Angiotensinogen M235T and chymase gene CMA/B polymorphisms are not associated with nephropathy in type II diabetes

Marcin J. Zychma, Ewa Zukowska-Szczechowska, Beata I. Lacka and Wladyslaw Grzeszczak

Department and Clinic of Internal Medicine and Diabetology, Silesian School of Medicine, Zabrze, Poland



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Several studies have suggested that the same genetic factors may be involved in the predisposition to both essential hypertension and diabetic nephropathy, but the molecular mechanism underlying this predisposition still remains unclear. In particular, the role of genes involved in blood-pressure regulation and angiotensin II action is still controversial. This study examines a possible association between angiotensinogen M235T and chymase gene CMA/B polymorphisms with the presence of nephropathy in type II diabetic Caucasians.

Methods. For the purposes of the study, 323 microalbuminuric and 127 overt proteinuric cases, together with 243 normoalbuminuric controls with long-duration diabetes were selected from a group of 941 type II diabetic patients with established renal status.

Results. No differences in the genotype distributions or allele frequencies of the examined polymorphisms between the study groups were observed. The study groups were also stratified by gender, diabetes duration, level of glycaemic control, body mass index, hypertension, and retinopathy status, but still no distortion in the distributions of genotypes of any of the examined polymorphisms in any of the strata was shown.

Conclusions. Our study provided evidence against an association between angiotensinogen M235T or chymase gene CMA/B polymorphisms and the presence of incipient or overt nephropathy in Caucasian patients with type II diabetes.

Keywords: angiotensinogen; chymase; genetics; nephropathy; NIDDM; polymorphisms



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The increasing prevalence of type II diabetes in the general population makes it the most common diagnosis among patients entering renal replacement therapy. Subsequently type II diabetes is the single most frequent cause of end-stage renal failure [1]. Compared with type I diabetes, only a proportion of type II diabetic patients develop overt nephropathy [2]. Glycaemic control, hyperlipidaemia, and glomerular as well as systemic hypertension are the major factors contributing to the development of diabetic nephropathy [3]. As demonstrated in several studies in both type I and type II diabetes, genetic predisposition increases the risk of diabetic nephropathy [49].

The association between elevated blood pressure (BP) and diabetic nephropathy has been well documented. A cohort study has found a significant increase in BP preceding the development of microalbuminuria [10]. In the observational study of macroalbuminuric type II diabetic patients, the decline in glomerular filtration rate correlated with systolic BP [11]. The Oklahoma Indian Diabetes Study also demonstrated that increased systolic BP was a significant predictor of renal failure in Oklahoma Indians with NIDDM [12]. Increased prevalence of hypertension, or raised arterial pressure among parents of type I diabetic patients with nephropathy suggest that the same genetic factors contribute to predisposition to essential hypertension and diabetic nephropathy [13,14]. Hypertensive individuals with a family history of hypertension, and siblings concordant for hypertension are more likely to exhibit the so-called non-modulating phenotype, which is an inappropriate decrease in renal blood flow in response to angiotensin II (Ang II) infusion [15]. This phenotype, characterized by increased salt retention and ‘salt sensitivity’ of BP [16], is postulated to be caused by the increased local formation of Ang II, which can be achieved by angiotensin-converting enzyme (ACE) and chymase (chymotrypsin-like serine proteinase) [17,18], and depends on plasma or tissue angiotensinogen expression [19,20]. Thus, genetic loci potentially implicated in the excess production of Ang II are promising candidates for the association with both essential hypertension and diabetic nephropathy.

Angiotensinogen M235T polymorphism was associated with the presence of essential hypertension, higher plasma angiotensinogen concentrations, and blunted renal vascular response to Ang II infusion [2123]. This polymorphism was recently found to be in tight linkage disequilibrium with a molecular variant of the gene promoter, which was shown to affect the transcription rate of the gene [24]. The examination of angiotensinogen M235T polymorphism for association with diabetic nephropathy in several studies produced conflicting results [2532].

Chymase gene CMA/B polymorphism (G/A transition at position -1903 of the 5' untranscribed region of the gene) was found to have the effect on phenotypic expression of hypertrophic cardiomyopathy, acting together with the ACE gene I/D polymorphism [33]. Therefore the widespread tissue chymase activity and immunoreactivity, also present in the kidney [18] as well as localization of the CMA/B polymorphic site close to the regulatory region of the gene, makes this polymorphism a plausible candidate for the conditions induced or mediated by Ang II action.

We have recently excluded the ACE gene as a candidate for diabetic nephropathy after studying a large group of patients with type II diabetes [34]. In the present study we examined the association between angiotensinogen M235T and chymase gene CMA/B polymorphism with the presence of renal complications in these patients.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Evaluation of study groups
The population of patients selected for this study has previously been described in detail [34]. Briefly, each day between 1 June and 30 November 1996, screening for microalbuminuria (as albumin : creatinine ratio in random urine samples) was performed in approximately the first 10 patients with type II diabetes attending the Outpatient Clinic for Diabetic Patients in Zabrze, and was repeated during consecutive visits of patients in the clinic. Only Caucasian residents of Zabrze were included in the study. Clinical and epidemiological data, as well as a family history were collected for all patients according to our standardized questionnaire. Patients evaluation also included eye and cardiac autonomic neuropathy examinations. Patients were considered hypertensive if they were on antihypertensive medication, or if systolic or diastolic BP taken on two consecutive visits in the clinic was >=160 or 95 mmHg respectively. Definition of microalbuminuria and overt nephropathy was based on the criteria proposed by Warram et al. [35]. Based on results of clinical examination and medical history, patients with secondary causes of albuminuria (mostly acute or chronic urinary-tract infection, rapid decline of renal function, or systemic symptoms of vasculitis) were excluded from the study. In total, 941 type II diabetic patients were included in the inception group; 335 patients with microalbuminuria and 127 with overt proteinuria were selected as the two groups of cases and a control group of 254 patients with normoalbuminuria and known diabetes duration of at least 10 years was selected. Among these patients, good quality DNA was available from 693 persons, resulting in final groups of 243 normoalbuminuric patients, 323 microalbuminuric patients, and 127 patients with overt nephropathy for the case-control comparisons.

The study protocol was approved by the Ethics Committee of the Silesian School of Medicine.

DNA analysis
Genomic DNA was extracted from peripheral blood leukocytes.

Angiotensinogen M235T polymorphism was genotyped with a method similar to that described by Doria et al. [27]: 354 bp of exon 2 were amplified with primers: 5'-GAT GCG CAC AAG GTC CTG TC, 5'-GCGCGC GCC AGC AGA GAG GTT TGC CT, 5 min of initial denaturation at 95°C were followed by 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. Ten microlitres of polymerase chain reaction (PCR) product were electrophoresed on a 10% polyacrylamide gel (acrylamide/bis–37.5 : 1) with a linear gradient of denaturants from 40 to 60% (100%=7 mol/l urea and 40% formamide), in 1xTAE buffer at 60°C, 10 V/cm for 4.5 h. MM homozygotes were detected as a single band at 48% of denaturant concentration, TT homozygotes as a single band at 48.5%, while heterozygotes as four bands—48 and 48.5%, with two heteroduplexes melting at lower denaturant concentrations.

Chymase gene CMA/B polymorphism was determined as described by Pfeufer et al. [33]. Amplification primers for the 285 bp fragment of the 5' untranscribed region were 5'-GGA AAT GTG AGC AGA TAG TGC AGT C and 5'-AAT CCG GAG CTG GAG AAC TCT TGT C. After 5 min of denaturation at 95°C, 39 cycles of 30 s at 94°C, 15 s at 51°C, and 30 s at 72°C were performed. Ten microlitres of the PCR product were digested with 2 U of BstXI (New England Biolabs). CMA/B G allele was presented on 3% agarose as 195 and 90 bp bands, whereas A allele remained uncut.

For both polymorphisms tested in the study, PCR was performed from 200 ng of genomic DNA in 50 µl volume, with 10 pmol of the specific primers, 20 U/ml of PrimeZyme DNA polymerase (Biometra), 1.5 mmol/l MgCl2, 2.5 mmol/l of each dNTP, in either a UNO II (Biometra) or a Mastercycler Gradient (Eppendorf) cycler.

Statistical analysis
For continuous variables, comparison between groups was performed by either ANOVA or Kruskal–Wallis test of variance. Non-continuous variables, allele frequencies, and genotype distributions were compared by {chi}2 test. P value <0.05 (two sided) was considered statistically significant. All calculations were performed with Statistica version 5.0 package. Hardy–Weinberg equilibrium was checked by a HWE program from the Linkage-Utility package (available from ftp://linkage.rockefeller.edu/software/utilities/).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Table 1Go shows the selected clinical data of the study groups. Observed difference in known diabetes duration results from the inclusion criteria for the control group. Age at examination, body mass index, and HbA1c levels were similar in all of our study groups. Prevalence of hypertension and diabetic retinopathy was higher in the two groups of cases, with a markedly increased proportion of preproliferative and proliferative retinopathy among patients with overt proteinuria or chronic renal failure.


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Table 1. Selected clinical characteristics of the study groupsa

 
Genotype distributions of the examined polymorphisms (Table 2Go), as well as allele frequencies (Table 3Go) did not differ significantly between the study groups. Analysis stratified by gender, body mass index (BMI) and HbA1c levels, known diabetes duration, the presence or absence of retinopathy, and hypertension status also did not reveal significant differences in the distributions of the examined polymorphisms between the study groups (data not shown). Genotype distributions of each polymorphism tested in the study were in Hardy–Weinberg equilibrium in all of our study groups. Each of the examined loci was also analysed jointly with the ACE gene insertion/deletion polymorphism, and distributions of genotype pairs were compared between the study groups, with no positive association found (data not shown).


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Table 2. Distributions of AGT M235T and CMA/B genotypes in the study groups

 

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Table 3. Frequencies of AGT M235T and CMA/B alleles in the study groups

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study we tested polymorphisms of the genes encoding for components of the renin–angiotensin system: angiotensinogen and chymase, as candidates for the predisposition to nephropathy in type II diabetes. None of the examined polymorphisms was associated with the presence of microalbuminuria or overt nephropathy in our study population, with similar genotype distributions and allele frequencies across the study groups.

Published reports on the role of angiotensinogen M235T polymorphism in diabetic nephropathy give inconsistent results. For type I diabetes the first positive report came from Fogarty et al. [26] who found that carriers of the TT genotype had a 2.7-fold increased risk of developing nephropathy, but there were no differences in allele frequencies between cases and controls. A subsequent report from Doria et al. [27] demonstrated a slight increase of TT homozygotes among nephropathy cases; however, the increase did not reach statistical significance (OR 1.6, 95% CI, 0.8–3.5). No differences in either M235T genotype distributions or allele frequencies between nephropathy cases and normoalbuminuric controls were found in two other studies [28,29]. Recently a family-based study using the transmission-disequilibrium test demonstrated an excess transmission of the T allele from parents to type I diabetic patients with nephropathy, but the effect was seen only in males [31]. A similar gender-specific association between the presence of the T allele and nephropathy was observed in 72 men with type II diabetes and overt proteinuria, vs 61 normoalbuminuric controls [32]. In a negative study performed on a larger number of patients with type II diabetes, no stratification by gender was performed [29]. In our study, analysis carried out separately for men and women showed a slight tendency towards the excess of the TT genotype among male microalbuminuric and macroalbuminuric cases (frequency of TT homozygotes, 19/24/27% for normo/micro/macroalbuminuria respectively), but statistical significance was not reached. With respect to the presence of hypertension in either normoalbuminuric controls or microalbuminuric and macroalbuminuric cases, no association with the T allele was found (data not shown).

Chymase gene CMA/B polymorphism was found in the 5' untranscribed region, and was reported to modify disease susceptibility in patients with hypertrophic cardiomyopathy [33], thus suggesting the impact of this sequence difference on the level of chymase activity, and possibly Ang II production. In normal human kidneys, at least about 40% of angiotensin I is converted to Ang II via pathways other than ACE, presumably via a chymase [36], while in disease states (e.g. diabetes mellitus), the contribution of these non-ACE pathways may be substantially larger [37]. Similar CMA/B genotype distributions in our study groups suggest no impact of this polymorphism on the risk of development of diabetic nephropathy in type II diabetes. Nevertheless the role of CMA/B polymorphism or other molecular variants of chymase gene in promoting different conditions contributing to the increased morbidity and mortality in type II diabetic patients, e.g. myocardial infarction or left ventricular hypertrophy, should be considered.

Our study included a large number of patients: enough to detect allelic association with the examined complications of type II diabetes (different stages of nephropathy or hypertension). However, the study is not free from the pitfalls of a case-control design or the difficulties with defining a phenotype of a complex disorder. In order to avoid biased selection of cases and controls, on average the first 10 patients attending the clinic each day were included in the initial study population. Nevertheless, patients who were seen more frequently, e.g. because of difficulty in controlling hyperglycaemia, presence of complications, etc., were possibly included more often. This bias, however, was unlikely to diminish any associations that might exist in the population. All patients were selected from the same local population of residents of one city to minimize the possibility of population stratification.

Unlike changes in type I diabetes, morphological changes seen on kidney biopsies in type II diabetes are heterogeneous, despite similar clinical features of microalbuminuria or overt proteinuria presented by these patients [38,39]. Analysis stratified by level of glycaemic control, diabetes duration, BMI, hypertension status, or the presence or absence of retinopathy should help select subsets of patients with similar mechanisms leading to renal damage, thus making the phenotype of the strata more homogeneous. It has recently been stressed that in type II diabetic patients one should diagnose diabetic nephropathy only in the presence of retinopathy [40]. However, for the purposes of this study, we did not select cases on the basis of the presence of retinal lesions in consideration of the possibility of detecting association with retinopathy rather than with nephropathy. In any case, stratification by retinopathy status did not change our negative results (data not shown). The course of diabetic nephropathy, and thus definition of normoalbuminuric phenotype can also be distorted by the impact of antihypertensive treatment [3]. Among our control subjects, 192 (79%) were not receiving ACE inhibitors, while 105 (43%) were not receiving any antihypertensive therapy. Distribution of the examined polymorphisms did not differ in any of these subgroups from that observed in patients with microalbuminuria or macroalbuminuria (data not shown).

In the evaluation of urinary albumin excretion rate as albumin : creatinine ratio, we used the criteria proposed by Warram et al. [35], which correspond to widely accepted thresholds of 24-h albumin excretion in type I diabetic patients. The study group was also stratified using the threshold for microalbuminuria of 2.5 and 3.5 mg of albumin per mmol creatinine for men and women respectively, as proposed by Viberti et al. [41], but that did not change the negative results (data not shown).

In conclusion, none of the examined polymorphisms, angiotensinogen M235T or chymase gene CMA/B, was associated with the increased risk of the development of incipient or overt nephropathy in our group of type II diabetic patients. Further investigations with other candidate genes implicated in BP regulation or angiotensin II action are required to explain the overlaping predisposition to diabetic nephropathy and essential hypertension.



   Acknowledgments
 
We gratefully acknowledge technical assistance of Dr Wanda Trautsolt, Ilona Szydlowska, and Sylwia Gorczynska.

This work was supported by the Polish National Scientific Committee grant NN-2-027/97. Preliminary results of the study were presented in part at the 11th Annual General Meeting of the European Diabetic Nephropathy Study Group, Rennes, 1998, and the 58th Annual Meeting of the American Diabetes Association, Chicago 1998.



   Notes
 
Correspondence and offprint requests to: Wladyslaw Grzeszczak, Department and Clinic of Internal Medicine and Diabetology, Silesian School of Medicine, 3-Maja 13-15, 41-800 Zabrze, Poland. Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 6. 5.99
Revision received 24. 7.00.



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