Prognostic relevance of angiotensinogen gene polymorphisms in hypertensive patients with type 2 diabetes mellitus

ABSTRACT

Studies upon role of certain gene-candidates in pathogenesis, onset and progression of various chronic cardiovascular diseases are extremely widespread with an explosion of data in recent years. But still, there was no prognostic model accepted or any gene candidate as gene-adjusted treatment strategy approved.

Aim. The study aimed to evaluate prognostic relevance and possible implication into the treatment strategy of AGT polymorphisms T174M and M235T in patients with hypertension and type 2 diabetes mellitus.

Patients and methods. 153 patients with type 2 diabetes mellitus and hypertension were enrolled into the study. All patients underwent standard clinical evaluation and were genotyped in order to obtain information upon two functional AGT polymorphisms - T174M and M235T.

Results. Significant differences in blood pressure parameters and indicators of kidney dysfunction were described for various M235T allelic variant. TT-genotype patients represented higher blood pressure and signs of advanced kidney dysfunction as GFR decrease and more eminent albuminuria. T-allele carriers of M235T also represented increased albuminuria. Combination of unfavorable polymorphic AGT variants 235TT with 174M-allele was associated with early onset of proteinuria and a significant GFR decrease. Conclusion. Genotyping for M235T AGT polymorphism and unfavorable polymorphisms combination detection, can be a good instrument for early diabetic nephropathy onset prediction, as well as early RAAS- blocker treatment administration.

Key words: type 2 diabetes mellitus, arterial hypertension, T174M and M235T polymorphic allelic variants of AGT, diabetic nephropathy.

Background: Renin-angiotensin-aldosterone system (RAAS) directly takes part in regulation of electrolyte balance, circulationg fluid volume, blood pressure, vascular reactions in response to alteration or inflammation. Excessive RAAS activation leads to hypertension (AH), thrombotic and atherogenous effects, which in long-term perspective results in target-organ damage. Numerous prospective studies confirmed that the RAAS blockage is protective in AH and chronic kidney disease (CKD) [1].

During last decades gene-candidates, encoding RAAS compounds, were actively studied in different populations. It was shown that certain gene polymorphisms can be associated not only with high risk of hypertension, target organ damage, cardiovascular events, but with metabolic disturbances as well, particularly obesity and impaired glucose tolerance [2, 3]. One of the functional polymorphisms of angiotensinogen gene (AGT), which remains controversial in hypertension risk prediction for European population is substitution of metionine (M) into threonine (T) in position 235. Studies showed that 235T- allele is associated with 10-20% higher serum AGT concentrations, comparing to patients with MM-variant. Two meta-analyses have drawn to conclusion of M235T polymorphism is associated with essential hypertension risk in European population [4]. 2009, Gil et al. showed no association between M235T polymorphism and cardiovascular complications in Korean hypertensive adolescents, but serum insulin, renin levels, body mass index and obesity index were significantly higher in those with the MM genotype, compared to patients with TT or MT genotypes [5].

G. Ragia et al., (2010) indicated that AGT 235 TT homozygous genotype was associated with coronary arteries disease in hypertensive patients [6]. Recent studies show presence of relation between high serum concentrations of cytokines IL-1 and TNF-a, which promote transcription and expression of angiotesinogene in hypertensive patients with 235T allele [7].

Another polymorphism-candidate that can influence AH onset and target organ damage is T174M [8]. Studies of this polymorphism are complicated due to the low frequency of M-allele in the European population [9]. Gu et al., 2012 showed high risk of essential hypertension development in 174M-allele carriers [10]. Recent meta-analysis, which summarized several studies in European and Asian populations didn’t confirm certain association between T174M AGT polymorphism in essential AH onset [11]. But at least several population studies, analyzed by W.Wang, have a common conclusion about higher risk of coronary arteries stenosis and myocardial infarction in white T-allele carriers. There is no confirmed mechanism, which could explain this association, however T-allele carriers represented higher serum concentrations of angiotensine [12].

Interactions between T174M and M235T AGT polymorphisms were also discussed.

Z. Junli et al. showed no association between M235T and T174M polymorphisms with left ventricle hypertrophy in essential hypertension patients [13]. In the 4-year follow-up study by A. Pilbrow et al., patients with heart failure (HF) had 2 times higher risk of death, being carriers of 235TT genotype and/or >1 174M allele. This study suggested that angiotensinogen variants M235T and T174M may provide prognostic information for long-term survival in HF patients [14].

Polymorphic genetic variants of RAAS compounds, except for disease course modification and high risk of complications, can be associated with suboptimal response to antihypertensive treatment with ACE-inhibitors and ARB-blockers [15].

Thus, role of various functional genetic polymorphisms of AGT gene requires further prospective research with direct implication into personalized tailored treatment strategy, especially in patients with high risk of target organ damage as in case of AH and type 2 diabetes mellitus combination.

Aim of the study. The study aimed to evaluate prevalence of T174M and M235T AGT functional allelic variants in patients with type 2 diabetes mellitus and hypertension and to validate their prognostic role in disease clinical course and complications, particularly, diabetic nephropathy.

Patients and methods: 153 hypertensive patients with type 2 diabetes mellitus patients were enrolled, aged 61,3±4,4 years. Patients were excluded if they had type 1 diabetes mellitus, history of congestive heart failure, malignant hypertension, malignancies, liver failure, renal artery stenosis and ESRD. All patients underwent detailed history, standard physical and laboratory evaluation for hypertension and type 2 diabetes mellitus. All of the enrolled patients signed an informed consent for genetic material collection and assay. At the moment of evaluation all patients were given standard treatment with oral hypoglycemic drugs and RAAS-inhibitors - enalapril or losartan.

Blood pressure was measured with mercury sphygmomanometer three times, with interval of 5 minutes, in a seated position after at least 10 minutes rest. Office blood pressure was estimated as the average of three measurements.

Glomerular filtration rate was estimated according to CKD-EPI formula. Glomerular filtration rate was calculated by the formula CKD-EPI [16].

Genomic DNAs was extracted out of venous blood derived leucocytes. Standard reagent kits «DNA-sorb-B» were applied (InterLabService, Russian Federation). Polymorphic variants of AGT were assessed via polymerase chain reaction (PCR) using amplification kits «SNP-express» (Litech, Russian Federation). PCR was carried out with BIO-RAD amplifier (USA). 100-150 ng of DNA was amplified. Primary denaturation at the temperature 95°С during 10 minutes. PCR during 40 cycles: denaturation at 95°С - 30 sec., annealing at 64°С - 30 sec., elongation at 72°С for 30 sec. and final elongation for 3 minutes at 72°C. PCR products were visualized by electrophoresis in 2% horizontal agarose gel, prepared with trisborate buffer.

Obtained data was statistically managed with Statistica 10.0 (StatSoft, USA). One-way ANOVA was used in order to analyze between-group and within-group differences.

Results: enrolled patients were homozygous for functionally weakened polymorphic variants in 28,77% of cases (22 patients) and only 2,61% (4 patients) for M235T and Т174М polymorphic AGT variants, correspondingly. Such allelic distribution is peculiar to general European population.

Mean values of systolic (SBP) and diastolic blood pressure (DBP) were significantly higher in patients with MT and TT genotypes of M235T polymorphism, p=0,001 (Table 1). In group of patients with TT-genotype valuable decrease in GFR (p=0,002), increased serum creatinine (p=0,032) and albuminuria (p=0,001) were observed . Decrease in GFR was also shown in the group of patients with MT-genotype (p=0,002). Such changes of BP and clinical signs of DN can be contributed to suboptimal antihypertensive and nephroprotective effects of RAAS-blockers in patients-T-allele carriers of M235T AGT polymorphism.

Number of patients, which were monozygous for T174M M-allele was too small to conduct any analysis of variances between groups. Further analysis was carried out only in groups with TT and MM genotype (Table 2). Veracious differences were estimated only in patients with TT-genotype, which tended to have higher levels of fasting glucose, than patients with MT-allelic variant (p=0,001).

19 individuals were identified as carriers of both functionally unfavorable allelic variants of AGT gene - they presented with TT genotype of M235 polymorphism and were carriers or monozygous for T174M M-allele. Such combination of allelic AGT variants was associated with early onset of diabetic nephropathy (average diabetes duration of diabetes was 8,2±4,1 years, р=0,032), also this subgroup presented with veraciously lower GFR (р=0,012).

CONCLUSIONS:

Thus, hypertensive patients with type 2 diabetes have the same allelic distribution of functionally weakened genotypes, as general European population. Singular contribution of Т174М polymorphism and its prognostic relevance in pathogenesis and treatment strategy of type 2 diabetes mellitus and hypertension should be estimated in bigger populations and prospective studies. Nevertheless, combination of M235T TT- genotype and being at least a carrier of T174M M-allele is a —risk genotype” for early DN onset in hypertensive individuals with type 2 diabetes mellitus. Interactions with other polymorphic gene variants and multiple modifiable risk factors can be a clue in type 2 diabetic patients, who are experiencing renal function decline despite adequate antihypertensive, lipid-lowering and hypoglycemic treatment.

Genotyping for functionally weakened AGT allelic variants can be a valuable step to personalized treatment adjustment and more strict metabolic control in patients with combination of type 2 diabetes mellitus and hypertension. As early as possible, administration of RAAS-blockers, furthermore, in combined antihypertensive and nephroprotective treatment could reduce renal function decline in genetically predisposed individuals.

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Table 1 - Clinical characteristics of hypertensive type 2 diabetes mellitus patients depending on M235T AGT genotype.

All data is given as mean values with standard declination, except values of albuminuria, which are represented as the median with interquartile range.

*р< 0,05

Table 2 - Clinical characteristics of hypertensive type 2 diabetes mellitus patients depending on T174M AGT genotype.

I M235T AGT genotype | ТТ | MT | MM^t | Р тт-мт ~|

*р< 0,05

'Taking into consideration small number of patients with MM genotype of T174M AGT, all values in this table are given as mean with standard declination, microalbuminuria is represented as median with interquartile ranges; analysis of variances was carried out only for groups of TT and MT genotype.