Genetic polymorphisms of angiotensin-2 type 1 receptor and angiotensinogen and risk of renal dysfunction and coronary heart disease in type 2 diabetes mellitus

Increased activation of the renin-angiotensin system (RAS) has been postulated to play a central role in the progression of chronic kidney disease (CKD) and coronary heart disease (CHD). This theory is supported by randomized clinical trials that have shown beneficial clinical effects of the blockade of production of angiotensin-II (AII) by angiotensin converting enzyme inhibitor (ACE-I) medications or antagonism of AII action through angiotensin receptor blockade (ARBs). For example, among diabetics, the ARB losartan significantly slowed progression of CKD [1], whereas the ACE-I medication ramipril significantly reduced CHD morbidity and mortality [2].

Although the D-allele of the ACE I/D polymorphism has been associated with diabetic nephropathy in two large meta-analyses of several thousand individuals [3, 4], the role of genetic polymorphisms in other components of the RAS in CKD and CHD in people with type 2 diabetes has been less well defined. Associations between these SNPs and renal and cardiovascular diseases have been reported in other sub-groups, however. For example, the homozygous AGT (angiotensinogen gene) 235-T/T genotype has been associated with faster progression to ESRD in patients with glomerulonephritis [5] and with susceptibility to nephropathy in patients with type I diabetes mellitus [6], although not in two populations with type 2 diabetes [7, 8].

Higher concentrations of plasma angiotensinogen are associated with the AGT 235 T-allele [9] and may be directly related to the number of Thr alleles present [10] thereby supporting the theory that components in the RAS are physiologically activiated and may promote glomerulosclerosis and interstitial fibrosis via accumulation of extracellular matrix [11]. Recent research has revealed that another more clinically relevant AGT polymorphism may be a linked promoter substitution that increases the rate of gene transcription; this G-6A polymorphism appears to be the one most strongly associated with hypertension [12]. Genetic variation in AGT genes, especially the AGT 235T and AGT (-6) G alleles have also been reported to be associated with CHD [10, 13, 14].

Angiotensin II type 1 receptor (AT1R) polymorphisms may influence intrarenal angiotensin II (AngII) activity. Healthy Caucasians carrying the A1166 C-allele (AC or CC) polymorphism showed lower basal GFR and basal renal plasma flow and had increases in GFR following treatment with the AT1R blocker losartan [15].

We therefore investigated associations of genetic polymorphisms in the AGT1R and AGT genes including AGT1R T573C, AGT1R A1166C, and AGT M235T and presence of CKD and CHD in type 2 diabetes.

AGT A(-6)G is in complete linkage disequilibrium with M235T and results were almost identical between the two SNPs; therefore, we present only the results for AGT SNPs from the M235T analyses. Prevalence of the disease-associated allele ranged from 47% for AGT1R 1166 C-allele to 68% for the AGT1R 573 C-allele. Prevalence of homozygosity for the disease-associated allele ranged from 9% for AGT1R 1166 C-allele to 20% for the AGT1R 573 C-allele. The SNPs were in Hardy-Weinberg equilibrium (all p-values for chi-square test ≥ 0.60).

For all SNPs, the magnitude of the fully adjusted odds ratios for the dominant models showed little difference when compared to the age-adjusted only models (Table 2). For the outcome of eGFR < 60 ml/min/1.73 m², a significantly elevated odds ratio was seen in the fully-adjusted model for the AGT1R 1166 C-allele in the HPFS men only (Table 2). Adjusted dominant models that did not include prevalent hypertension for the outcome of eGFR < 60 ml/min/1.73 m² were not different from the fully adjusted models. When the two cohorts were combined, the AGT1R 1166 C-allele remained significantly associated with eGFR < 60 ml/min/1.73 m² (adjusted OR 1.42 [1.02, 1.98]) (Figure 1); no significant associations were seen in recessive or additive models in the combined dataset. Results using CrCl<60 ml/min as the outcome of interest were similar to those using eGFR < 60 ml/min/1.73 m² (results not shown).

For the outcome of CHD, the AGT1R 1166 C-allele in dominant model was associated with a significantly higher odds ratio in men while the AGT SNP M235T was associated with a significantly higher odds ratio in women (Table 2). Recessive inheritance modeling was borderline significant only for the AGT M235T (adjusted OR 1.52 [0.98 to 2.35]) for the presence of CHD in women. No recessive models were significant for men, and all additive models were null for both groups. Multivariable models that did not include prevalent hypertension yielded the same results as the fully adjusted models. Excluding eGFR < 60 ml/min/1.73 m² as a covariate in the models for CHD also did not influence the associations seen.

The AGT 235 T-allele had a borderline association with history of CHD (OR 1.24 [0.96, 1.61] in the pooled data (Figure 1). The fully adjusted odds ratios for the combined data were not notably different from the age-adjusted only odds ratios (all differences resulted in < 10% change in OR). Results for CHD outcome did vary by sex; we observed significant interaction term p-values for the AGT1R 1166 C-allele (p = 0.008) and AGT 235 T-allele (p = 0.03) in relation to CHD (Table 2). In the combined data set, no significant associations were observed in recessive or additive models for CHD.

Because different allelic frequencies may be seen in different ethnic populations, we also analyzed these data with the few African-Americans excluded. Exclusion of these participants did not meaningfully change any of the results in the analyses stratified by sex or in the combined cohort. For example, the odds ratio for AGT1R A1166 C-allele was 1.70 [1.04, 2.78] for eGFR < 60 ml/min/1.73 m² and 1.56 [1.09, 2.21]for CHD when African-Americans were excluded from the HPFS analyses. Similarly in the combined cohort, the odds ratio for AGT1R A1166 C-allele and eGFR < 60 ml/min/1.73 m² was 1.42 [1.02 to 1.98].

The AGT1R 1166 C-allele was directly and significantly associated with renal dysfunction in the combined dataset. This SNP was also associated with higher odds ratio for prevalent CHD in men with type 2 diabetes whereas the AGT 235 T-allele was significantly associated with increased odds ratio for CHD in women with type 2 diabetes. In contrast to the majority of previous publications of AGT1R and AGT polymorphisms, this study only included adults with type 2 diabetes, which is a population especially at high risk for kidney dysfunction and CHD.

We also found that sex might modify the association between SNPs and presence of CHD, and recommend that future investigations consider stratifying analyses by sex. A biological basis for these observed sex differences may be indirectly supported by previous studies of the RAS. For example, a population study of several hundred Bavarians revealed higher renin and prorenin levels in men compared with women; the authors also noted the lowest renin and prorenin levels in women taking hormone replacement therapy and hypothesized that sex hormones may regulate expression of the renin gene [24]. Healthy men also exhibit greater decreases in plasma renin and angiotensin-II levels than healthy women when given angiotensin-receptor blocker medications [25]. Another investigation of 93 healthy young men and women also reported higher levels of angiotensin-(1–7) peptides in men compared to women, but did not detect significant differences in levels of A-I, A-II, or angiotensin-(1–7) between the sexes when stratified by AGT1R 1166 C-allele or AGT M235T genotypes [26]; however, the study had low power because many subgroups analyzed had less than 10 people.

We examined dominant, recessive, and additive genetic models for each of these four SNPs and eGFR< 60 ml/min/1.73 m² and CHD and did not detect significant associations with recessive models or additive models. Previous analyses of the AGT M235T SNP and CHD have reported an association with the homozygous T/T variant [10, 13, 14], but these relatively small studies may not have had enough power to effectively identify recessive genetic effects.

Our findings that the AGT1R 1166 C-allele was associated with renal dysfunction in men and women with type 2 diabetes is consistent with a smaller French study of 235 type 2 diabetics where the authors reported that the CC genotype was significantly associated with nephropathy (defined as presence of microalbuminuria) especially in men[27]. Although previous case-control studies demonstrated an increased frequency of the AGT1R 1166 C-allele in ESRD patients with [28] and without type 2 diabetes [29] compared to healthy controls, our study appears to be one of the few to examine an earlier stage of moderate kidney dysfunction. In contrast, other studies have not found association between A1166 C-allele and nephropathy in type 2 diabetes [30, 31].

Consistent with our findings, however, a study of 132 Japanese adults with type 2 diabetes [32], a Hong Kong study of 168 type 2 diabetics [33], a study from Germany of 301 type 2 diabetics [34], and a meta-analysis of several thousand people [35] reported no association between nephropathy and the AGT M235T polymorphism. In contrast, a recent investigation of 421 Asian Indian type 2 diabetics did find an association between the AGT 235 T-allele and renal insufficiency (defined as plasma creatinine ≥ 3 mg/dl and/or urinary albumin excretion rate > 200 mcg/min) (OR 2.68, 95%CI 2.01 to 3.57) [36].

Direct comparisons of conflicting reports in the literature, however, may be difficult and discrepancies could be due to many factors such different prevalence in genetic polymorphisms in different ethnic populations as well as varying definitions of nephropathy (impaired eGFR or CrCl in our study vs. microalbuminuria in other studies) and varying sample sizes. For example, the frequencies of the AGT 235 T-allele and the AGT (-6) G-allele appear to be much higher in Japanese compared with Caucasian populations [12].

Interestingly, the associations observed between these candidate RAS SNPs and renal dysfunction were not meaningfully changed after multivariable adjustment. In particular, excluding hypertension as a covariate from the model did not change results and support the concept that association between RAS SNPs (and presumed upregulation of the RAS) and renal and CHD outcomes may be mediated by other influences in besides hypertension, which likely include cellular hyperplasia, stimulation of hormonal pathways and direct pro-fibrotic effects on the kidneys and vasculature [11]. Other investigators have also reported that hypertension may not mediate associations between these SNPs and CHD [13] or progressive CKD [37]. Also notable is the lack of influence of including eGFR < 60 ml/min/1.73 m² as a covariate in models where CHD was the dependent variable, which may suggest that renal dysfunction has a weaker association with CHD in type 2 diabetes (who have often have multiple overlapping vascular risk factors) than in non-diabetic populations.

Some important limitations of this investigation need to be acknowledged. Data on albuminuria, another measure of kidney dysfunction in addition to eGFR or CrCl were not available in the men and the majority of the women. Kidney function was not directly measured but estimated by a single measurement of plasma creatinine in each participant. The participants were mostly Caucasians and the findings may not be generalizable to other ethnic groups. There are no measurements of plasma angiotensiongen levels or other markers of RAS activation available to correlate directly with the genetic polymorphisms investigated here. The relatively small sample sizes and few numbers of participants meeting criteria for kidney dysfunction has resulted in wide confidence intervals for many of the estimates for eGFR < 60 ml/min/1.73 m². Also, as the DNA was collected several years after the initiation of the cohort study, there is a possibility that participants who died prior to the blood collection may have had the candidate SNPs; this situation would be expected to bias results towards the null, however, whereas we did observe a number of significant associations.

Strengths of the current study include the examination of genetic polymorphisms in the RAS that have received less attention than the ACE-I/D genotype, which provide novel information concerning genetics and kidney and cardiac disease in adults with type 2 diabetes. Furthermore, by examining the SNPs separately in a cohort of men and one of women as well as in a combined dataset, these analyses revealed that sex may modify the association between RAS gene polymorphisms and coronary heart disease in type 2 diabetes.

In conclusion, among type 2 diabetics, the AGT1R 1166 C-allele is directly associated with kidney dysfunction in men and women but with CHD only in men. The AGT 235 T-allele is associated with CHD in women. Previous evidence suggests that these polymorphisms are associated with up-regulation of the RAS, and therefore, these data further support the central role of RAS activation in CKD and CHD.