ACE DD genotype is more susceptible than ACE II and ID genotypes to the antiproteinuric effect of ACE inhibitors in patients with proteinuric non-insulin-dependent diabetes mellitus

Sung-Kyu Ha1,, Seung Yong Lee1, Hong Su Park1, Jae Ho Shin1, Seung Jung Kim2, Do Hun Kim2, Kyung Rae Kim1, Ho Yung Lee1 and Dae Suk Han1

1 Department of Internal Medicine, Yongdong Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, and 2 Department of Internal Medicine, Ajou University College of Medicine, Suwon, Korea



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Methods
 Results
 Discussion
 References
 
Background. ACE polymorphism, especially genotype DD or D allele, may be involved in the progression of diabetic nephropathy. It may also have different effects on the reduction of proteinuria by ACE inhibitors in patients with proteinuria. We investigated the relationship between ACE gene polymorphism and antiproteinuric effect of ACE inhibitors (Benazepril 10 mg/day or Perindopril 4 mg/day) in 83 NIDDM patients with overt proteinuria (urinary protein excretion over 500 mg/day).

Methods. We recruited NIDDM patients with overt proteinuria from our renal clinic. Before entry, previously used ACE inhibitors were withdrawn for at least 2 weeks and baseline proteinuria and albuminuria were measured. Patients were classified into three groups in accordance with ACE genotypes (17 DD; 33 ID; 33 II) and prospectively followed up for 3 months. Various clinical parameters including age, DM duration, body mass index (BMI), 24-h urine sodium, protein and albumin, BUN, serum creatinine, creatinine clearance (Ccr), mean arterial pressure (MAP), and HbA1c were measured in the pre- and post-treatment periods. ACE genotypes were determined by polymerase chain reaction.

Results. There were no significant differences in the clinical parameters such as age, DM duration, BMI, BUN, serum creatinine, Ccr, MAP, HbA1c, and daily urinary excretion of sodium, protein and albumin among three groups (P>0.05). After the 3-month treatment period using ACE inhibitors, there were no significant differences in the reduction of MAP and Ccr among the three groups (P>0.05). However, the percentage reductions in urinary excretion of protein and albumin for DD genotype were significantly higher than in ID and II genotypes (50.9±19.2% vs 19.2±16.0%, 20.2±20.4%; 52.6±23.6% vs 13.5±51.8%, 24.8±23.9%, P<0.05). There were no statistically significant correlations between the levels of baseline proteinuria and albuminuria and the magnitudes of the reduction of proteinuria and albuminuria under ACE inhibition (P>0.05).

Conclusions. Our results suggest that the ACE gene polymorphism might have a role in determining the responsiveness to the antiproteinuric effect of ACE inhibition in proteinuric NIDDM patients.

Keywords: ACE gene; ACE inhibitors; antiproteinuric effect; NIDDM



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Methods
 Results
 Discussion
 References
 
Angiotensin-converting enzyme (ACE) inhibitors are the first line drugs for the therapy of hypertension associated with diabetes in that they not only lower systemic blood pressure but also affect renal haemodynamic changes. Imanishi et al. [1] showed that the mechanism by which an ACE inhibitor caused short-term decrease in albuminuria in early diabetic nephropathy involved a glomerular haemodynamic change, namely, a decrease in glomerular capillary pressure, and this property has an important role in the treatment of diabetic nephropathy [2,3]. Overt proteinuria itself is a risk factor that may adversely affect renal function, and it is associated with a faster rate of renal disease progression [4,5]. Therefore the reduction of proteinuria is an important tool for retarding the progression of renal disease in these patients. ACE inhibitors have been used widely in everyday clinical practice for the purpose of reducing proteinuria in patients with various renal diseases including diabetes mellitus. However, the antiproteinuric effect of ACE inhibitors on proteinuria is variable and the percentage of reducing proteinuria is in the range of 20–80% in a variety of renal diseases [68]. The antiproteinuric effect of ACE inhibitors may be related to a number of factors: the type or the dose of ACE inhibitors, the duration of therapy, the level of sodium intake, and the type of the patient's ACE genotype [912]. The ACE in the renin–angiotensin system affects the cardiovascular system through angiotensin II formation and inactivation of bradykinin. The ACE gene is located on the long arm of chromosome 17. Insertion or deletion of any of the 287 base pair fragments in the 16th intron of the ACE gene may determine its genotype. These genotypes in turn are used to characterize the polymorphism as II, ID or DD type. Recently it has been reported that each of these genotypes appeared to be a major determinant of plasma and tissue ACE activities [13,14]. Individuals with DD genotype have the greatest and those with II genotype the least ACE concentrations. It is expected that the differences in plasma and tissue ACE activities associated with ACE genotype might affect the antiproteinuric response to ACE inhibition. The antiproteinuric mechanism of ACE inhibitors are thought to decrease glomerular capillary pressure by reducing both efferent renal arteriolar resistance and systemic blood pressure [15,16].

In the present study we investigated the antiproteinuric effect of ACE inhibitors (benazepril or perindopril) in relation to ACE I/D polymorphism in an observational short-term follow-up study of NIDDM patients with overt proteinuria.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Methods
 Results
 Discussion
 References
 
Subjects
Eighty-three NIDDM patients with overt proteinuria (mean value of the urinary protein excretion over 500 mg/day in the baseline status) were recruited from the Renal Clinic of the Department of Medicine at Yongdong Severance Hospital, Yonsei University College of Medicine from September 1997 to August 1998. They were classified into three groups in accordance with their ACE genotypes (17 DD; 33 ID; 33 II). They were prospectively followed up for 3 months. All were Korean. Before enrollment in the study, all the patients signed informed consent forms. Previously used ACE inhibitors were withdrawn for at least 2 weeks before the study and baseline proteinuria and albuminuria were measured using a 24-h urine collection. Each patient provided two 24-h urine collections. The mean values of the two measurements were taken as the baseline data. We resumed previously used ACE inhibitors with fixed dose (benazepril 10 mg/day or perindopril 4 mg/day). Ten of the 17 DD patients, 15 of the 33 ID patients, and 18 of the 33 II patients were treated with benazepril. The remaining patients were placed on perindopril. There was no specific reason for choosing one ACE inhibitor over the other in a given patient. Blood pressures were measured by sphygmomanometer in a sitting position, and a mean of two consecutive blood pressure reading was recorded. Mean arterial pressure (MAP) was calculated as the sum of two-thirds of the diastolic and one-third of the systolic blood pressure. A diagnosis of hypertension was made if the patient had two consecutive blood pressure readings >140/90 mmHg or if mean arterial pressure was >105 mmHg. Various clinical parameters including age, gender, DM duration, BMI, 24-h urinary excretion of sodium, protein and albumin, BUN, serum creatinine, creatinine clearance (Ccr), mean arterial pressure (MAP), and HbA1c were measured in the pre- and post-treatment periods (3 months). During the 3-month study period, patient medication was not changed. Dietary intake for sodium was also not changed during the study period.



   Methods
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 Subjects and methods
 Methods
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Specimen collection
After a 12-h overnight fast, 5 ml of EDTA-anticoagulated blood samples were drawn from the patients, and centrifuged within 4 h. Buffy coat layers were refrigerated for DNA extraction at -20°C. BUN, serum creatinine and HbA1c were measured from the separated plasma. Urinary excretions of creatinine, protein, albumin, sodium and creatinine clearance were measured by 24-h urine collections for two samples at baseline and 3-month follow-up periods. The mean of the two measurements was taken.

Measurements of serum and urine parameters
BUN, serum creatinine and urinary excretions of creatinine, protein, and sodium were measured with standard laboratory techniques. Urinary albumin was measured by a nephelometric method (Behring Nephelometer 100, Marburg, Germany). HbA1c was measured by ion-capture assay.

Determination of ACE genotypes
For determination of I/D polymorphism of ACE gene, genomic DNA was extracted from peripheral blood leukocytes. A 287-bp I/D polymorphism in the intron 16 of the ACE was examined by polymerase chain reaction (PCR). PCR was performed according to the method of Tiret et al. [17]. The sequences of the sense and the antisense primers were 5'-CTGGAGAC-CACTCCCATCCTTTCT-3' and 5'-GATGTGGCCATCACAT-TCGTCAGAT-3' respectively. PCR was performed in a final volume of 20 µl that contained 200 ng of genomic DNA, 0.5 µmol of each primer, 0.25 mmol/l dNTP, 1.5 mmol/l MgCl2, 50 mmol/l KCl, 10 mmol/l Tris-HCl (PH 8.4), and 1 U Taq DNA polymerase. Amplification was carried out for 35 cycles with steps of denaturation at 94°C for 2 min, annealing at 58°C for 15 s, and extension at 72°C for 30 s. The PCR products were subjected to electrophoresis in 1.5% agarose gels and stained by ethidium bromide for visualization. To avoid mistyping of the ID genotype as DD, we repeated the genotyping procedure with DD genotype.

Statistical analysis
All data are presented as mean±SD except urinary protein and albumin. A one-way ANOVA test was used to analyse between-group and within-group differences. A paired Student's t-test was used to test for differences between the baseline values and those after ACE inhibition therapy. Variables with skewed distributions (e.g. BUN, creatinine) were logarithmically transformed and then tested by Student's t-test. Concomitantly, the non-parametric Mann–Whitney U tests were used to analyse variables with skewed distributions in spite of logarithmic transformations (e.g. urinary protein and albumin excretion). Statistical significance was assumed at a 5% level.



   Results
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 Subjects and methods
 Methods
 Results
 Discussion
 References
 
The ratio of ACE genotypes (DD : ID : II) for the 83 proteinuric NIDDM patients was 0.2 : 0.4 : 0.4, respectively and that of ACE allele, D and I, was 0.4 : 0.6. The distribution of ACE genotypes and allele frequencies were not different from a normal Korean population [18]. Table 1GoGo shows baseline characteristics of patients before initiating ACE inhibitors. There were no significant differences in the clinical parameters such as age, gender, body mass index (BMI), DM duration, BUN, serum creatinine, HbA1c, % of hypertension, mean arterial pressure (MAP), creatinine clearance (Ccr), and 24-h urinary excretions of sodium among three groups (P>0.05) (Table 1Go). However, the median values of 24-h urinary protein excretion in the three genotype groups were significantly different (1152 (182–6580) mg/day in DD vs 628 (204–4752) mg/day in ID and 666 (162–3600) mg/day in II, P<0.05). Also, there were statistically significant differences in the level of median albumin excretion among the three groups (1010 (18–4958) mg/day in DD vs 289 (13–3596) mg/day in ID and 330 (56–3358) mg/day in II, P<0.05). After the 3-month treatment using ACE inhibitors, there were no significant differences in the reduction of blood pressure and creatinine clearance among the three groups (P>0.05) (Table 2GoGo).


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Table 1. Clinical characteristics of NIDDM patients with nephropathy by ACE genotypes before treatment with ACE inhibitors

 

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Table 2. Comparison of cinical findings among the three different genotypes after treatment with ACE inhibitors

 
Short-term 3-month ACE inhibition resulted in significant reductions of proteinuria and albuminuria in all three genotype groups. The magnitude of the responses varied between individuals. The individual responses of proteinuria and albuminuria are shown in Figures 1Go and 2Go, respectively. There were statistically significant differences between baseline values of protein and albumin excretion and values of protein and albumin excretion after ACE inhibitions (P<0.05). Also, the percentage reduction of 24-h urinary excretion of protein and albumin for DD genotype was significantly greater than that of ID and II genotypes (50.9±19.2 vs 19.2±16.0, 20.2±20.4%; 52.6±23.6 vs 13.5±51.8, 24.8±23.9% respectively, P<0.05) (Table 2Go, Figure 3Go). To exclude the possibility that the patients with the greater baseline proteinuria and albuminuria had the greater decreases of proteinuria and albuminuria following ACE inhibition, we compared the baseline values of proteinuria and albuminuria and the percentage reduction of proteinuria and albuminuria after ACE inhibition. There was no statistically significant correlation between the levels of baseline proteinuria and albuminuria and the magnitudes of the reduction of proteinuria and albuminuria after ACE inhibition (Figure 4Go). These findings suggest that the amount of baseline proteinuria and albuminuria do not affect the magnitude of the percentage reduction of proteinuria and albuminuria after ACE inhibition.



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Fig. 1. Comparison of proteinuria before and after treatment with ACE inhibitors in the three different genotypes. *P<0.05 by paired Student's t-test (ACE inhibition vs baseline). Median values are indicated by the horizontal lines.

 


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Fig. 2. Comparison of albuminuria before and after treatment with ACE inhibitors in three different genotypes. *P<0.05 by paired Student's t-test (ACE inhibition vs baseline). Median values are indicated by the horizontal lines.

 


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Fig. 3. Per cent reduction of proteinuria and albuminuria according to ACE genotype before and after treatment of ACE inhibitors. *P<0.05, compared to ID and II genotypes by one-way ANOVA test.

 


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Fig. 4. Correlations between baseline proteinuria and albuminuria and percentage reduction of proteinuria and albuminuria. No significant correlations between baseline values and % reductions.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Methods
 Results
 Discussion
 References
 
In the present study, we evaluated whether the effects of short-term ACE inhibition on proteinuria, albuminuria, blood pressure, and renal haemodynamics were affected by the ACE genotypes in proteinuric NIDDM patients. We found that patients with DD genotype are more susceptible to the antiproteinuric effect of ACE inhibition than those with II or ID. Interest in the role of ACE genotype in the progression of renal disease has been raised by several recent studies and the rate of long-term renal function loss has been found to be more rapid in patients with the DD genotype than those with II or ID. This was found in patients with IgA nephropathy [19], diabetic nephropathy in IDDM [20], as well as NIDDM [21], and in a population of patients with non-diabetic renal disorders of diverse aetiology [22]. In IgA nephropathy, Yoshida et al. [19] reported that a significant decrease in urinary protein excretion was observed in patients with ACE gene DD genotype after 1 year of treatment with ACE inhibitors, but not in patients with the ID or II genotypes. Our present study in NIDDM patients with overt proteinuria concurs with the result of Yoshida et al. [19] in that the ACE gene DD may have a significant role in the antiproteinuric effect of the ACE inhibitors. Moriyama et al. [9] also studied 36 patients with various renal disorders, and found impaired antiproteinuric response to ACE inhibition in patients with the II genotype. This study, however, included a small number of patients with the DD genotype, a feature that is characteristic of Northern Asians. Not only the difference in study population, but also difference in study design preclude a straightforward comparison of their data with ours.

Recently the EUCLID trial examined the effect of the ACE I/D polymorphism on the antiproteinuric responsiveness to ACE inhibition in a randomized, double-blind, placebo-controlled study of lisinopril (10 mg/day) in 530 normotensive IDDM patients with normo- or microalbuminuria [23]. Patients with II genotype showed the fastest rate of albumin excretion rate (AER) progression on placebo but had an enhanced response to lisinopril. AER at 2 years was 51.3% lower on lisinopril than on placebo in the II genotype patients (P=0.01), 14.8% in the ID group (P=0.2), and 7.7% in the DD group (P=0.7). Jacobsen et al. [24] also reported the antiproteinuric effect of ACE inhibition in 60 IDDM patients with diabetic nephropathy. They examined the potential role of an I/D polymorphism of the ACE gene on the early antiproteinuric responsiveness in an observational follow-up study and concluded that hypertensive albuminuric IDDM patients with the II genotype were particularly susceptible to renoprotective therapy. Our present observation does not coincide with EUCLID and Jacobsen studies. More recent REIN trial reported that in non-diabetic proteinuric nephropathies, the ACE I/D polymorphism is a strong predictor of ACE inhibition-associated renoprotection, in that proteinuria, {Delta} GFR, and a progression to ESRD are effectively reduced in patients with the DD, but not in those with the II or ID genotype [25]. Our study concurs with the result of REIN trial, in that the ACE gene DD may have a significant role in the antiproteinuric effect of the ACE inhibitors.

The antiproteinuric effect of ACE inhibitors has been thought to be independent of its antihypertensive effect on the systemic blood pressure. In this study we did not find any significant differences in MAP fall among the three genotype groups. However, the antiproteinuric effect of ACE inhibition in DD genotype was pronounced compared to II or ID genotypes. An I/D polymorphism of the ACE gene has been shown to influence the ACE level in circulation and tissue. It has been thought that the ACE gene polymorphism correlates with the tissue ACE levels and thus determines the local angiotensin II and bradykinin availability [13,14]. Therefore we could expect that ACE gene D allele is associated with the higher availability of angiotensin II and the lower availability of bradykinin in the kidney. One can speculate that the ACE gene D allele is associated with a higher efferent arteriolar resistance, resulting in a higher intraglomerular pressure. In such a case, the vasodilatory effect of ACE inhibition on the efferent arteriole may be more pronounced. However, this speculation needs further clinical and experimental observation [1].

There was a possibility that the antiproteinuric effect of ACE inhibitors has also been shown to be strongly affected by the sodium intake, since a 50% reduction of proteinuria during 50 mmol sodium intake could be abolished almost completely by RAS suppression by a 200 mmol sodium intake [12,26]. In our study, dietary sodium intake was not changed during the study period and sodium excretion was similar among three genotype groups after ACE inhibition. There was another possibility that the higher percentage reductions of proteinuria and albuminuria in the DD genotype group was related to the higher baseline levels of proteinuria and albuminuria. To exclude this possibility, we compared the baseline values of proteinuria and albuminuria and the percentage reductions of proteinuria and albuminuria after ACE inhibitions in our study population. There were no statistically significant correlations between the level of baseline proteinuria and albuminuria and the magnitudes of the reduction of proteinuria, and albuminuria after ACE inhibitions (Figure 4Go). These findings suggest that the amount of baseline proteinuria and albuminuria does not affect the magnitude of the percentage reduction of proteinuria and albuminuria after ACE inhibition.

In conclusion, the results of the present study show that the antiproteinuric effect of ACE inhibitors in DD genotype is significantly higher than that in ID and II genotypes. As to the distribution of allele D and I, allele I is more frequent than allele D. This result corresponds with reports from China and Japan, unlike those of Europe or USA, suggesting an ethnic difference [18]. This study suggests a potential role of the ACE genotype DD as a determinant of responsiveness to the antiproteinuric effect of ACE inhibition in NIDDM patients with overt proteinuria. However, further long-term and large-scale studies are required to confirm the issues regarding the mechanisms underlying this observed association.



   Notes
 
Correspondence and offprint requests to: Sung-Kyu Ha MD, Department of Internal Medicine, Yongdong Severance Hospital, Yonsei University College of Medicine, Seoul Young Dong, PO Box 1217, Seoul, Korea. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Methods
 Results
 Discussion
 References
 

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Received for publication: 27.10.99
Accepted in revised form: 18. 5.00