Angiotensin-II type 1 receptor gene polymorphism and diabetic microangiopathy

Transcription

1 Nephrol Dial Transplant (1996) 11: Original Article Nephrology Dialysis Transplantation Angiotensin-II type 1 receptor gene polymorphism and diabetic microangiopathy Lise Tarnow, Francois Cambien 1, Peter Rossing, Flemming S. Nielsen, Birgitte V. Hansen, Sylvain Ricard 1, Odette Poirier 1 and Hans-Henrik Parving Steno Diabetes Center, Gentofte, Denmark and 'IERM SC7, Paris, France Abstract Background. Genotypic abnormalities of the reninangiotensin system have been suggested as risk factors for the development of hypertension, diabetic nephropathy and proliferative. Most of the known actions of angiotensin-ii are exerted through the angiotensin-ii type 1 receptor, which is present particularly in vascular smooth muscle cells, myocardium and the kidney. A transversion of adenine to cytosine at nucleotide position 1166 in the gene coding for the angiotensin-ii type 1 receptor has been associated with hypertension in the non-diabetic population. Methods. We studied the relationship between the A >C polymorphism in the angiotensin-ii type 1 receptor gene in patients with insulin dependent diabetes mellitus (IDDM) and diabetic nephropathy (121 men, 77 women, age 41 ± 10 years, diabetes duration years) and in normoalbuminuria (116 men, 74 women, age years, diabetes duration years). 16 patients (40%) had proliferative, 67 patients (17%) had no diabetic. Results. There was no difference in genotype distribution between diabetic nephropathy and normoalbuminuria: 103 (2%) / 81 (41%) / 14 (7%) vs. 97 (1%) / 80 (42%) / 13 (7%) had // genotypes, respectively. The allele frequencies (A/C) in patients with nephropathy (0.73/0.27) and patients with normoalbuminuria (0.72/0.28) were also similar. No difference in genotype distribution between proliferative and without diabetic was found either: 77 (0%) / 66 (42%) / 13 (8%) vs. 42 (63%) / 22 (33%) / 3 (4%) had // genotypes, respectively. Conclusions. The A >C polymorphism in the angiotensin-ii type 1 receptor gene does not contribute to the genetic susceptibility to diabetic nephropathy or proliferative in Caucasian IDDM patients. morphism; diabetic nephropathy; IDDM; proliferative Introduction A number of factors has been considered to contribute to the initiation and progression of diabetic nephropathy, including genetic and racial predispositions, glycaemic and other metabolic abnormalities, alterations in systemic and renai hemodynamics and various cytokines and growth factors, as reviewed by Parving etal. [ 1 ]. Abnormalities in the renin-angiotensin system have been suggested to play a role in the development of diabetic nephropathy and [2-]. Nearly all known actions of angiotensin-ii are mediated through the angiotensin-ii type 1 (AT t ) receptor, a G-protein coupled receptor, which is particularly present in vascular smooth muscle, myocardium, adrenal cortex and the kidney [6-8]. Thus the gene coding for the AT, receptor (AGT t R gene) could be a candidate gene for diabetic nephropathy. Recently the cdna encoding the human AT, receptor has been cloned [9,10]. A polymorphism in the AGTjR gene, corresponding to a transversion of adenine to cytosine (A-»C) at nucleotide position 1166 of the mrna sequence has been identified and associated with essential hypertension in a non-diabetic population [11]. Furthermore, genetic predisposition to hypertension has been suggested as a risk marker for diabetic nephropathy in patients with insulin dependent diabetes mellitus (IDDM) [12,13]. We have studied the relationship between the A >C polymorphism in the AGTiR-gene in two large groups of and without diabetic nephropathy, respectively. Since previous Key words: angiotensin-ii type 1 receptor gene poly- studies have suggested a potential role for the reninangiotensin system in the development of proliferative [14], we furthermore assessed the possible Correspondence and offprint requests to: Lise Tarnow, Steno Diabetes link between the AGT,R gene polymorphism and Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. proliferative diabetic European Dialysis and Transplant Association-European Renal Association

2 1020 Subjects and methods The patients participating in this study have been described in details previously [1,16]. All albuminuric IDDM patients attending the outpatient clinic at Steno Diabetes Center in 1993, who were more than 18 years of age and had their glomerular filtration rate measured during the same year were invited to participate in the study. Two hundred patients (83%) accepted the invitation and were enroled. The AGT X R gene polymorphism was determined in 198 nephropathic patients and in 190 persisting normoalbuminuria recruited from our outpatient clinic and matched for sex, age and duration of diabetes (Table 1). Lymphocytes were isolated from peripheral blood and DNA prepared by standard techniques [17]. The A >C polymorphism was assayed by allele-specific oligonucleotide hybridisations of PCR products [11]. The primers used to amplify the AGT X R region encompassing the A >C polymorphism were '-TGCTTGTAGAGTCAT-3' and '-GGCTTTGCTTTGTCTT-GTTG-3'. The hybridization procedure [11] was slightly modified: Each allele was detected by preincubating the amplification products for 2 h with 0 pmoles of unlabelled oligonucleotide probe specific for the other allele and then incubating for 4 h with 10 pmoles of labelled probe specific for the allele (sequences published previously [11]); the membranes were then washed and autoradiographed. Patients were classified as // according to the absence/presence of the A >C mutation. Urinary albumin concentration was determined by enzyme immunoassay [18] from 24 h urine collections. Arterial blood pressure was measured twice, on the right arm, after at least 10 min rest in the supine position and averaged. The measurements were performed with a Hawksley random zero sphygmomanometer (Hawksley & Sons Ltd, Lancing, Sussex, UK) and appropriate cuff size. Diastolic blood pressure was recorded at the disappearance of Korotkoff sounds (phase V). Hypertension was diagnosed according to the WHO criteria: systolic blood pressure > 160 mm Hg and/or diastolic blood pressure ^ 9 mm Hg, or if antihypertensive medication was being prescribed. Retinopathy was assessed by fundus photography after pupillary dilatation and graded: nihil, simplex or proliferative diabetic. L. Tarnow et al. Body mass index (BMI) was calculated as weight (kg)/height (m) 2. From venous blood samples haemoglobin A Jt.(HbA lc ) was determined by high performance liquid chromatography (DIAMAT Analyzer, BIO-RAD, CA, USA). Normal range of HbA ic in our laboratory is %. Serum creatinine concentration was assessed by a kinetic Jaffe' method. Smokers were defined as persons smoking more than one cigarette/cigar/pipe per day, all others being classified as non-smokers. Statistical analysis Urinary albumin excretion rate and serum creatinine were non-normally distributed and values are given as medians (range), all other values are given as means + SD. For normally distributed variables comparison between groups was performed by an unpaired Student's /-test or ANOVA. In non-normally distributed continuous variables a Mann-Whitney U-test or Kruskal-Wallis test were used for comparison between groups. A x 2 -square test was used to compare the distribution of AGT a R genotypes and alleles in and and was also used for comparison between groups of non-continuous variables. Allele frequencies were estimated by the gene counting method and Hardy-Weinberg equilibrium was checked by a x 2 -square test. A P value (two sided) <0.0 was considered statistically significant. All calculations were performed with a commercially available program, Statgraphics (STSC, Rockville, MD, USA). Results The group of patients with nephropathy and the normoalbuminuric group were well matched with regard to sex, age, and duration of diabetes (Table 1). Patients with nephropathy had higher systolic and diastolic blood pressure as compared to normoalbuminuric patients (P). The AGTjR genotype distribution was similar and the frequency of the A-/C-allele was also the same in and without Table 1. Clinical characteristics of 198 diabetic nephropathy and 190 normoalbuminuria Nephropathy (n=198) Normoalbuminuria (n = 190) P value Sex (men/women) Age (years) Duration of diabetes (years) Body mass index (kg/m 2 ) HbA lc (%) Smokers (%) Urinary albumin excretion rate* (mg/24 h) + Serum creatinine* (umol/1) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Prevalence of antihypertensive treatment (%) Retinopathy, (numbers [%]) nihil simplex proliferative 121/ % 796(16-144) 103 (4-684) % 0 61 (31%) 137 (69%) 116/ % 8(1-30) 76(40-116) ±10 11% 67 (3%) 104 (%) 19 (10%) Mean + SD, *Median (range), or n(%). + Some patients with previously persistent albuminuria receiving antihypertensive medication have UAER<30Omg/24h.

3 AGT,R-gene polymorphism and diabetic microangiopathy Table 2. Distribution of AGT,R genotypes in 198 IDDM patients with diabetic nephropathy and 190 normoalbuminuria 1021 Table 4. Distribution of AGT[R genotypes in 16 IDDM patients with proliferative and 67 out diabetic nephropathy normoalbuminuria proliferative no Genotype Allele frequency A/C 103 (2%) 81 (41%) 14 (7%) 0.73/ (1%) 80 (42%) 13 (7%) 0.72/0.28 Genotype Allele frequency A/C 77 (0%) 66 (42%) 13 (8%) 0.71/ (63%) 22 (33%) 3 (4%) 0.79/0.21 diabetic nephropathy (Table 2). The distribution of AGT t R genotypes in and were in Hardy-Weinberg equilibrium (). The prevalence of hypertension was 81% in patients with nephropathy. No difference in genotype distribution was observed between nephropathic patients with and without hypertension: 83 (2%) / 6 (40%) / 13 (8%) vs. 20 (4%) / 16 (43%) / 1 (3%) had // genotypes, respectively (). Furthermore, no difference in genotype distribution was observed, when the study population was dichotomized according to sex and above or below the median value either with respect to age, duration of diabetes or HbA lc, (). Patients with nephropathy had a much higher prevalence of proliferative (137 [69%]) compared to patients with normoalbuminuria (19 [10%], P). Thus, when patients were stratified according to status diabetic nephropathy was present in 137 (88%) of patients with proliferative. Clinical data for these patients are shown in Table 3. There was no difference in AGT X R genotype distribution and no difference in allele frequency between patients with proliferative and without diabetic (Table 4). Discussion In our cross-sectional case control study of Caucasian IDDM patients the genotype and allele frequencies of the AGT t R gene polymorphism did not differ between patients with and without diabetic nephropathy. In addition, no difference in AGT t R genotype distribution was observed between nephropathic patients with and without hypertension. The distribution of the AGT t R genotypes in and were in Hardy- Weinberg equilibrium, and furthermore in accordance with the allele frequencies (A/C: 0.71/0.29) found in the control group of Caucasian subjects (n = 723) in the Etude Cas-Temoin de l'lnfarctus du Myocarde study (ECTIM)[ 19]. Our cohorts were sufficiently large to yield 80% power to detect a 10% deviation of genotype frequency with / > <0.0 and had 99.% power to detect a 20% deviation. The negative finding is thus not explained by insufficient statistical power. Systemic and glomerular hypertension play a role in the initiation and progression of experimental and human diabetic glomerulopathy [20-22]. Predisposition. to systemic hypertension has been reported increased in some [12,13] but not all studies [23] dealing with IDDM patients suffering from diabetic nephropathy. In contrast, all studies reporting on presence or absence of familial clustering in diabetic nephropathy have found it to be present [24,2]. Some of the above mentioned abnormalities may be explained by alterations in the renin-angiotensin system. In addition, angiotensin-ii acts as a growth Table 3. Clinical characteristics of 16 proliferative and 67 patients with no signs of diabetic Proliferative (H=16) No signs of diabetic (n = 67) P value Sex (men/women) Age (years) Duration of diabetes (years) Body mass index (kg/m 2 ) HbA Ic (%) Smokers (%) Urinary albumin excretion rate* (mg/24 h) Serum creatinine* (umol/1) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Prevalence of antihypertensive treatment (%) 89/ ± % 733 (4-144) 99 (9-684) % 41/ % 9(2-30) 76 (40-103) % <0.01 Mean + SD, *Median (range), or «(%).

4 1022 promotor by enhancing mesangial cell proliferation and formation of mesangial extracellular matrix [26,27], abnormalities characteristically found in diabetic glomerulopathy [28,29]. The cellular effects of angiotensin-ii are mediated by two structurally distinct receptor subtypes, AT t and AT 2 [6,30]. In adult humans, most of the known actions of angiotensin-ii are exerted through the ATj receptor, which is present in vascular smooth muscle, myocardium and in the kidney (6-8). Therefore, the gene coding for AGT t R could be a candidate gene for diabetic nephropathy. Originally, Bonnardeaux et al. [11] identified five polymorphisms in the gene coding for the AI\ receptor, though no mutation that alters the encoded amino acid sequence was detected. A case control study, performed on white hypertensive and normotensive subjects, revealed a significant increase in frequency of the C 1166 allele in hypertensive non-diabetic subjects. Recently, the same group has demonstrated that the association between the DD-genotype of the E/ID polymorphism in the E gene and myocardial infarction is restricted to a subset of individuals, also carriers of AG^R C 1166 allele [19]. Though we have studied the E/ID polymorphism in the present patient population previously [1,16], no valid analysis of interaction between the E/ID and the AGT t R polymorphisms in relation to coronary heart disease in IDDM patients can be performed presently due to the small number of affected patients (n = 3/388). No interaction between the two above mentioned gene polymorphisms was found, comparing patients with and without diabetic nephropathy (Table ). Other genotypic abnormalities of the renin-angiotensin system have been suggested as risk factors for the development of diabetic nephropathy [31,32]: originally, Marre et al. [31] reported that the II genotype of the E/ID polymorphism is a marker for reduced risk for diabetic nephropathy in IDDM patients. Patients with the II genotype having the lowest level of plasma E [31]. Conversely, we found that raised plasma E concentration may play a role in the initiation and progression of diabetic nephropathy in Caucasian IDDM patients [1]. This suggestion is in Table. Distribution of AGT,R genotypes in 198 IDDM patients with nephropathy and 190 normoalbuminuria according to E/ID genotypes L. Tarnow et al. accordance with the beneficial effects of E inhibition on the development and progression of experimental and human diabetic glomerulopathy [33-38]. Subsequent studies have not been able to confirm the initial finding of an association between the E/ID polymorphism and diabetic nephropathy [1,39]. Also the gene coding for angiotensinogen could be implicated in the genetic basis of diabetic microangiopathy. Recent studies imply a strong linkage to essential hypertension of regions within or close to the angiotensinogen gene [40]. None of the investigated gene polymorphisms contributes to the genetic susceptibility to diabetic nephropathy in IDDM patients [41]. Studies suggest, that prorenin serve as a risk-factor/ marker for diabetic micro vascular disease [2]. Elevated plasma prorenin is associated with increased risk of development and presence of diabetic [2,3]. Furthermore, raised prorenin levels have been demonstrated in eyes with proliferative, thus indicating an activation of the intraocular reninangiotensin system [14]. Angiotensin-II is known to stimulate new vessel formation in the eye [42] and finally, diabetic have enhanced pressor response to intravenously infused angiotensin-ii [43], suggesting an enhanced AT t receptor mediated vascular responsiveness. Genetic abnormalities in the AGT X R gene could thus be responsible for a possible constitutive activation of the AT t receptor in patients with proliferative in IDDM. In the present study no difference in AGT X R genotype distribution was observed between IDDM patients with proliferative and patients without. Due to our method of patient selection there was a preponderance of patients with diabetic nephropathy among patients with proliferative. In the normoalbuminuric group the AGT t R genotype distribution did not differ significantly between 19 patients with proliferative as compared to patients without, but numbers were small. Thus, the A »-C polymorphism does not seem to be involved in the pathogenesis of proliferative in IDDM. We conclude that the A >C polymorphism in the AGT X R gene does not contribute to the genetic susceptibility to diabetic nephropathy or proliferative in Caucasian IDDM patients. Acknowledgements. We acknowledge the assistance of Ms B. R. Jensen and Ms U. 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