Angiotensin converting enzyme gene polymorphism and ACE inhibition in diabetic nephropathy

Angiotensin converting enzyme gene polymorphism and ACE

inhibition in diabetic nephropathy

P

J

, K

R

, P

R

, L

T

, C

M

, O

P

,

F

C

, H

-H

P

Steno Diabetes Center, Gentofte, Denmark and Institut National de la Sante´ et de la Recherche Me´dicale SC7, Paris, France

Angiotensin converting enzyme gene polymorphism and ACE inhibition in diabetic nephropathy.The antiproteinuric effect of angiotensin con-verting enzyme (ACE) inhibition in insulin-dependent diabetes mellitus (IDDM) patients with diabetic nephropathy varies considerably. There-fore, we tested the potential role of an insertion (I)/deletion (D) polymor-phism of the ACE gene on this early antiproteinuric responsiveness in an observational follow-up study. Sixty (II, N5 13; ID, N 5 26 and DD, N 5 21) young hypertensive IDDM patients suffering from diabetic nephrop-athy were investigated during three months before and for the initial six month period during ACE inhibition [captopril 44 (SD22) mg/24 hr, no differences in drug dose between groups]. Blood pressure (MABP) and albuminuria (ELISA) were measured three (1 to 6) times before and three (1 to 13) times during ACE inhibition. At baseline the groups (II/ID/DD) had comparable (1) mean arterial blood pressure (MABP mm Hg) of 1136 10/108 6 9/114 6 8, (2) albuminuria (geometric mean with 95% CI) 1394 (747 to 2608)/1176 (844 to 1797) and 1261 (827 to 2017) mg/24 hr, and (3) serum creatinine (geometric mean with 95% CI), 80 (68 to 93)/85 (76 to 97)/103 (85 to 119)mmol/liter, respectively. Angiotensin converting enzyme inhibition induced a significant reduction in MABP, albuminuria and kidney function in all three groups (II/ID/DD; P, 0.05): (1) MABP (mean 6 SD) 126 7/5 67/8 6 9 mm Hg (ANOVA, P 5 0.02); (2) albuminuria [mean (95%CI)] 61 (34 to 77)/22 (3 to 37)/31 (13 to 46) %, (ANOVA, P, 0.01); and (3) increasing serum creatinine [mean (95%CI)] 8 (4 to 12)/9 (3 to 16)/8 (0 to 16) % (ANOVA, NS), respectively. Adjusting for differences in reduction in MABP did not change the association between decrease in albuminuria and ACE/ID genotypes (P, 0.01). A multiple linear regression analysis revealed that the ACE/ID polymor-phism, albuminuria and MABP at baseline independently influenced the decline in albuminuria after initiation of ACE inhibition (R2_{5 0.21, P ,} 0.01). A significant association between changes in MABP and albumin-uria was demonstrated (R2 _{5 0.16, P , 0.01). Our data show that} hypertensive albuminuric IDDM patients with the II genotype are partic-ularly susceptible to commonly advocated renoprotective treatment.

Albuminuria is a risk factor for progression of diabetic ne-phropathy [1, 2]. Antihypertensive treatment, especially angioten-sin converting enzyme (ACE) inhibition, has been shown to reduce albuminuria and to ameliorate progression of diabetic nephropathy in IDDM patients [3]. Studies of diabetic and

non-diabetic kidney disease suggest that reduction in albuminuria during the initial six-month treatment period after the start of ACE inhibition predicts an attenuated rate of decline in glomer-ular filtration rate [4–6]. As a consequence, early reduction in albuminuria during ACE inhibition can be regarded as a surro-gate end point for the prognosis of diabetic nephropathy. The antiproteinuric response to ACE inhibition varies considerably between patients. Individual differences in the renin-angiotensin and kallekrein-kinin systems may influence this variation. Re-cently, an insertion(I)/deletion(D) polymorphism of the ACE gene (ACE/ID) has been shown to influence the antiproteinuric efficacy of ACE inhibition in non-diabetic kidney disease [7–10] and the deterioration in kidney function in both diabetic and non-diabetic kidney disease [7, 8, 11–14].

We tested the potential role of the ACE/ID polymorphism on the antiproteinuric responsiveness to ACE inhibition during the initial six-month treatment period in an observational follow-up study of IDDM patients with diabetic nephropathy.

METHODS Research design

We examined the records of all patients with albuminuria attend-ing the outpatient clinic at Steno Diabetes Center in 1993 who had IDDM and diabetic nephropathy, and who had their glomerular filtration rates (GFRs) measured during the same year (before and/or after antihypertensive treatment). A total of 242 Caucasian patients more than 18 years of age were identified. Angiotensin converting enzyme genotyping was performed in 198 of them as described in detail previously [15]. Lymphocytes were isolated from blood, and DNA was prepared by standard techniques. Polymerase chain reaction (PCR) was used to detect the two alleles of the ID polymorphism. DNA was amplified using primers and PCR-cycling conditions as described previously [16]. In our laboratory, this method yielded no discrepancy with the results obtained using a new procedure with an additional allele-specific primer in the insertion sequence, when tested on 50 samples (11 DD, 30 ID, 9 II) from the ECTIM study [17]. Subjects were classified according to the presence (I) or absence (D) of a 287 base pair insertion in intron 16 of the ACE gene into II, ID or DD genotypes. We identified all patients who had never received antihypertensive treatment (N5 55) or had their antihypertensive treatment withdrawn (N5 16) for at least one month before the initiation of ACE inhibition. Eleven of the previously untreated

Key words: ACE gene and inhibition, diabetic nephropathy, IDDM, polymorphism in ACE gene, proteinuria, albuminuria, progression of diabetes, glomerular filtration rate.

Received for publication May 2, 1997 and in revised form November 6, 1997 Accepted for publication November 7, 1997

© 1998 by the International Society of Nephrology

patients could not be evaluated in the present study since data on albuminuria and serum creatinine during the initial six-month treatment period were lacking (see below). The remaining sixty patients (II, N 5 13; ID, N 5 26; DD, N 5 21) fulfilled the inclusion criteria and all gave fully informed consent. The dose of captopril, given b.i.d., was titrated to achieve a reduction in blood pressure to , 140/90 mm Hg or at least 10/5 mm Hg below baseline. In an attempt to achieve this goal or because of edema 20 patients had a diuretic added. Apart from this addition, no other antihypertensive drugs were used. The long-term effect of ACE inhibition in relation to the ACE/ID polymorphism has previously been described in 24 of the current previously-un-treated patients and in the excluded eleven patients mentioned above [11]. The experimental design was approved by the local ethical committee. The patients had diabetic diet (45 to 55% carbohydrates, 30 to 35% fat, and 15 to 20% protein) without restriction in sodium or protein intake. Measurements of albu-minuria and blood pressure were carried out one to six (median 3) times within three months before the start of treatment with an ACE inhibitor in the previously untreated patients (N5 44, NS between groups), and three times one month after withdrawal of other antihypertensive treatment in the previously treated pa-tients (N 5 16). During the first six months of ACE inhibitor treatment albuminuria and blood pressure were measured 1 to 13 (median 3) times (NS between groups). The mean duration after ACE inhibition of albuminuria assessment was the same in the three groups [II, 3 months (range 1 to 6 months); ID, 3 months (1 to 6); and DD 3 months (2 to 6)]. Urinary albumin concentration was determined by an enzyme immunoassay from all 24-hour urine collections made at home and during the four-hour clear-ance period when GFR was measured [18]. More than 90% of all albuminuria measurements in each of the three groups were based on 24-hour urine collections (NS between groups). Arterial blood pressure was measured with a standard clinical mercury sphygmomanometer (cuff 25 cm3 12 cm) on the right arm while the patient was sitting after 10 minutes of rest. Diastolic blood pressure was recorded at the disappearance of the Korotkoff sounds (phase V). Mean arterial blood pressure (MABP) was calculated as diastolic blood pressure plus one third of the pulse amplitude. Serum creatinine concentration was assessed by a kinetic Jaffe method before and six months after the start of ACE inhibition. Forty-seven patients had their GFR measured before and 48 after the initiation of ACE inhibition by applying a single injection, 51_{Cr-EDTA (3.7 MBq) four-hour plasma clearance}

technique [19]. Only 38 patients had their GFRs measured both before and during ACE inhibition (data not shown). Hemoglobin A1C (HbA1C) concentration from venous blood samples was

determined by high performance liquid chromatography (DIAMAT Analyzer; Bio-Rad, Richmond, CA, USA). Serum cholesterol concentrations were measured by a conventional laboratory technique. Diabetic retinopathy was assessed by fundus photography after pupillary dilation.

Statistical analysis

Values are given as means 6 SD, except for urinary albumin

concentration and serum creatinine, which are expressed as geometric means with a 95% confidence interval (CI) owing to the skewed distribution. The calculations of baseline values of albu-minuria and arterial blood pressure for each patient include all values measured within three months prior to initiation of ACE

inhibitor treatment in previously untreated patients and values measured one month after the withdrawal of other antihyperten-sive drugs in previously treated patients. Calculations of albumin-uria and blood pressure during ACE inhibition for each patient include all values measured from one to six months after initiation of ACE inhibitor treatment. For normally distributed variables, including logarithmically transformed values of urinary albumin concentration and serum creatinine, the three groups were com-pared by a one-way analysis of variance (ANOVA). Significant ANOVAs were followed by a pair-wise comparison uring Schef-fe’s test. For non-normally distributed continuous variables the three groups were compared by the Kruskal-Wallis test. A paired Student’s t-test was used to compare albuminuria, blood pressure and serum creatinine before and after the start of ACE inhibition within the groups. A linear regression analyses of albuminuria change (logarithmical) versus blood pressure change for each of the groups and all 60 patients were performed. A multivariate stepwise linear regression analysis of putative risk factors influ-encing the change in albuminuria after start of ACE inhibition was performed with backward selection. Angiotensin converting enzyme I/D genotype was entered as an ordered categorical variable (II5 1, ID 5 2, DD 5 3) due to the relationship between genotype and serum ACE activity. The R2_{value is adjusted for the}

number of variables introduced into the model. A P value (two sided) of, 0.05 was considered to be significant. All calculations were performed using SPSS (SPSS, Chicago, IL, USA).

RESULTS

At baseline the three groups with different genotypes were similar with regard to sex, age, duration of diabetes, body mass index, insulin dose, frequency of retinopathy, glycemic control, MABP, albuminuria, serum creatinine and serum cholesterol (Table 1). The dose of captopril (mg/24 hr) [42 (22)/43 (23)/47 (21)], and the number of patients receiving a diuretic after starting ACE inhibition (N5 5/7/8) were also similar in the patients with II, ID and DD genotypes, respectively (NS).

Initiation of treatment with ACE inhibition induced a signifi-cant drop in albuminuria in all three groups (P, 0.05), but the reduction was significantly greater in patients with II genotype, 61% (34 to 77)% [mean (95% CI)], compared to patients with either ID, 22% (3 to 37%), or DD genotype, 31% (13 to 46%; P, 0.01; Table 2). The MABP declined during ACE inhibition within each group (P , 0.01), however, a more pronounced reduction was observed in the patients with II genotype compared to patients with ID and DD genotype (P5 0.02; Table 2). An almost identical (P 5 0.95 between groups) increase in the serum creatinine was seen in all groups after the initiation of ACE inhibition (Table 2).

Plotting change in Log10(albuminuria) versus change in mean

arterial blood pressure for each of the groups (Fig. 1) revealed the following regression slopes (95% CI): II, 0.044 (0.024 to 0.064; R2

5 0.65, P 5 0.0005); ID, 20.003 (20.017 to 0.012; R2_{5 0.01, P 5}

0.67); and DD, 0.008 (20.003 to 0.019; R2_{5 0.06, P 5 0.14). A}

simple factorial ANOVA of the change in albuminuria including all 60 patients adjusted for change in MABP revealed an inde-pendent effect of the ACE genotype (P 5 0.004). The MABP adjustment was carried out within each of the three groups based on the above-mentioned regression slopes.

genotype, baseline albuminuria and baseline MABP indepen-dently influenced the decline in albuminuria after the start of ACE inhibition (R2 _{5 0.21, P , 0.01), while baseline serum}

creatinine, HbA1Cand captopril dose did not contribute to the

decline. A linear regression analysis revealed a significant corre-lation between changes in MABP and albuminuria (R2_{5 0.16,}

P, 0.01).

DISCUSSION

During the initial six months after the beginning of antihyper-tensive treatment, our observational follow-up study revealed that ACE inhibition has an antiproteinuric effect in hypertensive IDDM patients with diabetic nephropathy, and that this beneficial effect is most pronounced in patients with the insertion allele of the ACE/ID polymorphism. The latter group also had a slightly greater reduction in MABP during this initial period of ACE inhibition, but adjusting for this difference in blood pressure reduction within each of the three groups did not change the association between decline in albuminuria and ACE/ID

geno-types. Furthermore, it should be stressed that other factors influencing albuminuria such as the dose of ACE inhibitor, glycemic control, and kidney function were comparable between groups. Thus, our study indicates that the ACE/ID polymorphism, albuminuria, and MABP at baseline independently influence the decline in albuminuria after initiation of ACE inhibition. Since the present data are not based on a randomized, double-blind treatment trial of the same dose of ACE inhibition, bias must be taken into account. During the last 10 years ACE inhibition has been considered the drug of choice in treating IDDM patients with nephropathy at the Steno Diabetes Center. Treatment with ACE inhibitors was independent of genotype and none of the patients stopped ACE inhibition during the study, thus accounting for all enrolled patients. Furthermore, the distribution of geno-types in patients enrolled in the present study did not differ from the genotype distribution observed among hypertensive patients in the original cohort [20]. Finally, baseline clinical and laboratory data were the same in the different genotype groups.

Recently, the Euclid Study Group examined the effect of the ACE/ID polymorphism on the antiproteinuric responsiveness to ACE inhibition in a randomized, double-blind placebo controlled trial of lisinopril (10 mg/day) in 530 normotensive IDDM patients with normo- or microalbuminuria [21]. At two years, urinary albumin excretion rate was 57% lower on lisinopril in the II group, 19% lower in the ID group, and 19% higher in the DD group as compared to placebo. In contrast, previous short-term studies dealing with a variety of non-diabetic kidney diseases have yielded conflicting results regarding the antiproteinuric effect during ACE inhibition in relation to the ACE/ID polymorphism [8–10, 22]. However, long-term studies dealing with larger numbers of patients with different non-diabetic nephropathies have revealed that neither ACE inhibition nor ab-blocker lowered the degree of proteinuria in the DD group, while the ID and II groups were both sensitive to the antiproteinuric effect [7].

Originally, Rigat et al [23] demonstrated that plasma ACE

Table 2. Effect of ACE inhibition in 60 IDDM patients with diabetic nephropathy according to ACE/ID genotypes

Genotype P value II ID DD Number 13 26 21 Decline in MABP mm Hg 12 (7) 5 (7) 8 (9) 0.02b Decline in albuminuria %a 61 (34–77) 22 (3–37) 31 (13–46) , 0.01 c Increase in serum creatinine %a 8 (4–12) 9 (3–16) 8 (0–16) NS Values are means (SD).

a_{mean (95% C.I.)}

b_{P, 0.05 for II versus ID, NS between other pairwise comparisons} c_{P, 0.05 for II versus ID and II versus DD}

Table 1. Clinical and laboratory data at baseline in 60 IDDM patients with diabetic nephropathy according to insertion (I)/deletion (D) polymorphism in the ACE gene (ACE/ID genotypes)

II genotype ID genotype DD genotype P value

No. of men/women 7/6 17/9 17/4 NS

Age years 35 (5) 39 (9) 40 (8) NS

Duration of diabetes years 20 (7) 19 (5) 22 (9) NS

No. of patients with retinopathy

Simplex 3 10 10 NS

Proliferative 10 16 11 NS

Insulin dose IU/kg/24 hr 0.7 (0.1) 0.7 (0.2) 0.6 (0.2) NS

Body mass index kg/m2 _{24.2 (4.0) 23.5 (2.1) 24.4 (2.9) NS}

HbA1C% 9.3 (0.9) 9.3 (1.4) 9.1 (1.0) NS

MABP mm Hg 113 (10) 108 (9) 114 (8) 0.07

Albuminuria mg/24 hra _{1394 (747–2608) 1176 (844–1797) 1261 (827–2017) NS}

Serum creatininemmol/litera _{80 (68–93) 85 (76–97) 103 (85–119) 0.08}

GFR ml/min/1.73m2b _{94 (22) 96 (27) 85 (26) NS}

Serum cholesterol mmol/liter 5.4 (0.9) 5.5 (1.2) 5.6 (1.0) NS

Antihypertensive treatment during study

Captopril dose mg/24 hr 42 (22) 43 (23) 47 (21) NS

Frusemide mg/24 hrc _{80 (40–1000) 100 (40–200) 50 (40–160) NS}

No. of patients taking

frusemide 5 7 8 NS

Values are means (SD). a_{geometric mean with 95% CI}

b_{Values based on 47 patients (II, ID, DD: N5 10, N 5 18, N 5 19, respectively)} c_{median (range)}

levels in DD subjects were twice that of II subjects, with ID subjects having intermediate levels. We found a similar associa-tion in IDDM patients with diabetic nephropathy [15]. The patients studied at present constitute a subset of this former study. Angiotensin converting enzyme is of importance in regulating systemic and glomerular circulation by converting angiotensin I into angiotensin II and inactivating bradykinin. Increased synthe-sis of angiotensin II may play a role in the progression of albuminuria and glomerular filtration rate in diabetic nephropa-thy by hemodynamic mechanisms, that is, systemic and glomerular hypertension and by growth promoting effects on glomerular cells, particularly mesangial cells [24]. Enhanced systemic pressure response to intravenous infusion of angiotensin I, in normotensive non-diabetic subjects homozygous for the DD allele of the ACE gene as compared to the II genotype, has been reported [25]. The same group demonstrated in a double blind, cross-over design that the attenuation of the intravenous angiotensin I pressor response by 0.01 mg of enalapril was significantly greater and lasted longer in non-diabetic normotensive subjects homozygous for the II ACE genotype, suggesting that the ACE I/D polymor-phism may differentiate responses to ACE inhibition [26]. Addi-tional explanations for the deleterious effect of the deletion polymorphism on albuminuria may be elevated intrarenal angio-tensin II formation and/or insufficient angioangio-tensin converting enzyme inhibition.

The antiproteinuric effect during ACE inhibition is in part dependent on the reduction of systemic blood pressure and particularly on the degree of transmission of the systemic pressure to the glomerular capillaries. The systemic blood pressure reduc-tion was greater in the II group as compared to the two other groups, but even after an adjustment for this difference the data still suggest that the II genotype is particularly susceptible to ACE inhibition. In addition to the hemodynamic effect of ACE inhibi-tion on albuminuria, non-hemodynamic effects such as reducinhibi-tion in pore size in the glomerular capillaries have been documented [27]. Furthermore, the maximum antiproteinuric effect of ACE inhibition usually occurs after two to four weeks, which suggests contribution from non-hemodynamic mechanisms [28]. Protein and sodium restriction was not applied in our study and

approx-imately the same number of patients (27 to 40%) within the three genotype groups received diuretics. A previous study has docu-mented that severe sodium restriction potentiates the antiprotein-uric effect of ACE inhibition in non-diabetic kidney diseases [29]. None of our patients received non-steroidal anti-inflammatory drugs or other compounds known to influence albuminuria during the study period. These findings and the multiple linear regression analysis suggest that hypertensive macroalbuminuric IDDM pa-tients with the II/ACE genotype are particularly susceptible to the commonly advocated renoprotective therapy.

Previous studies in diabetic [4, 6, 30] and non-diabetic kidney disease [5] have indicated that the initial degree of reduction in albuminuria after the start of antihypertensive treatment with or without ACE inhibition is highly predictive for future progression in GFR, that is, patients with the largest initial reduction in albuminuria have the smallest subsequent rate of decline in GFR. All previous studies in non-diabetic and diabetic nephropathies have demonstrated that the deletion polymorphism of the ACE gene, particularly the homozygote DD, is a risk factor for an accelerated loss of kidney function [7, 11, 12, 22]. Furthermore, the deletion polymorphism in the ACE gene reduces the long-term beneficial effect of ACE inhibition on progression of dia-betic and non-diadia-betic kidney disease [7, 11].

Our data show that hypertensive albuminuric IDDM patients with the II genotype are particularly susceptible to the commonly advocated renoprotective treatment.

Reprint requests to Dr. Hans-Henrik Parving, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark.

APPENDIX

Abbreviations used in this article are: ACE, angiotensin converting enzyme; D, deletion genotype; GFR, glomerular filtration rate; HbA1C, hemoglobin A1C; I, insertion genotype; IDDM, insulin-dependent diabe-tes mellitus; MABP, mean arterial blood pressure; PCR, polymerase chain reaction.

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