Angiotensin.-Converting Enzyme Polymorphism in Patients with ...

Angiotensin.-Converting with Terminal Penal

Enzyme Failure1

Polymorphism

Alice Schmidt, Hans Peter Kiener, Ursula Barnas, Ingrid Arias, Winfried Graninger, Alexandra Kaider, and Gert Mayer

Arno

H.P.

Kiener.

ogy.

W.

KA

Rudolfstiftung.

Austria

Vienna,

Austria

KH Lainz.

Vienna.

A.

Department

of

University

(J. Am.

Soc.

DepartVienna,

tween

Medical

of Vienna.

Nephrol.

T

Austria Computer

Vienna.

1996;

be

Sci-

insertion/deletion

7:314-317)

polymorphism

has

been

de-

scribed for the gene that encodes the angiotensinconverting enzyme. The deletion allele is associated

with higher levels,

giotensin mediator

mined

anglotensin-converting

which

ultimately

enzyme

might

II concentrations.

lead

plasma

to increased

Because

an-

anglotensin

II is a

for progressive renal injury, this study deterthe frequency of distribution of the angioten-

sin-converting enzyme phism in 106 hemodialysis

insertion/deletion patients and

polymorin a group of

95 healthy

control patients. There was no difference between the two groups as far as the distribution of the insertion and deletion allele was concerned. Of the total hemodialysis population, 26.4% exhibited the deletion/deletion genotype, as compared with 37.9% of the healthy control population. Also, when patients

with

terminal

renal

failure

as a result

of gb-

analyzed separately, the frequency of the deletion/deletion genotype was identical to that of the control group. Furthermore, the frequency of hypertension, coronary artery disease, left ventricular hypertrophy, and dilated cardiomyopathy, were analyzed according to the angiotensinmerular

disease

were

converting enzyme genotype, but the deletion allele could not be defined as a risk factor in the study’s hemodialysis population. It was therefore concluded that the angiotensin-converting letion polymorphism is not 1

ReceIved

2

corraspondence

VIenna,

October

Wahrlnger

14, 1994. Accepted

to Dr. G. Mayer, Gurtel

18-20,

enzyme

a major

September

Department

A-1090

Vienna,

314

for

3, 1995.

Austria.

1046.6673/0702-03 14$03.00/0 of the American Society of Nephrology copydght C 1996 by the American Society of Nephrology Journal

insertion/de-

risk factor

of Nephrology,

Martin

Auinger,

renal

patients,

failure.

there

is no

risk for cardiovascular

diseases

enzyme

Kidney failure chronic disease, risk factors,

he variability enzyme (ACE) associated with

of plasma concentrations a variation

Additionally,

association

be-

and

UnIversity

of

the

genotype. genetics. hypertension. insertion-deletion angiotensin-converting has been shown to found in the gene that

encodes the ACE ( 1). An insertion (1)/deletion polymorphism situated in intron 1 6 of the ACE ( 1 7q23) accounts for 50% of the interindividual

Austria

ABSTRACT An

the

Key Words: cardiovascular

of Rheumatol-

Vienna,

M. Auinger. Kaider.

G. Mayer. Vienna,

Department

of Vienna,

ences.

of

in hemodialysis angiotensin-converting

Graninger.

University

I. Arias,

A. lllievich, University

Illievich,

of end-stage

development

A. Schmidt, U. Barnas, ment of Nephrology, Austria

in Patients

(D) locus ACE-

level differences (2). and the association of the DD genotype with significant elevated ACE plasma levels was shown by Tiret et al. ( 1). ACE plays a key role in the production of angiotensin II (All) and maintenance of intrarenal All levels (3). All itself contributes to the regulation of the GFR, because All increases the resistance ofthe efferent glomerular artery (4). All is also supposed to act as a renal growth promotor and a modulator of mesangial cell function (5). All seems to be involved In the pathogenesis of progressive renal disease, and it can be speculated that the ACE polymorphism might contribute to the development of ESRD via increased All levels. We therefore determined the frequency distribution of the lID polymorphism in a population of patients on regular hemodialysis (HD) and compared the data with those obtained in healthy controls. Furthermore, we evaluated the frequency of different cardiovascular diseases in HD patients according to genotype. as recent publications report an association of the DD genotype with an increased risk for myocardial infarction, hypentrophic cardiomyopathy. and

sudden

cardiac

hypertrophy

death,

as

well

as

left

ventricular

(6,7,8).

SUBJECTS AND METHODS Patients

and

Controls

The ACE genotype was determined in 106 Caucasian patients with end-stage renal failure (45 women, 61 men, mean age 55. 1 ± 1 7.3 yr; mean time on dialysis, 3.7 ± 4.3 yr). The mean age at the first manifestation ofrenal disease in this group was 40.9 ± 20.5 yr and was therefore similar to the age of a healthy control group of 95 Caucasians (46 women, 49 men, mean age 37.5 ± 1 1 yr) at the study time. All control subjects were normotensive (blood pressure measured after 5 mm rest ofbelow 140/90 mm Hg on at least two different occasions) and showed no signs of renal (normal serum creatinine level and a negative dipstick test for proteinuria, hematunia, and glucosuria) or cardiac disease (negative medical history and normal electrocardiogram.

Volume

7

.

Number

2

.

1996

Schmidt

In 62 patients. the native kidney disease that led to terminal renal failure was of gbomerular origin (including 24 patients with Type I and 13 patients with Type II diabetes). and 25 patients suffered from tubulointerstitial nephropathy. In 19 patients, the underlying renal diseases could not be determined retrospectively. HD patients were diagnosed as hypertensive when blood pressure-lowering agents had to be administered, or when blood pressure after hemodialysis was repeatedly above 140/90 mm Hg although the patient had reached an optimum dry weight on clinical assessment. The presence of coronary artery disease was proven by angiography in nine patients, 1 7 patients had a history of myocardial infarction with pathological changes in the electrocardiogram. and an additional 1 7 patients had a positive medical history of nitrosensitive angina. Furthermore, the prevalence of other risk factors for coronary heart disease, such as nicotine abuse (in a total of 23 patients. and in 10 patients who had coronary artery disease) and hyperchobesterobemia (total serum cholesterol values above 5.2 mmol/L, with low-density lipoprotein cholesterol levels above 3.9 mmol/L. or highdensity lipoprotein cholesterol levels less then 0.9 mmol/L, detectable in a total of34 patients, including 12 patients with coronary artery disease) was determined. All HD patients underwent

cardiac

ultrasonography,

and

pertrophy was diagnosed in 30 of the patients had a reduced left-ventricular out regional wall motion disturbance, classified with dilated cardiomyopathy. patients an ischemic etiology cannot

left-ventricular

hy-

106 patients. Fourteen ejection fraction withand were therefore although in these be excluded.

PCR Analysis Genomic DNA from leukocytes was prepared by standard techniques. A set of primers was used to encompass the polymorphic region in intron 16 of the ACE gene. The pobymerase chain reaction (PCR) contained 1 jig of DNA template. 140 nmol of each primer, 200 tmol of 4dNTPs, 2.5 units of Taq DNA Polymerase. and 3.0 mmol MgCl2. DNA was amplifled for 30 cycles (denaturation at 95#{176}C for 1 mm. annealing at 58#{176}C for 1 mm, extension at 72#{176}C for 1 min) with a final extension time of 7 mm (9). The two PCR products, a 190base pair (bp) fragment in case of deletion, and a 490-bp fragment in case of insertion, were separated on 2% agarose gel and identified by ethidium-bromide staining.

Statistics

patients

gene

and

a group

frequency distribution in hemodialysis

of healthy

controls Genotype

ACE Group

DD Hemodialysis Patients (N= 106) Healthy Controls

(N

=

ID

II

28 (26%)

51 (48%)

27 (26%)

36 (38%)

38 (40%)

21 (22%)

95)

served by Marian et a!. (7). Nonetheless, we compared the data obtained in our HD patients population with the data obtained by Cambien et al. in the largest published healthy-control population of approximately 730 individuals (6). Although the frequency of the DD alelbe in this study was only 27% and was therefore comparable with a recent control population published by Schunkert et al. (8), our HD patient population was not different. Patients with gbomerubar disease formed the largest subgroup in our study population. and especially in this disease entity. the renin-angiotensin system (RAS) has been reported to be of importance. Still, no higher frequency of the DD genotype could be found in this population when compared with the control group (see Table 2). Table 3 shows the frequency of hypertension, coronary artery disease, left-ventricular hypertrophy, and dilated cardiomyopathy in our patient group according to the ACE genotype. There was no difference in the distribution of cardiovascular the different I/D groups. In logistic statistical influence of the genotype risk factors such as nicotine abuse olemia could be seen. However, smoking and hyperchobesterobemia patients) significantly increased the onary heart disease independent of

diseases between regression. no or of other single or hypercholestera combination of (obtained in five incidence of corthe ACE genotype.

There

is increasing

evidence

TABLE 2. Absolute confidence controls,

with

the

RAS

and

relative frequency, the DD genotype on hemodialysis, and

Intervals,

for

in patients

terminal

that

renal

failure

as a result

DD Genotype was

no

significant

difference

in

tion of the I and D allele in the HD patients when compared with our healthy controls. percent of the patients showed the DD

the

did 38% of the healthy controls (see Table 1). The prevalence of the DD genotype was quite high in our control population. but was comparable to that ob-

of the

American

Society

of Nephrology

and 95% in healthy in patients

of glomerular

95% Confidence

Group Absolute

distribu-

as a whole Twenty-six genotype, as

is involved

disease Frequency

RESULTS

Journal

and relative polymorphism

.

of the ACE

DISCUSSION

Statistical analysis was done first by chl-squared test. In a second step, 95% confidence intervals were evaluated to describe the magnitude of prevalence of DD genotype. compared with ID and II genotypes in the whole patient popubation and in patients with primary glomerular disease. In a third stage, marginal and partial (adjusted for the other factors) logistic regression was used to evaluate the strength of the prognostic factors (genotype, hypercholesterolemia, and nicotine abuse) for the cardiovascular diseases.

There

TABLE 1 Absolute

et al

Healthy Controls (N = 95) All Patients (N = 106) Hemodialysis Patients with Glomerular (N = 62)

36 28 13

Relative 38% 26% 21 %

Interval 28 to 48% 1 8 to 36% 1 2 to 33%

Disease

315

ACE Polymorphism

and

TABLE 3. Absolute gene polymorphism cardiovascular

Renal

and

Disease

relative frequency of ACE in hemodialysis patients with

diseases AC E Genotype

Cardiovascular

Hypertension Coronary

(N

=

Disease

(N = 67) Artery Disease

ID

II

1 6 (24%) 14 (33%)

34 (51 %) 18 (42%)

1 7 (25%) 1 1 (25%)

7 (23%)

14 (47%)

9 (30%)

7 (50%)

4 (29%)

43)

Left Ventricular Hypertrophy (N = 30) Dilated Cardiomyopathy (N=

DD

3 (21

%)

14)

in the pathogenesis ical and experimental

of progressive trials that

renal disease. Clinuse ACE inhibitors

and All antagonists have shown the beneficial effects of decreasing All availability in various experimental and clinical renal diseases. ACE inhibitors and All antagonists act by influencing intrarenal hemodynamic parameters (10) and by reducing proteinuria ( 1 1 ). All antagonists have also been suggested to slow the progression of renal disease independent of the hemodynamic factors by reducing the mesangial influx and the tubular concentration of plasma proteins ( 1 2) via restoration of gbomerular size selectivity (13). Furthermore, All is supposed to act as an intrarenal growth hormone (5), inducing proliferation of mesangial cells and an increase of mesangial matrix. both of these being factors that contribute to an increase in gbomerular volume, which has been shown to lead to subsequent glomerulosdlerosis ( 14). A variability of the activity of the RAS, because of genetic variations of the constituents, may therefore predispose people to kidney disease. because all components of the RAS are expressed within kidney tissue (3). Systemic variations were found to be partly the result ofan I/D polymorphism, which is the reason for the interindividual variability ofACE plasma levels (2). The DD genotype deserves special attention, because it is correlated with higher plasma ACE levels ( 1 ). We therefore analyzed the distribution frequency of the ACE gene polymorphism in patients with terminal renal failure. Compared with healthy controls, there was no significant difference in the variation of the gene in the overall HD group and In our largest subgroup ofpatients with primary gbomerular disease. However, our patient sample size did not allow further subanalysis of specific gbomerular diseases, and we therefore cannot exclude, e.g. , that in diabetic nephropathy, the DD genotype might constitute a risk factor for the development ofESRD. Recently. Marre et al. associated the I /1 genotype with a lower incidence of diabetic nephropathy ( 1 5), whereas Doria et a!. found a significant correlation between the DD genotype and diabetic nephropathy ( 16). These results, however, have recently been questioned by Schmidt et at. (17).

316

In patients with cardiovascular diseases, the ddetion genotype was found to be an Independent risk factor for the development of myocardlal infarction, hypertrophlc and dilated cardiomyopathy, as well as left-ventricular hypertrophy and the incidence of sudden cardiac death (6,7,8), whereas no correlation of the ACE polymorphism with essential hypertension could be observed ( 18). Cardiovascular diseases are the most frequent cause of mortality in hemodialysis patients and are increased fivefold when compared with the general population ( 19). We therefore analyzed the frequency of cardiovascular diseases in our study population with regard to the ACE genotype, and also considered other risk factors such as nicotine abuse and hypercholesterolemia. In our HD population, there was no significant difference in the prevalence of cardiovascular diseases among the three genotype groups. The ACE polymorphism therefore does not seem to be associated with a higher risk for cardiovascular disease in the dialysis population, perhaps because of the influence of other potent risk factors. We concluded that the ACE polymorphism, and especially the DD genotype, is not a major risk factor for the development of terminal renal failure in an unselected patient population. We also found no influence of the ACE polymorphism on the frequency of cardiovascular disease in our patient population.

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8. Schunkert H, Hense HW, Holmer SR. et at.: Association between an deletion polymorphism of the angiotensin converting enzyme gene and left ventricular hypertrophy. N EngI J Med 1994;330:1634-1638. 9. Saiki mc, Gelfand DH, Stoffel 5, et aL: Primer directed enzymatic amplification ofDNA with a thermostable DNA pobymerase. Science 1988;239:487-49 1. 10. Anderson 5, Meyer TW, Rennke HG, Brenner BM: Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass. J Clin Invest 1985;76: 612-619.

1 1 . Weidmann

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de

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Number

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2

of

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