| IntroductionPrimary aldosteronism (PA) is caused by autonomous aldosterone hypersecretion in the absence of excess angiotensin leading to sodium retention and potassium excretion with various degrees of hypertension, hypokalemia and renin suppression. A large body of evidence has established that PA is one of the most common form of secondary hypertension, with a prevalence of>10% among unselected hypertensives and 20% among patients with resistant hypertension. The most common clinical subtypes of PA are aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA), which respectively accounts for 60% and 30% of PA patients. But the pathogenesis of APA or IHA still remains unclear. Mineralocorticoid receptor antagonists are recommended for IHA while APA usually needs surgical treatment (unilateral laparoscopic adrenalectomy). Surgery has been reported to abolish aldosterone hypersecretion and hypokalemia in most patients with APA, however, it cures hypertension in only one in every two cases. Part of patients with APA show postoperative persist hypertension.The renin-angiotensin system (RAS) mediates several classic physiologies including body water and electrolyte homeostasis, blood pressure and generation of aldosterone. These functions appear to be mediated by the angiotensinⅡtype 1 and type 2 receptors (AT1R, AT2R) subtype system. In the adrenal cortex, renin, angiotensinogen, AT1R and AT2R together compose a local angiotensinⅡsystem regulated independently from the circulating RAS. AT2R appear to modulate tissue development and repair and to counterbalance the effects of the angiotensinⅡmediated by AT1R, including cell growth, vasoconstriction, cardiovascular fibrosis and so on. More and more studies suggest that polymorphisims in AT1R and AT2R genes are associated with hypertension, myocardial disease, stroke even femal breast cancer. Genetic or environmental factors potentially disturb the expression and activity of AT1R and AT2R, which may lead to disorders of their regulating function. On the basis of the above hypothesis, this study aims to investigate the associations of polymorphisms in AT1R/AT2R genes with the risk and postoperative prognosis of APA.Methods1. Genomic DNA was extracted by DNeasy Blood & Tissue DNA purification Kit from adenoma tissues of 148 APA patients and peripheral blood samples of 192 normal people as controls, and stored at-20℃.2. Total RNA was extracted by Trizol from 50 cases of APA tissues and 50 normal adrenal tissues. The mRNA expression of AT1R and AT2R were examined through reverse transcriptase polymerase chain reaction (RT-PCR). The difference of AT1R/AT2R protein expression in APA and normal adrenal tissue was detected by immunohistochemical stain. Fluorescent quantitative real-time RCR was performed for the quantitative determination of AT2R mRNA expression.3. Based on the public SNP databases (http://ncbi.nlm.nih.gov/SNP/and http://www.hapmap.org/) and published documents, four DNA polymorphism loci at AT1R/AT2R genes with a minor allele frequency≥0.05 and potential associations between their variations and hypertension or other cardiovascular disease were selected for our study. The 4 SNPs are rs5182 (573T/C) in exon 4, rs5186 (1166A/C) in 3'untranslated region (UTR) of AT1R gene and rs5194 (2274G/A) in 3'-UTR, rs1403543 (1675G/A) in intron 1 of AT2R gene.4. Genotypes of the 4 SNP loci were detected by MGB-Taqman probe method.5. Collect clinical data of the 148 APA patients, including preoperative BP, biochemical index and tumor size et al. The postoperative recovery of BP was followed up since 6 months after surgery. Patients were categorized as cured if they had no hypertension [defined as systolic blood pressure (SBP)<140 mm Hg and diastolic blood pressure (DBP) <90 mm Hg] and were not taking any antihypertensive medications, or they were categorized as having persistent hypertension.6. Hardy-Weinberg equilibrium (HWE) of each polymorphism locus was determined by SNPassoc in R statistics program 2.7.0 software package (P>0.05 was considered that genotype distribution was in accordance with HWE). SPSS 16.0 and R statistics program were used for statistical analyzing the associations between polymorphisms in AT1R/AT2R genes and the risk of APA,postoperative recovery of BP and gene expression. Odds ratio (OR) and 95% confidence interval (CI) were calculated to show the relative risk。A P value <0.05 or odds ratio (OR) 1.00 is not in the range of 95% CI implicating statistical significant.Results1. Genotyping and distribution:Four SNP loci including rs5182, rs5186, rs5194 and rs1403543 at AT1R/AT2R genes were successfully detected. The distribution of genotypes of each locus was in accordance with Hardy-Weinberg Equilibrium (HWE) in APA and control group (P> 0.05).2. Association between AT1R/AT2R gene polymorphisms and risk of APA:The sex, age and BMI of APA patients and normal peoples have no significant difference (P>0.05). Of the 4 loci, rs5194 SNP at AT2R gene was most significantly associated with APA in additive (OR=1.64,95%CI=1.21-2.20, P=0.001), dominant (OR=1.94,95%CI=1.23-3.06, P=0.003), and recessive model (OR=2.01,95%CI=1.17-3.45, P=0.01). The A allele frequency at rs5194 was significantly higher in APA group (0.49) than that in normal controls group (0.35) (χ2=12.08, P=0.001). Homozygotic genotype GG and heterozygotic genotype GA had an increased risk of APA compared to AA genotype (OR=2.66,95% CI=1.45-4.87; OR=1.67,95% CI=1.02-2.74).3. Association between AT1R/AT2R gene polymorphisms and postoperative BP recovery of APA:Total of 128 APA patients have been followed up including 72 patients with idea postoperative BP control and 56 with postoperative persist hypertension. Except for amounts of antihypertensive medications for preoperative BP control, no significant differences were found in sex, age, BMI, tumor size, preoperative BP level and duration of hypertension between the two groups. The distribution of genotypes of each locus was in accordance with Hardy-Weinberg Equilibrium (HWE) in the above two groups (P> 0.05). Of the 4 SNPs, only rs 1403543 locus in AT2R gene was found associated with postoperative persist hypertension of APA patients. APA patients with homozygotic genotype GG had an increased risk of persist postoperative hypertension compared to patients with AA or GA genotype (OR=5.00,95% CI=1.31-19.15, P=0.01).4. Relationship between polymorphisms in angiotensinⅡreceptor gene and its expression variation:Semiquantitative analysis found that mRNA expression of AT1R in adenoma and normal tissues of adrenal gland showed no apparent differences (P>0.05). But the mRNA expression of AT2R in APA tissue is lower than that in normal adrenal gland tissues (P<0.05). Further more, immunohistochemical stain showed AT2R protein expression was also down regulated in APA tissue compared with normal adrenal tissue. But the AT1R protein was positive expressed in both APA and normal adrenal tissue. Fluorescent quantitative real-time RCR further verified the result of down regulation of AT2R mRNA expression in APA tissue. Alle A in rs5194 was associated with down regulation of AT2R mRNA expression. The ratio of AT2R mRNA expression in AA and GA genotypes to GG genotype is about 1/8 (P=0.000). No statistical significance was found in AT2R mRNA expression between different genotypes of rs1403543 locus.Conclusions1. DNA polymorphisms within AT2R gene were associated with the risk of APA and postoperative BP recovery of APA patients. Detection of polymorphisms within AT2R gene may provide useful genetic information for prognosing the risk of APA, anl also could be a predictor for APA patients with postoperative persist hypertension.2. DNA polymorphisms within AT2R gene may be able to down regulate AT2R mRNA expression, which could be a potential pathogenesis of APA. But the exact molecular mechanism needs to be further investigated.3. Genetic variants within AT1R gene, which is closely relative to hypertension disease, maybe not accociated with the risk and clinical phenotype of APA.
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