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Positive association between KCNJ5 rs2604204 (A/C) polymorphism a n d plasma aldosterone levels, but also plasma renin a n d angiotensin I
作者:陈慧[1] 
单位:福建省立医院[1]  
文章号:W122741  
2017/8/21 14:29:42    
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INTRODUCTION 365医学网 转载请注明
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  The physiological role of aldosterone (ALD) is to regulate blood pressure (BP), a n d to control water homoeostasis a n d electrolytes balance. However, high levels of adrenal ALD secretion can cause hypertension, that is, primary aldosteronism, a well-known fo rm of secondary hypertension. 365医学网 转载请注明
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  Hyperaldosteronism is revealed to be a much mo re common cause of hypertension than previously thought. Even within the no rmal BP range, higher ALD level was also associated with incidence of hypertension.1,2 Framingham Offspring Study3 strongly suggested that increasing ALD levels contributed impo rtantly to the development of hypertension. 365医学网 转载请注明
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  As fo r cardio a n d cerebrovascular diseases, ALD is also a key ho rmonal mediato r.4 In primary aldosteronism, there is an increased incidence of cardio a n d cerebrovascular events such as left ventricular hypertrophy, co ronary artery disease, nonfatal myocardial infarction, heart failure, atrial fibrillation, stroke a n d proteinuria, compared with essential hypertensive controls.5,6 Increased ALD was also associated with higher BP a n d greater left ventricular mass,7,8 cardiometabolic a n d metabolic syndrome risk.9–11 In a large coho rt study,12 variation in median plasma ALD levels within the no rmal range was related to increased all-cause a n d cardiovascular disease mo rtality, independent of majo r established cardiovascular risk facto rs. 365医学网 转载请注明
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  Of greater focus during the last several years has been the impo rtance of the gene encoding the G protein-activated inward rectifier K+ channel, KCNJ5, as a central acto r in both the spo radic a n d the familial fo rms of primary hyperaldosteronism.13 The KCNJ5 gene variants affect the Na+ permeability of the channel, which is thought to lead to depolarization of zona glomerulosa cells in the adrenal co rtex thereby activating ALD release. KCNJ5 rs2604204 single-nucleotide polymo rphism (SNP) was shown to be associated with spo radic primary aldosteronism in Chinese male. In addition, KCNJ5 rs2604204 SNP was common (mino r allele frequency = 32.5%) in Chinese patients with essential hypertension (EH).14 However, the relationship between KCNJ5 rs2604204 SNP a n d plasma ALD levels in EH patients has not been repo rted previously. 365医学网 转载请注明
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  In this study, we investigated the association between plasma ALD a n d KCNJ5 rs2604204 SNP in newly diagnosed, never-treated EH patients. 365医学网 转载请注明
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SUBJECTS a n d METHODS 365医学网 转载请注明
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Ethics statement 365医学网 转载请注明
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  The study was reviewed a n d approved by the Ethic Committee of the Fujian Provincial Hospital. All procedures were conducted acco rding to the 365医学网 转载请注明
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  sta n dards of the Declaration of Helsinki. All study participants provided signed info rmed consent. 365医学网 转载请注明
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Subjects 365医学网 转载请注明
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  All the 229 subjects were recruited from October 2011 to January 2014 in the Department of Cardiovascular Diseases of Fujian Provincial Hospital. Case subjects were outpatients with newly diagnosed mild-to-moderate EH, without any antihypertensive agents. Hypertension diagnosis was made acco rding to the criteria published by 2010 Chinese guidelines fo r the management of hypertension.15 Inclusion criteria included 418 years old, at least three occasions of systolic BP (SBP) 140 mm Hg a n d/o r diastolic BP (DBP) 90 mm Hg, no rmal serum potassium, not taking any antihypertensive agents o r affecting blood glucose a n d insulin levels drugs, no histo ries of hypokalemia a n d diabetes, a n d sleep-diso rdered breathing. To exclude primary aldosteronism, included subjects also had no rmal plasma potassium a n d sta n ding ALD renin ratio, no abno rmalities in adrenal gla n d, a n d renal vascular ultrasound o r computed tomography angiography (CTA), a n d negative result in saline infusion test fo r those plasma ALD 423.9 ng dl− 1 (n = 50). Patients were excluded if they were diagnosed with any secondary fo rms of hypertension such as renal arterial hypertension, primary aldosteronism, Cushing’s syndrome, o r computed tomography imaging demonstrated an adrenal hyperplasia o r adenoma; if they had hypertension combined with pregnancy, substance abuse (including alcohol), congestive heart failure, acute myocardial infarction, serious heart valve disease, renal function insufficiency o r cerebrovascular lesions; if they had severe hepatic insufficiency, blood disease, severe infections, trauma o r malignant tumour; a n d if they were day a n d night shift wo rkers. 365医学网 转载请注明
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Data collection 365医学网 转载请注明
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  Acco rding to the median sta n ding plasma ALD (18 (14–23) ng dl − 1) (no rmal range: 6.3–24), the patients enrolled were divided into two groups, high-ALD (n = 114) group a n d control (n = 115) group. 365医学网 转载请注明
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  Anthropometric measurements a n d BP were obtained by physical examination. The anthropometric indices of height a n d weight were measured while the patients were barefoot a n d in light clothing. Body weight a n d height were measured to the nearest 0.1 kg a n d 0.1 cm, respectively, by using sta n dardized equipment a n d procedures. Body mass index was calculated as weight in kg divided by height squared in metres. Waist circumference was measured to the nearest 0.1 cm at the level of the iliac crest, whereas the subject was at minimal respiration. BP was measured in the right arm in the sitting position with a regular mercury sphygmomanometer after resting fo r at least 5 min. The mean of three consecutive BP readings was used a n d BP of the selected patients must be 140 mm Hg a n d/o r 90 mm Hg. All of the subjects underwent 24 h ambulato ry BP monito ring (24 h ABPM) on a day of daily activity. A proper cuff was selected acco rding to the size of subject´s arm a n d placed on the non-dominant arm. The subjects were asked to keep their arms still at the time of measurements. The 24 h BP was reco rded automatically using a Holter a n d ABPM Combiner CB-1804-B (BIOX Instruments Co. Ltd., Jiangsu, China). The daytime BP monito ring was from 0600 to 2159 hours, measured automatically every 30 min, a n d during the night time, from 2200 to 0559, the BP was measured once an hour. The subjects had to fulfil 80% of SBP a n d DBP successful reco rdings during the daytime a n d night-time periods, 24 h duration. Twenty-four hour mean SBP (24 h SBP) a n d DBP (24 h DBP), daytime mean SBP a n d DBP, a n d night-time mean SBP a n d DBP were reco rded. Sho rt-term BP variability (BPV) was evaluated through 24 h s.d. of SBP a n d DBP, a n d 24 h coefficient of variance (coefficient of variance = s.d./mean SBP o r DBP values). 365医学网 转载请注明
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Ho rmonal a n d biochemical assays 365医学网 转载请注明
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  Following an overnight fast, blood samples were drawn from an indwelling catheter positioned in an antecubital vein after 2 h sta n ding position between 0800 a n d 1000 hours. Serum o r plasma from each blood sample was separated within 1 h after blood collection, a n d all samples were sto red at − 20 °C until application. In addition, isolated blood cells were sto red at − 80 °C until subsequent genotyping. 365医学网 转载请注明
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  Sta n ding plasma ALD a n d angiotensin Ⅱ (Ang Ⅱ) were measured with an iodine [125I] ALD a n d an iodine [125I] Ang Ⅱ radioimmunoassay kit (No rthern Biotechnology Institutes, Beijing, China), respectively. Plasma renin activity was measured with an iodine [125I] angiotensin I (Ang I) radioimmunoassay kit (No rthern Biotechnology Institutes). All intra- a n d inter-assay coefficients of variation were o10% a n d 15%, respectively. 365医学网 转载请注明
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  Ra n dom co rtisol (Co r) was measured by ACCESS automatic chemilumines-cence immunoassay system (Beckman, USA). Fasting insulin (In) was measured by chemiluminescence method (Roche Cobas 6000, Roche Diagnostics, Holliston, MA, USA). Fasting plasma glucose, lipids profiles (total cholesterol, triglycerides (TGs), high-density lipoprotein cholesterol a n d low-density lipoprotein cholesterol), serum sodium (Na), potassium (K), creatinine (Cr) a n d uric acid were measured with a Beckman CX9 PRO automated biochemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA). Glycated haemoglobin (HbAc1) was measured with an ADAMS A1c HA-8180 automatic A1C analyzer (Arkray, Kyoto, Japan). HOMA insulin resistance index (HOMA-IR) was calculated acco rding to the fo rmula: fasting glucose (mmol l − 1) × insulin (mIU l − 1)/22.5. Creatinine clearance using Cockcroft–Gault fo rmula: (140 age in years) × (weight in kg) × (0.85 if female)/(72 × Cr in mg dl − 1)). 365医学网 转载请注明
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Genotyping 365医学网 转载请注明
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  The genetic variant rs2604204 of KCNJ5 were selected from the National Center fo r Biotechnology Info rmation dbSNP a n d the International HapMap Project databases (http://www.ncbi.nlm.nih.gov a n d http://www. hapmap.o rg, respectively). Genomic DNA was isolated from peripheral blood mononuclear cells using Blood Genome DNA Extraction Kit (Takara Biotechnology Co., Ltd, Dalian, China). SNP genotyping was perfo rmed using PCR (Labnet MultiGene Optimax PCR, Edison, NJ, USA) a n d ABI 3700 Automated DNA Sequencer (Applied Biosystems, Foster City, CA, USA). PCR conditions included an initial denaturation of 95 °C fo r 5 min, followed by 30 cycles of 95 °C fo r 30 s, 57 °C fo r 30 s, 72 °C fo r 30 s a n d then a final extension at 72 °C fo r 7 min. The primer sequences were 5′-GCCAACTGGCTCATCATGTC-3′ (fo rward primer) a n d 5′-CTGCTGTGTT CTGTGTGTCC-3′ (reverse primer). 365医学网 转载请注明
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Statistical analysis 365医学网 转载请注明
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  All statistical analyses were perfo rmed with (SPSS, Inc., Chicago, IL, USA) statistical software, version 16.0 (USA). The data are expressed as mean ± s.d., mean ± s.e.m. o r median (interquartile range). Inter-group comparisons were made with the unpaired t-test, Mann–Whitney U-test o r χ2-test. Inter-group differences of SNP genotype o r allele frequency were analysed by the χ2-test a n d χ2 segmentation. Hardy–Weinberg equilibrium test was selected. Binary logistic regression analysis was used to calculate odds ratios (o rs) with 95% confidence intervals (CIs). In this model, high-ALD group o r control group was a dependent variable, a n d TG, uric acid, Co r, In, HOMA-IR, plasma renin activity, Ang I, Ang Ⅱ, C allele (AC+CC genotypes) of KCNJ5 were independent variables. P-values o0.05 were considered to indicate statistical significance. 365医学网 转载请注明
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RESULTS 365医学网 转载请注明
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  Baseline characteristics of control a n d high-ALD patients Hypertensive patients were divided into two groups depend on the median of sta n ding ALD (18.3 ng dl − 1): high-ALD group (n = 114) a n d control group (n = 115). Baseline characteristics of two groups are summarized in Table 1. There was no significant difference in gender, age, body mass index, waist circumference, fasting plasma glucose, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, K, Na, Cr a n d creatinine clearance between two groups. Compared with control group, high-ALD group had higher TG, In, HOMA-IR, uric acid, renin–angiotensin–ALD a n d Co r levels (P o0.05–P o0.001). 365医学网 转载请注明
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Comparison of ABPM results between two groups 365医学网 转载请注明
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  Compared with control group, 24 h SBP a n d daytime mean SBP (Table 2) were increased in high-ALD group (P o0.05). Parameters of sho rt-term BPV by ABPM such as 24 h s.d. (DBP), s.d. (SBP), coefficient of variance (SBP) a n d coefficient of variance (DBP) (Table 3) were higher in high-ALD group than in control group (P o0.05). There was no significant difference in 24 h DBP, daytime mean DBP, night-time mean SBP a n d night-time mean DBP between the two groups. 365医学网 转载请注明
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Genotype a n d allele frequencies 365医学网 转载请注明
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  The KCNJ5 rs2604204 genotypes in hypertensive patients were in Hardy–Weinberg equilibrium (P40.05). Table 4 presents the rs2604204 (A/C) genotypes a n d allele frequencies in two groups. The high-ALD group a n d the control group showed significant differences in the distributions of genotypes a n d alleles of rs2604204 (P o0.05). The distribution frequencies of rs2604204 AC a n d CC genotypes were higher in the high-ALD group (χ2 = 11, P = 0.004), so was the C allele frequency (χ2 = 9.8, P = 0.002). There was no significant difference between AC a n d CC genotypes 365医学网 转载请注明
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  in two groups.Compared with hypertensive patients with AA genotype, patients with the C allele showed increased high-ALD risk (χ2 = 9.8, o r: 2.36, 95% CI: 1.4–4.0, P = 0.002). 365医学网 转载请注明
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Logistic regression analysis 365医学网 转载请注明
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  Binary logistic regression analyses were carried out to examine risk facto rs of increased plasma ALD level in newly diagnosed, never-treated EH patients. In this model, high-ALD group o r control group were seen as dependent variables, influencing facto rs including TG, uric acid, Co r, In, HOMA-IR, plasma renin activity, Ang I, Ang Ⅱ, C allele (AC+CC genotypes) of KCNJ5 were as independent variables. 365医学网 转载请注明
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  As a result, the rs2604204 C allele (o r 2.2; 95% CI 1.2–4.1; P = 0.008), which was found to be the most significant 365医学网 转载请注明
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DISCUSSION 365医学网 转载请注明
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  In this study, we observed that plasma ALD levels showed a significant association with the HOMA-IR index, which was consistent with previous results.16 Another 10-year prospective study17 demonstrated that plasma ALD levels could predict the development of insulin resistance. The current theo ries regarding the effects of ALD on glucose metabolism primarily focus on insulin resistance, which was mainly induced by inflammation, oxidative stress, hypokalemia, disturbance of insulin signalling a n d hepatic gluconeogenesis.18 One could also postulate that Ang Ⅱ induces ALD synthesis through an AT1R-dependent mechanism a n d, at least partly, through a MR-dependent mechanism in human peripheral blood mononuclear cells.19 Rutkowska-Zapała20 found human monocyte subsets exhibit divergent Ang I-converting activity, that might be involved in the regulation of vessel wall homoeostasis. 365医学网 转载请注明
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  In addition, our study demonstrated a significant co rrelation between the variation in plasma ALD levels within the no rmal range a n d untreated 24 h ABPM levels. We found that the ALD variation was significantly related to 24 h a n d daytime SBP levels, as well as the sho rt-term BPV, such as s.d. a n d coefficient of variance during the 24 h period. The sho rt-term BPV is usually seen as one cause of hypertension-related target o rgan damage a n d cerebral cardiovascular complications.21 Therefo re, indepen-dent of mean BP, 24 h BPV has prognostic value fo r cardiovascular diseases a n d mo rtality.22 As local RAS activity in the kidney had a role in BPV23 a n d ABPM-derived sho rt-term BPV was found to be increased in primary aldosteronism.24 The variation in plasma ALD might have an impo rtant role in regulating BPV. 365医学网 转载请注明
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  The reason why KCNJ5 rs2604204 SNP was associated with increased plasma ALD levels in EH patients is yet to be discovered. So far, multiple KCNJ5 gene variants have been found to be related to ALD synthesis, but the mechanisms how mutant KCNJ5 stimulating production of ALD have not been fully characterized. In 2011, Choi et al.25 identified two somatic variants (G151R, L168R) of KCNJ5 (encoding a potassium inwardly rectifying channel) in 8 out of 22 spo radic aldosterone-producing adenomas (APA) patients. Because of KCNJ5 variants resulting in a loss of K+ channel selectivity a n d constitutive ALD production, KCNJ5 variant carriers might have higher ALD level.26 Acco rding to the study by Zheng et al.,27 in Chinese APAs, 76.8% had somatic KCNJ5 variants. Interestingly, in our study those in the high-ALD group also had higher renin, Ang I a n d Ang II level, which is in opposition to autonomous ALD production in primary aldosteron-ism (PA). Given the result of logistic regression analysis, the rs2604204 C allele was the most significant independent variable as a facto r that might affect plasma ALD level. This mutation might be associated with the regulation of ALD synthesis in both APAs a n d none APAs; the underlying mechanisms are yet to be discovered. The rs2604204 SNP could have a role in modifications in tubular feedback a n d lead to elevation of RAAS activity, as shown in a previous study of another SNP.28 Previous studies have indicated that insulin resistance was associated with inappropriate activation of the renin–angiotensin–ALD system with subsequent elevations in Ang II a n d ALD.29,30 These could be other reasons 365医学网 转载请注明
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  why those in high-ALD group had higher renin–angiotensin–ALD activities. Besides of KCNJ5, there might also be SNPs in renin– angiotensin–ALD (RAAS) genes, that we did not test in these patients. 365医学网 转载请注明
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  KCNJ5 variants were also found to be associated with insulin resistance (rs11221497)31 a n d metabolic syndrome32 (rs11221497, rs6590357, rs4937391 a n d rs2604204, a n d M210I) in the Uygurian 365医学网 转载请注明
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  population. Both insulin resistance a n d metabolic syndrome could affect renin–angiotensin–ALD activities.33 Recently, KCNJ5 (T158A) variant was found to increase CYP11B2 expression a n d production of ALD, co rticosterone a n d hybrid steroids by upregulating both acute a n d chronic regulato ry events in ALD production.33 However, the function effect of rs2604204 is remain unknown. Thereby, further validation with a larger sample size a n d studies on determining the rs2604204 SNP function a n d association with ALD level a n d EH, are needed in the future. A limitation of our studies is that patient’s sodium o r potassium intake a n d 24 h urinary excretion of sodium o r ALD could not be monito red, given that all participants in this study were newly diagnosed EH outpatients that did not require hospitalization. 365医学网 转载请注明
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CONLUSION 365医学网 转载请注明
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  Our findings indicated that the variation of plasma ALD might be associated with increased IR a n d BPV. Mo reover, KCNJ5 rs2604204 polymo rphism was associated with increased plasma ALD level, as well as plasma renin a n d Ang I a n d II levels in newly diagnosed, never-treated EH patients. 365医学网 转载请注明
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CONFLICT OF INTEREST 365医学网 转载请注明
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  The autho rs declare no conflict of interest. 365医学网 转载请注明
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ACKNOWLEDGEMENTS 365医学网 转载请注明
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  We would like to express their sincere gratitude to all the study subjects fo r their voluntary participation a n d to all the staff of Hypertension Labo rato ry in Fujian Provincial Cardiovascular Disease Institute fo r their excellent assistance in data collection a n d conservation of samples. This wo rk was suppo rted by the Natural Science Foundation of Fujian Province (no 2014Y01010261) a n d Health Innovation Fund of Fujian (no 2012-CXB-5). The funders had no role in study design, data collection a n d analysis, decision to publish, o r preparation of the manuscript. 365医学网 转载请注明
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AUTHo r CONTRIBUTIONS 365医学网 转载请注明
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  Conceived a n d designed the experiments: HC. Perfo rmed the experiments: HW, CW a n d HC. Analysed the data: HW a n d CW. 365医学网 转载请注明
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  Contributed reagents/materials/ analysis tools: HW a n d CW. Wrote the paper: HW, CW a n d HC.

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作者简介
陈慧
单位:福建省立医院
简介: 从医35年,福建省立医院.省心血管病研究所内科二级主任医师、高血压研究室主任、福建医科大学省立临
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