

croalbuminunia. Age, BMI, LDL cholesterol, and ACE

genotype significantly influence A/C levels and to-

100

50-

0 10

� 5.

(I,0001

8.5

5.2 5.2

Pontremoli et al

Journal of the American Society of Nephrology 2553

TABLE 1 . Clinical characteristics of patients according to ACE gene polymorphisma

CharacteristicACE Gene Polymorphism

DD ID II pb

Number of Patients 29 53 24Male/Female 18/1 1 28/25 15/9 NSAge 43.6 ± 2.2 49.7 ± 1.3 47.0 ± 1.9 NSDuration of Disease (months) 47.6 ± 9.3 60.7 ± 8.2 59.2 ± 13.0 NSFamily History of Hypertension (%) 84 65 57 NSCurrent Smokers (%) 52 38 40 NSSBP (mm Hg) 164 � 3 161 ± 2.9 154 � 2.9 NSDBP (mm Hg) 106 ± 1.3 103 ± 1.0 102 ± 1 . 1 NSMBP(mmHg) 125± 1.6 122± 1.4 119t 1.5 NSBody Mass lndexc 27.7 ± 0.83 27.0 ± 0.57 26.6 � 0.9 NSFasting Blood Glucose (mg/dL) 87.5 ± 1.7 91 .0 ± 1.8 94.7 ± 2.5 NSUric Acid (mg/dL) 5.3 ± 0.3 5.4 ± 0.2 5.3 ± 0.4 NSTriglycerides (mg/dL) 128 � 11 130 ± 9 107 ± I 1 NSTotal Cholesterol (mg/dL) 220 ± 10 212 ± 6 214 ± 7 NSHDL Cholesterol (mg/dL) 46 ± 3.7 49 ± 2.0 50 ± 2.7 NSLDL Cholesterol (mg/dL) 154 ± 9.9 133 ± 5.2 143 ± 6.5 NSUrinary Na (mmol/day) 173 ± 17 156 ± 14 147 ± 20 NSA/C (mg/mmol) 3.6 ± 0.9 2.6 ± 0.7 0.9 ± 0.2 0.027Prevalence of Microalbuminuria (%) 37 26 6 0.034Prevalence of Retinopathy (%) 83 80 44 0.009LVMI (g/m2) 154 ± 8.0 151 � 6.0 133 � 5.7 0.132Prevalence of LVH (%) 74 83 33 0.015

a Data are mean ± SE.ACE. angiotensin-converting enzyme; SBP. systolic blood pressure; DBP, diastolic blood pressure; MBP. mean blood pressure;HDL high-densitylipoprotein; LDL low-densityiipoprotein; A�. albumin/creatinine ratio; LvMl. left ventricular mass. corrected for body surface area;LVH, left ventricular hypertrophy; NS, not statistically significant.b p values were calculated by one-way analysis of variance or chi-squared test as appropriate.C Calculated by dividing the weight in kilograms by the square of the height in meters.

0.1Mlcroalbumlnuria Retinopathy LVH

Figure 1 . Relative risk of microalbuminurla, retinopathy, andleft ventricular hypertrophy in patients with essential hyper-tension and ACE DD and ID genotypes. Patients with ACE 0genotypes (00 + ID) are more likely to have target organdamage than patients with ACE II genotype. LVH, left yen-tricular hypertrophy.

gether account for over 75% of variations in albumin-

uria (Table 3).

Hypertensive retinopathy was present in patients

with DD and ID genotypes significantly more often(DD, 83%; ID, 80%; II, 44%; chi-squared, 9.47. P =

0.009: chi-squared for trend, 6.73. P 0.009; Table 1)

than in II patients. The odds ratio for the presence ofhypertensive retinopathy was 5. 19 in patients with DD

and ID genotypes (95% confidence Interval, 1 .7 1 to

15.75, P = 0.005; Figure 1). The frequency ofD allele

was significantly higher In patients with hypertensive

retinopathy (0.59 versus 0.37; odds ratio, 2.44: 95%

confidence interval, 1.21 to 4.90, P = 0.015; Table 2).

Multiple regression analysis performed among the

entire cohort of patients demonstrated that ACE ge-

notype (P <0.01), age (P <0.01), and A/C (P <0.02)

significantly and independently influence the pres-

ence of retinopathy and together account for about

30% of variations In retinal status (r� = 0.292, Table

4).

DD and ID genotypes were associated with a signif-

icantly higher prevalence of left ventricular hypertro-

phy (DD. 74%; ID. 83%; II. 33%; chi-squared. 8.96. P =

0.0 1 ; Table 1 ). Patients with DD and ID genotypes

showed increased LVMI ( 1 52 ± 5 versus 1 33 ± 6

g/m2. p o.oi. Table 1; odds ratio for the presence of

left ventricular hypertrophy. 5.22; 95% confidence

interval, 1.52 to 17.94, P = 0.016; FIgure 1) compared

with II patients. Multiple regression analysis demon-

strated that ACE genotype (P < 0.03) and total cho-

besterob (P < 0.05) sIgnificantly and independently

influence the presence of left ventricular hypertrophy

ACE Gene and Organ Damage in Hypertension

2554 Volume 7 - Number 12 - 1996

TABLE 2. 0-! allele frequency in essential hypertensive patients with and without target-organ damage#{176}

ParameterAllelic Frequency

Alb+ AIb- Ret+ Ret- LVH+ LVH-

0 0.69 0.46 0.59 0.37 0.60 0.50I 0.31 0.54 0.41 0.63 0.40 0.50Odds Ratio 2.59 2.44 1.5295% Confidence Interval 1 . 24 to 5.41 1.21 to 4.90 0.71 to 3.26PValue 0.013 0.015 0.330

a Alb+ and AIb- indicate patients with and without microalbuminuria. Ret+ and Ret- indicate patients with and without retinopathy. LVH+ andLvH- indicate patients with and without left ventricular hypertrophy. The relative risk for organ damage in patients with 0 allele was calculated byFisher’s exact test.

TABLE 3. Significant determinants ofmicroalbuminuria in essential hypertension(piecewise linear regression analysis)a

IndependentVariables

Regression Coefficient

Below AboveBreakpoint Breakpoint

AgeBMI

-0.00120.0239

0.41500.4876

LDLChoI -0.0005 0.0514ACE -0.1843 3.206

a Dependent variable: albuminuria (A/C); independent variables:Age. BMI, LDL Chol. ACE genotype. Breakpoint = 2.4 Regressiondegrees of freedom = 4-16. F = 3.oo, P -0.026. r� = 0.775. A/Cindicates urinary albumin-to-creatinine ratio (mg/mmol); BMI, bodymass index; LDL Chol. LDL cholesterol (mg/dL); ACE, angiotensin-converting enzyme genotype (II = 0. ID + DO = 1).

TABLE 4. Multiple regression analysis of retinopathy0

IndependentVariables

/3 SE� Partial F P

ACE 0.307 0.116 10.80 0.010Age 0.296 0.111 8.63 0.010A/C 0.268 0.116 7.98 0.024

a Dependent variable: retinopathy: independent variables = ACEgenotype. Age. A/C. Regression degrees of freedom = 3-58. F =7.977. p = 0.00015. Intercept = -0.247; r� = 0.292. ACE indicatesangiotensin-converting enzyme genotype (II = 0. ID + DO = 1); A/C.urinary albumin-to-creatinine ratio (mg/mmol).

and together account for about 25% of its variations

(r2 = 0.236, Table 5).

Patients with DD and ID genotypes showed a re-

markabby higher percentage of either at beast two

(odds ratio, 9.333; 95% confidence interval, 2.373 to

36.717. P = 0.0006: data not shown) or all three

cardiovascular abnormalities investigated in this

study (odds ratio. 15.9; 95% confidence interval,

0.9038 to 280.05, P = 0.007; data not shown). Fur-

thermore, the D allele entails a threefold risk of having

either at least two (odds ratio, 2.483: 95% confidence

interval. 1 .26 1 to 4.888. P < 0.0 1 1 ; data not shown) or

all three of the above-mentioned abnormalities (odds

ratio. 3.333; 95% confidence interval. 1.425 to 7.795,

TABLE 5. Multiple regression analysis of leftventricular hypertrophy0

IndependentVariables 13 SEI3 Partial F P

ACE 0.361 0.160 4.40 0.031Chol 0.334 0.160 4.62 0.04

a Dependent variable: left ventricular hyperfrophy; independent van-

ables: ACE genotype. chol. Regression degrees of freedom = 2 to 30.F = 4.623, P = 0.178. Intercept = -0.290; r� = 0.236. ACE indicatesangiotensin-converting enzyme genotype (II = 0. ID + DD = 1); Chol,serum total cholesterol (mg/dL).

TABLE 6. Multi-way, between-groups analysis ofvariance and contrast analysis of the prevalence oftarget-organ damage and ACE genotypes (I! = 0,DD + ID = 1) in patients with essential hypertensiondivided into three clusters according to K-meanscluster analysisa

Variable df F P

Retinopathy 2 to 59 3.944 0.025Microalbuminuria 2 to 59 4.414 0.016ACE Genotype 2 to 59 4.330 0.017Left Ventricular Hypertrophy 2 to 59 19.330 0.005

a The three clusters differ significantly for the presence/absence of

target organ damage and ACE genotype. df. degrees of freedom.

P = 0.006; data not shown). K-means cluster analysis

of the entire cohort of patients allowed us to identify

three subgroups that differ significantly (Table 6) for

the presence (or absence) of retinopathy, microabbu-

minuria, left ventricular hypertrophy, and the fre-

quency ofDD or ID genotype (Figure 2), while showing

no difference as far as other clinical and biochemical

parameters are concerned. Patients in Cluster 2 (N =

15) show a lower than average frequency of ACE DD

and ID genotypes as well as bow prevalence of mi-

croabbuminuria and left ventricular hypertrophy. Pa-

tients in Cluster 1 (N = 3 1 ) show a frequency of DD

and ID ACE genotypes similar to the average of the

entire population and are characterized by a lower

prevalence of microabbuminuria but higher preva-

bence of left ventricular hypertrophy. Finally, patients

100

75

�‘\ ...., -o-clusterl(n31)

50 “� .�.‘ “\ -.- clusteq’ 2 (n15)

2: ‘\.,%, � �.\.\\.:i.ciuster 3 (n=16)

Pontremoli et al

Journal of the American Society of Nephrology 2555

ret. ME ACE. LVH

Figure 2. Plot offrequency oftarget organ damage and ACE0 genotype in patients with essential hypertension dividedaccording to K-means cluster analysis. K-means cluster anal-ysis was performed among the entire cohort of patients.Three different subgroups were identified accordin9 to the

prevalence of target organ damage and the frequency ofACE genotype. Ret + indicates the presence of hypertensiveretinopathy; Mi, microalbuminuria; ACE +, angiotensin-

converting enzyme genotypes DO and ID: LVH, left ventricu-lar hypertrophy. The three clusters differ significantly for eachvariable examined (see Table 6).

in Cluster 3 are characterized by a very high frequency

of ACE DD and ID genotypes associated with a high

prevalence of retinopathy. microalbuminuria, and left

ventricular hypertrophy (Figure 2).

DISCUSSION

The principal finding of this study is that essential

hypertensive patients with DD and ID genotypes of the

ACE gene show a greater prevalence of target organ

damage. The D allele seems to confer an increased risk

for the development of microalbuminuria and retmnop-

athy independently of blood pressure values as well as

other commonly acknowledged cardiovascular risk

factors, namely age. smoking habits, family history

and known duration of hypertension, body mass in-

dex, lipid profile, uric acid, and fasting blood glucose.

Although the DD genotype has been reported to be a

risk factor for left ventricular hypertrophy (3,4) and

ischemic and degenerative heart disease (22) In the

general population, and for a more rapid progression

of renal failure in nondiabetic renal disease (23,24),

this Is, to our knowledge. the first time that It has been

reported in association with the development of mul-

tiple organ damage in essential hypertension. Our

findings also support previous evidence that the re-

nmn-angiotensin-aldosterone system may play an im-portant role in the pathophysiobogy of vascular and

tissue damage in essential hypertension (1.25).In the study presented here, the prevalence of ml-

croalbuminurla, retinopathy. and left ventricular hy-

pertrophy was comparable with what has been re-

ported in the literature (26-29).

Earlier studies have shown a high prevalence of

retinopathy in patients with essential hypertension

(27). Retinal changes are thought to reflect vascular

and tissue damage and thereby the risk of future

vascular events. In fact, hypertensive retinopathy has

been previously reported to be an independent indica-

tor of all-causes mortality (30). In the study presented

here, patients with DD and ID genotypes had a fivefold

greater risk for retinopathy (Figure 1) compared with II

patients. The presence of the D allele entails a twofold

greater risk for retinopathy (Table 2). Patients with

Grades 1 and 2 lesions were analyzed together be-

cause of the substantial overlapping of the retinal

features described (Table 1 ). However, when the sever-

ity of retinal changes was taken into consideration, a

significant assocIation with DD and ID genotypes was

still present (chi-squared, 10.9, P = 0.028; data not

shown). Furthermore, the frequency of D allele was

higher in patients with more severe degrees of retmnop-

athy (chi-squared, 10.3, P = 0.006; chl-squared for

trend, 4.55, P 0.033: data not shown). Multipleregression analyses demonstrated that the ACE poly-

morphism Independently contributes to the presenceof retinopathy and accounts for a significant part of

variation in retinal status (Table 4).

Increased left ventricular mass is a webb-known,

powerful, independent predictor of cardiovascular

morbidity and mortality in hypertension (3 1-33) and

is thought to be a consequence of left ventricular

pressure overload (34-36). The relatively high preva-

bence of left ventricular hypertrophy In our study

(Table 1 ) Is comparable to the one previously reported

in the literature when similar sensitive and specific

techniques were used (37.28). In the study presented

here, patients with DD and ID genotypes had a higher

probability ofhaving left ventricular hypertrophy corn-

pared with II patients (Figure 1 . Table 1 ). When ana-

lyzed together, DD and ID patients showed increased

LVMI compared with II patients (151.9 ± 5 versus

133.2 ± 5 g/m2, P = 0.01; data not shown). Multiple

regression analyses demonstrated that ACE genotype

is an independent predictor of the development of left

ventricular hypertrophy and accounts for a significant

part ofvariation in cardIac mass (Table 5). At present.

data on the relationship between ACE gene and left

ventricular hypertrophy are controversial. A recent

study on a barge group of subjects from the Framing-

ham Heart Study failed to show a role of the ACE gene

in influencing left ventricular mass (38). However, the

results presented here are in accordance with previ-

ous studies reporting an association between the DD

genotype of ACE and the risk of left ventricular hyper-

trophy In the general population including patients

with hypertension (4.5). These contrasting results can

be explained at least in part by the nonuniform genetic

background of study populations and by the possible

confounding effect of antihypertensive treatment.

Although hypertensive retinopathy and left ventric-

ubar hypertrophy have long been regarded as lrnpor-

tant complications of hypertension and predictors of

cardiovascular death, the robe of microalbuminuria as

an important and independent cardiovascular risk

factor or more likely as a marker itself of diffuse

ACE Gene and Organ Damage in Hypertension

2556 Volume 7 - Number 12 - 1996

endothelial vascular damage has only been acknowl-

edged over the last few years. Several investigators

have shown that microalbuminuria is a predictor ofcardiovascular events in hypertensive patients

(29,39). In the 10-yr Goteborg follow-up study (40),

the predictive power of abbuminuria exceeded that of

total serum cholesterol and other cardiovascular risk

factors. More recently, other authors have shown that

In essential hypertension, microalbuminuria is asso-

elated with a higher degree of left ventricular hyper-

trophy (29,4 1 ) as well as hypertensive retinopathy

(27). These data support the conclusion that ml-

croalbuminuria is a marker for early end-organ and

cardiovascular damage in such patients. In the study

presented here. patients with DD and ID genotypes not

only showed higher levels of albuminuria but also a

significantly higher prevalence of microalbuminuria

compared with II patients (Table 1 ). DD and ID geno-

types were associated with an 8.5 times greater prob-

ability of microabbuminuria compared with II patients

(Figure 1 ). Piecewise linear regression analysis dem-

onstrated that ACE genotype, together with age, BMI,

and LDL cholesterol. strongly and independently in-

fluence variations in albumin excretion (Table 3). In-

terestingly, all these independent variables are posi-

tively related to A/C only above the breakpoint of the

regression line, thus indicating that their contribution

to variations In albumin excretion changes and is

more evident in patients with persistent microabbu-

rninuria. When taken together. these data strongly

suggest a robe for ACE gene polymorphism as a poten-

tial genetic risk factor for cardiovascular damage.

Several reasons make the ACE gene, among the

renin-anglotensin genes, one of the major etiobogical

candidates for the development of cardiovascular in-jury. First, the gene is highly expressed in endothelial

cells and in the heart. Second, the bevels of ACE in

plasma and inside the cells show quantitative varia-

tions related to gene polymorphism. An increased

enzyme concentration, associated with the DD and ID

genotypes, may lead to an increase in All generation

and bradykinin degradation. Bradykmnin acts on en-

dothelial �2-kinin receptors and is able to stimulate

nitric oxide formation by endotheliab nitric oxide-syn-

thetase and prostacycbmn synthesis. Furthermore. ex-

perirnental studies suggest that All may stimulate

cardiac protein synthesis (42-44) whereas bradykinin

may have an antiproliferative effect (45.46). Moreover,

lID ACE polymorphism was shown to modulate the

intracellular bevel of ACE in T lymphocytes (47). In

addition, recent studies have demonstrated elevated

cardiac expression of ACE messenger RNA and ACE

activity in experimental animals with pressure-over-

load left ventricular hypertrophy (46,48) and in pa-

tients with heart failure (49). Therefore it is tempting

to speculate that, in patients with DD genotype. higher

ACE activity In cardiac and vascular tissues might

facilitate cellular hypertrophy, extraceblubar matrix

formation (50.5 1 ), and sympathetic neurotransmis-

slon (52). All of the above might contribute to the

development of vascular and tissue damage in essen-

tial hypertension. The results of the study presented

here are in accordance with previous observations,suggesting that increased activity of the renin-angio-

tensin-aldosterone system might play a robe in the

development of target organ damage in essential hy-

pertension. Erley et at. (53) demonstrated the pres-

ence of altered renal hemodynamics and higher prey-

alence of left ventricular hypertrophy, retinopathy,

and microalbuminuria in young essential hyperten-

sive patients with hyperresponsivity of the renin-an-

giotensin-abdosterone system . Accordingly. Alderman

et at. (25) showed that plasma renin activity is an

important and independent risk factor for cardiovas-

cubar complications in a barge number of patients with

essential hypertension, prospectively followed-up for

several years. The fact that in our study the associa-

tion of microalbuminuria, retinopathy. and left yen-

tricular hypertrophy was found far more often in

patients with the ACE D allele is intriguing, because

these three different expressions of early organ dam-

age may be regarded as the end point of pathophysi-

obogical processes, promoted at beast in part by in-

creased angiotensin II bevels. This conclusion seems to

be supported by results of K-means cluster analysis.

which identifies three distinct subgroups differing

significantly in both the frequency of ACE genotype

and in the prevalence of organ damage. Patients with

a high frequency ofDD and ID ACE genotypes (Cluster

3, Figure 2 and Table 6) show a remarkably higher

prevalence of retinopathy, microalbuminuria, and left

ventricular hypertrophy, whereas those with a bow

frequency of the D allele (Cluster 2) seem to be pro-

tected from developing organ damage.

Finally, salt intake has been shown to be related to

both left ventricular hypertrophy and microalbumin-

uria in hypertensive patients (54). In the study pre-

sented here, there was no difference in urinary Na

excretion among the three groups of patients accord-

Ing to ACE genotypes (Table 1 ). However, the study

design does not allow us to exclude a contribution of

dietary salt intake in the development of target organ

damage.

Although the data presented need to be interpreted

with some caution because of the limited number of

patients studied. both the homogeneity of the genetic

background (all patients were of Caucasian origin)

and the frequency of different ACE genotypes, which

was almost identical to the one previously reported in

larger studies on Caucasian patients, led us to believe

that our patients are representative of a larger popu-

bation with essential hypertension.

In conclusion, the results of this study suggest that

the deletion polymorphism of the ACE gene might be

an independent risk factor for the development of

organ damage in hypertensive patients. From a clini-

cab perspective. this implies the possibility of early

identification of patients at increased risk for develop-

ment of target organ damage, by determining ACE

polymorphism. Therefore, a greater effort can be made

Pontremoli et al

Journal of the American Society of Nephrology 2557

to prevent cardiovascular complications and to treat

hypertension and other modifiable risk factors in

these patients. Whatever the underlying molecular

mechanisms linking the ACE-gene polymorphism to

the development of target organ damage may be. the

determination of the ACE genotype could be helpful in

the assessment of cardiovascular risk and in planning

treatment strategies in patients with essential hyper-

tension. Large longitudinal trials are needed to actu-

ably determine the incidence of target organ damage

and cardiovascular complications in patients with

high-risk genotypes and to clarify whether antihyper-

tensive agents that directly exert their action on the

renmn-angiotensmn system may provide additional or-

gan protection beyond blood pressure control.

ACKNOWLEDGMENTWe are grateful to Giacomo Morreale for technical assistance.

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