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Aldose reductase C-106T polymorphism is associated with the risk of essential hypertension
Gene 591 (2016) 65–68
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Aldose reductase C-106T polymorphism is associated with the risk of essential hypertension Yaqin Wang a,b, Min Yu a,b, Long Mo c, Zhenyu Li c, Junjie Wang a,b,c, Hong-hao Zhou a,b, Dong-Sheng Ouyang a,b,⁎ a b c
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, PR China Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, PR China Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410078, PR China
a r t i c l e
i n f o
Article history: Received 29 March 2016 Received in revised form 7 June 2016 Accepted 21 June 2016 Available online 23 June 2016 Keywords: Essential hypertension Aldose reductase Polymorphism
a b s t r a c t Aldose Reductase (AR), encoded by AKR1B1, is a member of NADPH-dependent aldo-keto reductase superfamily. The C-106T polymorphism of AKR1B1 is closely related to the diabetic complications. Our previous studies have indicated that the expression of AR was increased in spontaneously hypertensive rats, suggesting the effect of AR in hypertension. Here we investigated whether AKR1B1 C-106T polymorphism was associated with essential hypertension (EH). AKR1B1 C-106T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the direct sequencing methods. 383 healthy subjects and 383 essential hypertensive patients were recruited in this study. The polymorphism of AKR1B1 C-106T in EH and normal tensive (NT) groups was in agreement with Hardy-Weinberg equilibrium. -106T allele of AKR1B1 C-106T variants was more frequent in EH patients compared with normal tensive subjects, indicating that -106T allele was a risk factor of EH (OR = 1.841, 95%CI = 1.366–2.481). In male patients, C-106T polymorphism was associated significantly with decreased serum high density lipoprotein cholesterol and higher systolic blood pressure levels. Our results suggest that -106T allele of AKR1B1 C-106T polymorphism may be associated with increased risk for EH in Chinese Han population. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Essential hypertension (EH) is an independent risk factor for cardiovascular diseases, such as myocardial infarction and stroke. Aldose reductase (AR), a member of the aldo-keto reductase superfamily, is the first rate-limiting enzyme of the polyol pathway and catalyzes the NADPH-dependent reduction of glucose to sorbitol (Wang et al., 1993). Our previous studies showed that the expression level of AR was increased in spontaneously hypertensive rat (SHR; Gu et al., 2011; Li et al., 2013), demonstrating that AR may be involved in the development and progression of hypertension. Proinflammatory conditions and inflammation are associated with the pathophysiology of EH (González et al., 2014). AR is a redoxsensitive enzyme and acts as a key mediator in the oxidative and Abbreviations: AR, aldose reductase; BMI, body mass index; CI, confidence interval; DBP, diastolic blood pressure; EH, essential hypertension; FBG, fasting blood glucose; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NT, normal tensive; OR, odds ratio; PCR, polymerase chain reaction; PCR-RFLP, polymerase chain reactionrestriction fragment length polymorphism; SBP, systolic blood pressure; SNP, single nucleotide polymorphism; TC, total cholesterol; TG, triglyceride. ⁎ Corresponding author. E-mail address: [email protected] (D.-S. Ouyang).
http://dx.doi.org/10.1016/j.gene.2016.06.043 0378-1119/© 2016 Elsevier B.V. All rights reserved.
inflammatory signaling pathways that are associated with cardiovascular disease (Maccari et al., 2015). Previous studies have indicated that AR plays a role in causing glomerular fibrosis through mesangial cell proliferation and extracellular matrix biosynthesis (Li et al., 2014; Jing et al., 2015). Vascular fibrosis is the pathological basis for the development of hypertension. Therefore, we believe that AR plays an important role in EH by accelerating the inflammatory response and vascular fibrosis. EH is considered to be a multifactorial disease resulting from a combination of environmental and genetic factors. Increasing evidence has shown the effects of genetics on the high prevalence of EH (Luo et al., 2014; Vimaleswaran et al., 2014). Human AR is a monomeric protein (36 kDa) comprising 315 amino acids that is encoded by AKR1B1, which maps to chromosome region 7q35. The C-106T (rs759853) single nucleotide polymorphism (SNP), which is located at the promoter of the AKR1B1 gene, has been reported to be associated with the risk of diabetic complications, including nephropathy (Cui et al., 2015), retinopathy (dos Santos et al., 2006; Katakami et al., 2011) and macroangiopathy (Watarai et al., 2006). Our pre-experiment has explored the association between AKR1B1 gene polymorphisms and EH, but we failed to obtain positive results that may be due to limited sample size. Therefore, we increased sample size to investigate the association between AKR1B1 gene polymorphisms and EH in the present study.
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Y. Wang et al. / Gene 591 (2016) 65–68
2. Material and methods 2.1. Subject This study protocol was approved the Independent Ethics Committee of the Institute of Clinical Pharmacology, Central South University (Hunan, China). Written informed consent was obtained from each participant. From February 2014 to May 2014, a total of 766 Chinese Han volunteers, including 383 patients diagnosed with EH and 383 healthy subjects, were recruited by the Physical Examination Center of the Third Xiangya Hospital, Central South University. Blood pressures were measured twice at 5-minute intervals, according to the guidelines of the European Society of Hypertension (Mancia et al., 2007). Hypertension was diagnosed in participants exhibiting a mean clinic systolic blood pressure (SBP) ≥140 mm Hg and/or a mean clinic diastolic blood pressure (DBP) ≥ 90 mm Hg, as well as in participants receiving ongoing treatment for hypertension. Subjects with a history of secondary hypertension, diabetes mellitus, primary renal disease or other serious diseases were excluded from this study. Information regarding patient sex, age, height, weight, and family history, as well as triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDL-C) and fasting blood glucose (FBG) levels, was collected.
2.2. Genotyping Blood samples were placed in EDTA-containing containers and stored at − 20 °C. Genomic DNA was extracted from peripheral whole blood using a Qiagen DNA Isolation Kit (Valencia, CA, USA). The C-106T polymorphism of AKR1B1 was identified by the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) method using the primers and conditions described by Kao et al. (1999). PCR products were digested using the restriction enzyme Bfa I and fractionated via a 3% agarose gel. The amplified PCR product was 263 bp, and two fragments of 57 bp and 206 bp were generated after restriction digestion of the sequence corresponding to the CC genotype. Samples obtained from subjects who were heterozygous for the mutant allele were cut into four fragments (57, 59, 147 and 206 bp) due to the presence of an additional cutting site, while three fragments (57, 59 and 147 bp) were generated for samples obtained from subjects who were homozygous for the mutant allele, as shown in Fig. 1. To confirm PCR-RFLP genotyping results, 5% of the DNA samples were randomly selected to perform direct sequencing (Fig. 2), and we obtained consistent results using the two detection methods.
Fig. 1. A, the PCR results of the polymorphism of AKR1B1 gene. M, DL2000 DNA marker (100, 250, 500, 750, 1000, 2000 bp). Lanes 1–10 are amplification products of samples obtained from subjects. B, the PCR-RFLP results of polymorphisms of AKR1B1 gene. M, DL2000 DNA marker (100, 250, 500, 750, 1000, 2000 bp). Lanes 1 and 2 are CT genotype (57, 59, 147 and 206 bp); Lanes 3 and 4 are TT genotype (57, 59 and 147 bp); Lanes 5 and 6 are CC genotype (57 and 206 bp).
3. Results 3.1. Characteristics of the participants The clinical and biochemical characteristics of all subjects are shown in Table 1. There were no differences in the distributions of age and
2.3. Statistical analysis SPSS 19.0 software was used for data analysis. Comparisons between the groups regarding quantitative data were performed using an independent samples t-test, and a chi-square test was used to analyse categorical data. Differences in genotype and allele frequencies between groups and deviations from Hardy-Weinberg equilibrium were calculated by chi-square test. The associations between AKR1B1 genetic variants and EH were analyzed via binary logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (95%CI), which were adjusted for age and sex. Linear regression analysis and Bonferroni correction were used to analyse the relationships between AKR1B1 genetic variants and EH-related phenotypes, which were adjusted for age and sex, while comparisons between males and females were adjusted for age only. PASS software 11.0 was used to calculate the power of the tests. A two-tailed p-value b 0.05 was considered statistically significant.
Fig. 2. Genotyping results according to direct sequencing methods. Arrows indicated the polymorphism loci.
Y. Wang et al. / Gene 591 (2016) 65–68
DBP levels in a dominant model (p = 0.024). In female patients, there was no association between the C-106T polymorphism and blood pressure or biochemical indices.
Table 1 The clinical and biochemical characteristics of EH and NT groups. Variables Gender Male (%) Female (%) Age (years) Family history (%) BMI (kg/m2) FBG (mmol/l) TG (mmol/l) TC (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) SBP (mm Hg) DBP (mm Hg) a
NT group (n = 383)
EH group (n = 383)
192(50.1) 191(49.9) 55 ± 11 23 (6.0) 23.7 ± 3.0 5.1 ± 0.5 1.5 ± 1.5 5.2 ± 1.0 1.6 ± 0.4 2.9 ± 0.8 122 ± 13 76 ± 10
193(50.4) 190(49.6) 56 ± 8 74 (25.0) 25.1 ± 3.0 6.2 ± 2.3 1.7 ± 1.3 4.4 ± 1.1 1.2 ± 0.4 2.5 ± 0.8 152 ± 18 96 ± 12
67
p 0.942
0.128 b0.001a b0.001a b0.001a 0.125 b0.001a b0.001a b0.001a b0.001a b0.001a
p b 0.001 compared with NT group.
gender between the EH and NT groups. Compared with the NT subjects, the EH patients showed significantly higher body mass indices (BMIs) and rates of hypertensive family history, as well as significantly higher FBG, TC, HDL-C and LDL-C levels (p b 0.001). 3.2. Distribution of genotype The genotype distributions of AKR1B1 C-106T were in agreement with Hardy-Weinberg equilibrium in the EH and NT groups (EH: χ2 = 0.205, p = 0.651; NT: χ2 = 1.392, p = 0.238). Genotype distributions and allele frequencies were significantly different between the EH and NT groups, as shown in Table 2 (χ2 = 16.071, p1 = 0.000), and the -106T allele was more frequent in EH patients (χ2 = 17.034, p1 = 0.000). In addition, the -106T allele and CT + TT genotype were associated with an increased risk of EH (OR = 1.841, 95%CI = 1.366–2.481, p2 = 0.000; OR = 1.908, 95%CI = 1.367–2.663, p2 = 0.000). 3.3. Association between C-106T polymorphism of AKR1B1 and blood pressure, biochemical indices To further evaluate the association between the AKR1B1 C-106T polymorphism and EH, we performed relative analyses of the associations between the C-106T polymorphism and blood pressure, as well as the associations between the C-106T polymorphism and biochemical indices, including TC, TG, HDL-C, and LDL-C levels (Table 3). In EH patients, the C-106T polymorphism was associated with decreased serum HDL-C levels in a recessive model (p = 0.009). In male patients, the C-106T polymorphism was also found to be associated with decreased serum HDL-C levels in a recessive model (p = 0.024) and higher
4. Discussion In the present study, we found that the T allele of the AKR1B1 C-106T polymorphism was associated with an increased risk of EH. Our study provides new evidence supporting the relationship between inflammation and EH, as well as a new explanation regarding the pathogenesis of EH and a new target for the prevention of EH. The effect of the AKR1B1 C-106T polymorphism on the expression of AR has been studied by other researchers. Watarai et al. (2006) reported that erythrocyte AR protein content was increased in patients with the CT and TT genotypes compared with CC carriers, which was supported by our research. However, a functional reporter gene assay reported by Yang et al. (2003) indicated that a construct containing the C allele showed higher promoter activity than a construct containing the T allele in transfected HepG2 cells. Further studies are required to clarify the effects of the C-106T polymorphism on the expression of AR. In recent years, many studies have reported the associations between polymorphisms of AKR1B1 and diseases, especially diabetes complications, but the results of these studies are ambiguous. Some verified that the T allele was a risk factor for diabetes complications (So et al., 2008; Watarai et al., 2006), while others obtained contrasting results (dos Santos et al., 2006; Katakami et al., 2011). The discrepancies among those studies may be due to differences in participant ethnic, sampling, and inclusion and exclusion criteria, as well as experimental bias. Our pre-experiment, which included 148 EH volunteers and 137 NT volunteers, failed to obtain positive results (power = 0.175; Li et al., 2012). In the present study, we enrolled 383 EH and 383 NT volunteers, and the power to detect associations for the C-106T polymorphism at the 5% significance level was sufficient (power N 0.800), indicating that the sample size was large enough to obtain reliable results. HDL-C has been linked to reductions in EH events (Halperin et al., 2006). In the present study, we found that the C-106T polymorphism was associated with decreased HDL-C levels in EH patients, indicating that -106T carriers may have lower HDL-C levels and thus increased susceptibility to EH. However, we failed to observe an association between HDL-C levels and the C-106T polymorphism in females. Further studies are needed to address this issue. There were some limitations to this study. First, only one SNP of AKR1B1 was studied. An (AC)n dinucleotide repeat polymorphism in the promoter region is another common SNP of AKR1B1 (Xu et al., 2008). Therefore, further research is needed to investigate the effects of the above two SNPs on EH. Second, only a Han Chinese population
Table 2 Distribution of AKR1B1 C-106 T in EH and NT groups. NT group (n = 383) n (%)
EH group (n = 383) n (%)
χ2
p1
OR (95%CI)
p2
Genotype CC CT TT
319 (83.3) 59 (15.4) 5 (1.3)
262 (68.4) 108 (28.2) 13 (3.4)
16.071a
0.000
1 1.830 (1.336–2.427) 1.835 (1.082–3.110)
0.001b 0.024
Allele C T
687 (89.7) 79 (10.3)
631 (82.4) 135 (17.6)
17.034
1 1.841 (1.366–2.481)
0.000
319 (83.3) 74 (16.7) 378 (98.7) 5 (1.3)
262 (68.4) 121 (31.6) 370 (96.6) 13 (3.4)
15.197
0.000
3.641
0.056
Model Dominant Recessive a b
CC CT + TT CC + CT TT
p values are obtained from χ2 test. p values are obtained from binary logistic analysis.
0.000
1 1.908 (1.367–2.663) 1 1.610 (0.956–2.712)
0.000 0.074
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Y. Wang et al. / Gene 591 (2016) 65–68
Table 3 Results of association between AKR1B1 C-106T and blood pressure, biochemical indices in EH patients. Groups
All
Males
Females
Variables
SBP DBP TC TG HDL-C LDL-C SBP DBP TC TG HDL-C LDL-C SBP DBP TC TG HDL-C LDL-C
Dominant model
Recessive model
β (95%CI)
p
β (95%CI)
p
−0.029 (−4.897 to 2.706) 0.077 (−0.619 to 4.639) 0.027 (−0.366 to 0.495) −0.105 (−0.546 to 0.492) −0.071 (0.198 to 0.086) 0.086 (−0.170 to 0.485) 0.024 (−4.744 to 6.681) 0.162 (0.589 to 8.306) 0.038 (−0.451 to 0.622) 0.149 (−0.212 to 0.953) −0.193 (−0.305 to 0.028) 0.000 (−0.337 to 0.338) −0.110 (−8.937 to 1.165) −0.014 (−3.900 to 3.229) −0.054 (−0.898 to 0.617) −0.196 (−1.678 to 0.329) 0.122 (−0.149 to 0.364) 0.088 (−0.455 to 0.860)
0.571 0.134 0.768 0.917 0.434 0.343 0.743 0.024a 0.751 0.209 0.101 0.997 0.131 0.853 0.711 0.184 0.404 0.540
0.052 (−2.303 to 7.391) −0.071 (−5.727 to 0.986) −0.104 (−0.789 to 0.202) −0.082 (−0.877 to 0.319) −0.229 (−0.376 to −0.055) 0.047 (−0.278 to 0.480) 0.031 (−3.749 to 5.799) 0.111 (−0.715 to 5.765) −0.002 (−0.451 to 0.443) 0.096 (−0.290 to 0.687) −0.263 (−0.293 to −0.021) 0.017 (−0.261 to 0.301) 0.088 (−2.654 to 11.637) −0.089 (−8.128 to 1.897) −0.134 (−1.228 to 0.406) −0.084 (−1.422 to 0.769) −0.164 (−0.446 to 0.108) 0.024 (−0.653 to 0.782)
0.303 0.166 0.243 0.358 0.009a 0.599 0.672 0.126 0.985 0.420 0.024a 0.890 0.217 0.222 0.317 0.544 0.227 0.858
a p b 0.025 was considered to be significantly different. Because two independent hypothesis tests were performed, and the statistical significance level should be 1/2 times based on Bonferroni correction.
was included in our study; therefore, it is unclear if our results are applicable to other populations. In conclusion, our results suggest that the -106T allele of the AKR1B1 C-106T polymorphism may be associated with an increased risk of EH. Further investigation is necessary to explore the underlying mechanisms of the relationship between AR and EH. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements This work was supported by National Development of Key Novel Drugs for Special Projects of China (2012ZX09303014001) and the Special Fund for scientific Research in the Public Interest (201507004-4-2). We also acknowledge the Scientific Research Fund of Hunan Provincial Education Department (12B117), Natural Science Foundation of Hunan (2016JJ6142) and Postgraduate Innovation Project of Central South University (2016zzts127). References Cui, W., Du, B., Cui, Y., Kong, L., Wu, H., Wang, Y., et al., 2015. Is rs759853 polymorphism in promoter of aldose reductase gene a risk factor for diabetic nephropathy? A metaanalysis. Eur. J. Med. Res. 20, 14. dos Santos, K.G., Canani, L.H., Gross, J.L., Tschiedel, B., Souto, K.E., Roisenberg, I., 2006. The -106CC genotype of the aldose reductase gene is associated with an increased risk of proliferative diabetic retinopathy in Caucasian-Brazilians with type 2 diabetes. Mol. Genet. Metab. 88 (3), 280–284. González, J., Valls, N., Brito, R., Rodrigo, R., 2014. Essential hypertension and oxidative stress: new insights. World J. Cardiol. 6 (6), 353–366. Gu, J., Wang, J.J., Yan, J., Cui, C.F., Wu, W.H., Li, L., et al., 2011. Effects of lignans extracted from Eucommia ulmoides and aldose reductase inhibitor epalrestat on hypertensive vascular remodeling. J. Ethnopharmacol. 133 (1), 6–13. Halperin, R.O., Sesso, H.D., Ma, J., Buring, J.E., Stampfer, M.J., Gaziano, J.M., 2006. Dyslipidemia and the risk of incident hypertension in men. Hypertension 47 (1), 45–50. Jing, X., Huang, W.H., Tang, Y.J., Wang, Y.Q., Li, H., Tian, Y.Y., et al., 2015. Eucommia ulmoides Oliv. (Du-Zhong) lignans inhibit Angiotensin II-stimulated proliferation by
affecting P21, P27, and Bax expression in rat mesangial cells. Evid. Based Complement. Alternat. Med. Kao, Y.L., Donaghue, K., Chan, A., Knight, J., Silink, M., 1999. A novel polymorphism in the aldose reductase gene promoter region is strongly associated with diabetic retinopathy in adolescents with type 1 diabetes. Diabetes 48 (6), 1338–1340. Katakami, N., Kaneto, H., Takahara, M., Matsuoka, T.A., Imamura, K., Ishibashi, F., et al., 2011. Aldose reductase C-106T gene polymorphism is associated with diabetic retinopathy in Japanese patients with type 2 diabetes. Diabetes Res. Clin. Pract. 92 (3), e57–e60. Li, Z.Y., Deng, X.L., Huang, W.H., Li, L., Li, H., Jing, X., et al., 2014. Lignans from the bark of Eucommia ulmoides inhibited Ang II-stimulated extracellular matrix biosynthesis in mesangial cells. Chin. Med. 9 (1), 8. Li, Z.Y., Gu, J., Yan, J., Wang, J.J., Huang, W.H., Tan, Z.R., et al., 2013. Hypertensive cardiac remodeling effects of lignan extracts from Eucommia ulmoides Oliv. bark—a famous traditional Chinese medicine. Am J Chin Med 41 (4), 801–815. Li, L., Li, Z., Cheng, H., Yan, J., Hu, K., Wang, J., et al., 2012. Racial difference in aldose reductase C-106T genetic polymorphism and association with essential hypertension. Zhong Nan Da Xue Xue Bao Yi Xue Ban 37 (2), 156–160. Luo, Q.G., Zhang, J.Y., Cheng, W.W., Audibert, F., Luo, Z.C., 2014. Is gestational hypertension protective against perinatal mortality in twin pregnancies? PLoS One 9 (4), e94865. Maccari, R., Ottanà, R., 2015. Targeting aldose reductase for the treatment of diabetes complications and inflammatory diseases: new insights and future directions. J. Med. Chem. 58 (5), 2047–2067. Mancia, G., De Backer, G., Dominiczak, A., Cifkova, R., Fagard, R., Germano, G., et al., 2007. Guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J. Hypertens. 25 (6), 1105–1187. So, W.Y., Wang, Y., Ng, M.C., Yang, X., Ma, R.C., Lam, V., et al., 2008. Aldose reductase genotypes and cardiorenal complications: an 8-year prospective analysis of 1,074 type 2 diabetic patients. Diabetes Care 31 (11), 2148–2153. Vimaleswaran, K.S., Cavadino, A., Berry, D.J., Jorde, R., Dieffenbach, A.K.L., Alves, C., et al., 2014. Association of vitamin D status with arterial blood pressure and hypertension risk: a Mendelian randomisation study. Lancet Diabetes Endocrinol 2 (9), 719–729. Wang, K., Bohren, K.M., Gabbay, K.H., 1993. Characterization of the human aldose reductase gene promoter. J. Biol. Chem. 268, 16052–16058. Watarai, A., Nakashima, E., Hamada, Y., Watanabe, G., Naruse, K., Miwa, K., et al., 2006. Aldose reductase gene is associated with diabetic macroangiopathy in Japanese Type 2 diabetic patients. Diabet. Med. 23 (8), 894–899. Xu, M., Chen, X., Yan, L., Cheng, H., Chen, W., 2008. Association between (AC)n dinucleotide repeat polymorphism at the 5′-end of the aldose reductase gene and diabetic nephropathy: a meta-analysis. J. Mol. Endocrinol. 40 (5), 243–251. Yang, B., Millward, A., Demaine, A., 2003. Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphism of the aldose reductase gene may account for the association with diabetic microvascular complications. Biochim. Biophys. Acta 1639 (1), 1–7.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Aldose reductase C-106T polymorphism is associated with the risk of essential hypertension Yaqin Wang a,b, Min Yu a,b, Long Mo c, Zhenyu Li c, Junjie Wang a,b,c, Hong-hao Zhou a,b, Dong-Sheng Ouyang a,b,⁎ a b c
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, PR China Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, PR China Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410078, PR China
a r t i c l e
i n f o
Article history: Received 29 March 2016 Received in revised form 7 June 2016 Accepted 21 June 2016 Available online 23 June 2016 Keywords: Essential hypertension Aldose reductase Polymorphism
a b s t r a c t Aldose Reductase (AR), encoded by AKR1B1, is a member of NADPH-dependent aldo-keto reductase superfamily. The C-106T polymorphism of AKR1B1 is closely related to the diabetic complications. Our previous studies have indicated that the expression of AR was increased in spontaneously hypertensive rats, suggesting the effect of AR in hypertension. Here we investigated whether AKR1B1 C-106T polymorphism was associated with essential hypertension (EH). AKR1B1 C-106T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the direct sequencing methods. 383 healthy subjects and 383 essential hypertensive patients were recruited in this study. The polymorphism of AKR1B1 C-106T in EH and normal tensive (NT) groups was in agreement with Hardy-Weinberg equilibrium. -106T allele of AKR1B1 C-106T variants was more frequent in EH patients compared with normal tensive subjects, indicating that -106T allele was a risk factor of EH (OR = 1.841, 95%CI = 1.366–2.481). In male patients, C-106T polymorphism was associated significantly with decreased serum high density lipoprotein cholesterol and higher systolic blood pressure levels. Our results suggest that -106T allele of AKR1B1 C-106T polymorphism may be associated with increased risk for EH in Chinese Han population. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Essential hypertension (EH) is an independent risk factor for cardiovascular diseases, such as myocardial infarction and stroke. Aldose reductase (AR), a member of the aldo-keto reductase superfamily, is the first rate-limiting enzyme of the polyol pathway and catalyzes the NADPH-dependent reduction of glucose to sorbitol (Wang et al., 1993). Our previous studies showed that the expression level of AR was increased in spontaneously hypertensive rat (SHR; Gu et al., 2011; Li et al., 2013), demonstrating that AR may be involved in the development and progression of hypertension. Proinflammatory conditions and inflammation are associated with the pathophysiology of EH (González et al., 2014). AR is a redoxsensitive enzyme and acts as a key mediator in the oxidative and Abbreviations: AR, aldose reductase; BMI, body mass index; CI, confidence interval; DBP, diastolic blood pressure; EH, essential hypertension; FBG, fasting blood glucose; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NT, normal tensive; OR, odds ratio; PCR, polymerase chain reaction; PCR-RFLP, polymerase chain reactionrestriction fragment length polymorphism; SBP, systolic blood pressure; SNP, single nucleotide polymorphism; TC, total cholesterol; TG, triglyceride. ⁎ Corresponding author. E-mail address: [email protected] (D.-S. Ouyang).
http://dx.doi.org/10.1016/j.gene.2016.06.043 0378-1119/© 2016 Elsevier B.V. All rights reserved.
inflammatory signaling pathways that are associated with cardiovascular disease (Maccari et al., 2015). Previous studies have indicated that AR plays a role in causing glomerular fibrosis through mesangial cell proliferation and extracellular matrix biosynthesis (Li et al., 2014; Jing et al., 2015). Vascular fibrosis is the pathological basis for the development of hypertension. Therefore, we believe that AR plays an important role in EH by accelerating the inflammatory response and vascular fibrosis. EH is considered to be a multifactorial disease resulting from a combination of environmental and genetic factors. Increasing evidence has shown the effects of genetics on the high prevalence of EH (Luo et al., 2014; Vimaleswaran et al., 2014). Human AR is a monomeric protein (36 kDa) comprising 315 amino acids that is encoded by AKR1B1, which maps to chromosome region 7q35. The C-106T (rs759853) single nucleotide polymorphism (SNP), which is located at the promoter of the AKR1B1 gene, has been reported to be associated with the risk of diabetic complications, including nephropathy (Cui et al., 2015), retinopathy (dos Santos et al., 2006; Katakami et al., 2011) and macroangiopathy (Watarai et al., 2006). Our pre-experiment has explored the association between AKR1B1 gene polymorphisms and EH, but we failed to obtain positive results that may be due to limited sample size. Therefore, we increased sample size to investigate the association between AKR1B1 gene polymorphisms and EH in the present study.
66
Y. Wang et al. / Gene 591 (2016) 65–68
2. Material and methods 2.1. Subject This study protocol was approved the Independent Ethics Committee of the Institute of Clinical Pharmacology, Central South University (Hunan, China). Written informed consent was obtained from each participant. From February 2014 to May 2014, a total of 766 Chinese Han volunteers, including 383 patients diagnosed with EH and 383 healthy subjects, were recruited by the Physical Examination Center of the Third Xiangya Hospital, Central South University. Blood pressures were measured twice at 5-minute intervals, according to the guidelines of the European Society of Hypertension (Mancia et al., 2007). Hypertension was diagnosed in participants exhibiting a mean clinic systolic blood pressure (SBP) ≥140 mm Hg and/or a mean clinic diastolic blood pressure (DBP) ≥ 90 mm Hg, as well as in participants receiving ongoing treatment for hypertension. Subjects with a history of secondary hypertension, diabetes mellitus, primary renal disease or other serious diseases were excluded from this study. Information regarding patient sex, age, height, weight, and family history, as well as triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDL-C) and fasting blood glucose (FBG) levels, was collected.
2.2. Genotyping Blood samples were placed in EDTA-containing containers and stored at − 20 °C. Genomic DNA was extracted from peripheral whole blood using a Qiagen DNA Isolation Kit (Valencia, CA, USA). The C-106T polymorphism of AKR1B1 was identified by the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) method using the primers and conditions described by Kao et al. (1999). PCR products were digested using the restriction enzyme Bfa I and fractionated via a 3% agarose gel. The amplified PCR product was 263 bp, and two fragments of 57 bp and 206 bp were generated after restriction digestion of the sequence corresponding to the CC genotype. Samples obtained from subjects who were heterozygous for the mutant allele were cut into four fragments (57, 59, 147 and 206 bp) due to the presence of an additional cutting site, while three fragments (57, 59 and 147 bp) were generated for samples obtained from subjects who were homozygous for the mutant allele, as shown in Fig. 1. To confirm PCR-RFLP genotyping results, 5% of the DNA samples were randomly selected to perform direct sequencing (Fig. 2), and we obtained consistent results using the two detection methods.
Fig. 1. A, the PCR results of the polymorphism of AKR1B1 gene. M, DL2000 DNA marker (100, 250, 500, 750, 1000, 2000 bp). Lanes 1–10 are amplification products of samples obtained from subjects. B, the PCR-RFLP results of polymorphisms of AKR1B1 gene. M, DL2000 DNA marker (100, 250, 500, 750, 1000, 2000 bp). Lanes 1 and 2 are CT genotype (57, 59, 147 and 206 bp); Lanes 3 and 4 are TT genotype (57, 59 and 147 bp); Lanes 5 and 6 are CC genotype (57 and 206 bp).
3. Results 3.1. Characteristics of the participants The clinical and biochemical characteristics of all subjects are shown in Table 1. There were no differences in the distributions of age and
2.3. Statistical analysis SPSS 19.0 software was used for data analysis. Comparisons between the groups regarding quantitative data were performed using an independent samples t-test, and a chi-square test was used to analyse categorical data. Differences in genotype and allele frequencies between groups and deviations from Hardy-Weinberg equilibrium were calculated by chi-square test. The associations between AKR1B1 genetic variants and EH were analyzed via binary logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (95%CI), which were adjusted for age and sex. Linear regression analysis and Bonferroni correction were used to analyse the relationships between AKR1B1 genetic variants and EH-related phenotypes, which were adjusted for age and sex, while comparisons between males and females were adjusted for age only. PASS software 11.0 was used to calculate the power of the tests. A two-tailed p-value b 0.05 was considered statistically significant.
Fig. 2. Genotyping results according to direct sequencing methods. Arrows indicated the polymorphism loci.
Y. Wang et al. / Gene 591 (2016) 65–68
DBP levels in a dominant model (p = 0.024). In female patients, there was no association between the C-106T polymorphism and blood pressure or biochemical indices.
Table 1 The clinical and biochemical characteristics of EH and NT groups. Variables Gender Male (%) Female (%) Age (years) Family history (%) BMI (kg/m2) FBG (mmol/l) TG (mmol/l) TC (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) SBP (mm Hg) DBP (mm Hg) a
NT group (n = 383)
EH group (n = 383)
192(50.1) 191(49.9) 55 ± 11 23 (6.0) 23.7 ± 3.0 5.1 ± 0.5 1.5 ± 1.5 5.2 ± 1.0 1.6 ± 0.4 2.9 ± 0.8 122 ± 13 76 ± 10
193(50.4) 190(49.6) 56 ± 8 74 (25.0) 25.1 ± 3.0 6.2 ± 2.3 1.7 ± 1.3 4.4 ± 1.1 1.2 ± 0.4 2.5 ± 0.8 152 ± 18 96 ± 12
67
p 0.942
0.128 b0.001a b0.001a b0.001a 0.125 b0.001a b0.001a b0.001a b0.001a b0.001a
p b 0.001 compared with NT group.
gender between the EH and NT groups. Compared with the NT subjects, the EH patients showed significantly higher body mass indices (BMIs) and rates of hypertensive family history, as well as significantly higher FBG, TC, HDL-C and LDL-C levels (p b 0.001). 3.2. Distribution of genotype The genotype distributions of AKR1B1 C-106T were in agreement with Hardy-Weinberg equilibrium in the EH and NT groups (EH: χ2 = 0.205, p = 0.651; NT: χ2 = 1.392, p = 0.238). Genotype distributions and allele frequencies were significantly different between the EH and NT groups, as shown in Table 2 (χ2 = 16.071, p1 = 0.000), and the -106T allele was more frequent in EH patients (χ2 = 17.034, p1 = 0.000). In addition, the -106T allele and CT + TT genotype were associated with an increased risk of EH (OR = 1.841, 95%CI = 1.366–2.481, p2 = 0.000; OR = 1.908, 95%CI = 1.367–2.663, p2 = 0.000). 3.3. Association between C-106T polymorphism of AKR1B1 and blood pressure, biochemical indices To further evaluate the association between the AKR1B1 C-106T polymorphism and EH, we performed relative analyses of the associations between the C-106T polymorphism and blood pressure, as well as the associations between the C-106T polymorphism and biochemical indices, including TC, TG, HDL-C, and LDL-C levels (Table 3). In EH patients, the C-106T polymorphism was associated with decreased serum HDL-C levels in a recessive model (p = 0.009). In male patients, the C-106T polymorphism was also found to be associated with decreased serum HDL-C levels in a recessive model (p = 0.024) and higher
4. Discussion In the present study, we found that the T allele of the AKR1B1 C-106T polymorphism was associated with an increased risk of EH. Our study provides new evidence supporting the relationship between inflammation and EH, as well as a new explanation regarding the pathogenesis of EH and a new target for the prevention of EH. The effect of the AKR1B1 C-106T polymorphism on the expression of AR has been studied by other researchers. Watarai et al. (2006) reported that erythrocyte AR protein content was increased in patients with the CT and TT genotypes compared with CC carriers, which was supported by our research. However, a functional reporter gene assay reported by Yang et al. (2003) indicated that a construct containing the C allele showed higher promoter activity than a construct containing the T allele in transfected HepG2 cells. Further studies are required to clarify the effects of the C-106T polymorphism on the expression of AR. In recent years, many studies have reported the associations between polymorphisms of AKR1B1 and diseases, especially diabetes complications, but the results of these studies are ambiguous. Some verified that the T allele was a risk factor for diabetes complications (So et al., 2008; Watarai et al., 2006), while others obtained contrasting results (dos Santos et al., 2006; Katakami et al., 2011). The discrepancies among those studies may be due to differences in participant ethnic, sampling, and inclusion and exclusion criteria, as well as experimental bias. Our pre-experiment, which included 148 EH volunteers and 137 NT volunteers, failed to obtain positive results (power = 0.175; Li et al., 2012). In the present study, we enrolled 383 EH and 383 NT volunteers, and the power to detect associations for the C-106T polymorphism at the 5% significance level was sufficient (power N 0.800), indicating that the sample size was large enough to obtain reliable results. HDL-C has been linked to reductions in EH events (Halperin et al., 2006). In the present study, we found that the C-106T polymorphism was associated with decreased HDL-C levels in EH patients, indicating that -106T carriers may have lower HDL-C levels and thus increased susceptibility to EH. However, we failed to observe an association between HDL-C levels and the C-106T polymorphism in females. Further studies are needed to address this issue. There were some limitations to this study. First, only one SNP of AKR1B1 was studied. An (AC)n dinucleotide repeat polymorphism in the promoter region is another common SNP of AKR1B1 (Xu et al., 2008). Therefore, further research is needed to investigate the effects of the above two SNPs on EH. Second, only a Han Chinese population
Table 2 Distribution of AKR1B1 C-106 T in EH and NT groups. NT group (n = 383) n (%)
EH group (n = 383) n (%)
χ2
p1
OR (95%CI)
p2
Genotype CC CT TT
319 (83.3) 59 (15.4) 5 (1.3)
262 (68.4) 108 (28.2) 13 (3.4)
16.071a
0.000
1 1.830 (1.336–2.427) 1.835 (1.082–3.110)
0.001b 0.024
Allele C T
687 (89.7) 79 (10.3)
631 (82.4) 135 (17.6)
17.034
1 1.841 (1.366–2.481)
0.000
319 (83.3) 74 (16.7) 378 (98.7) 5 (1.3)
262 (68.4) 121 (31.6) 370 (96.6) 13 (3.4)
15.197
0.000
3.641
0.056
Model Dominant Recessive a b
CC CT + TT CC + CT TT
p values are obtained from χ2 test. p values are obtained from binary logistic analysis.
0.000
1 1.908 (1.367–2.663) 1 1.610 (0.956–2.712)
0.000 0.074
68
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Table 3 Results of association between AKR1B1 C-106T and blood pressure, biochemical indices in EH patients. Groups
All
Males
Females
Variables
SBP DBP TC TG HDL-C LDL-C SBP DBP TC TG HDL-C LDL-C SBP DBP TC TG HDL-C LDL-C
Dominant model
Recessive model
β (95%CI)
p
β (95%CI)
p
−0.029 (−4.897 to 2.706) 0.077 (−0.619 to 4.639) 0.027 (−0.366 to 0.495) −0.105 (−0.546 to 0.492) −0.071 (0.198 to 0.086) 0.086 (−0.170 to 0.485) 0.024 (−4.744 to 6.681) 0.162 (0.589 to 8.306) 0.038 (−0.451 to 0.622) 0.149 (−0.212 to 0.953) −0.193 (−0.305 to 0.028) 0.000 (−0.337 to 0.338) −0.110 (−8.937 to 1.165) −0.014 (−3.900 to 3.229) −0.054 (−0.898 to 0.617) −0.196 (−1.678 to 0.329) 0.122 (−0.149 to 0.364) 0.088 (−0.455 to 0.860)
0.571 0.134 0.768 0.917 0.434 0.343 0.743 0.024a 0.751 0.209 0.101 0.997 0.131 0.853 0.711 0.184 0.404 0.540
0.052 (−2.303 to 7.391) −0.071 (−5.727 to 0.986) −0.104 (−0.789 to 0.202) −0.082 (−0.877 to 0.319) −0.229 (−0.376 to −0.055) 0.047 (−0.278 to 0.480) 0.031 (−3.749 to 5.799) 0.111 (−0.715 to 5.765) −0.002 (−0.451 to 0.443) 0.096 (−0.290 to 0.687) −0.263 (−0.293 to −0.021) 0.017 (−0.261 to 0.301) 0.088 (−2.654 to 11.637) −0.089 (−8.128 to 1.897) −0.134 (−1.228 to 0.406) −0.084 (−1.422 to 0.769) −0.164 (−0.446 to 0.108) 0.024 (−0.653 to 0.782)
0.303 0.166 0.243 0.358 0.009a 0.599 0.672 0.126 0.985 0.420 0.024a 0.890 0.217 0.222 0.317 0.544 0.227 0.858
a p b 0.025 was considered to be significantly different. Because two independent hypothesis tests were performed, and the statistical significance level should be 1/2 times based on Bonferroni correction.
was included in our study; therefore, it is unclear if our results are applicable to other populations. In conclusion, our results suggest that the -106T allele of the AKR1B1 C-106T polymorphism may be associated with an increased risk of EH. Further investigation is necessary to explore the underlying mechanisms of the relationship between AR and EH. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements This work was supported by National Development of Key Novel Drugs for Special Projects of China (2012ZX09303014001) and the Special Fund for scientific Research in the Public Interest (201507004-4-2). We also acknowledge the Scientific Research Fund of Hunan Provincial Education Department (12B117), Natural Science Foundation of Hunan (2016JJ6142) and Postgraduate Innovation Project of Central South University (2016zzts127). References Cui, W., Du, B., Cui, Y., Kong, L., Wu, H., Wang, Y., et al., 2015. Is rs759853 polymorphism in promoter of aldose reductase gene a risk factor for diabetic nephropathy? A metaanalysis. Eur. J. Med. Res. 20, 14. dos Santos, K.G., Canani, L.H., Gross, J.L., Tschiedel, B., Souto, K.E., Roisenberg, I., 2006. The -106CC genotype of the aldose reductase gene is associated with an increased risk of proliferative diabetic retinopathy in Caucasian-Brazilians with type 2 diabetes. Mol. Genet. Metab. 88 (3), 280–284. González, J., Valls, N., Brito, R., Rodrigo, R., 2014. Essential hypertension and oxidative stress: new insights. World J. Cardiol. 6 (6), 353–366. Gu, J., Wang, J.J., Yan, J., Cui, C.F., Wu, W.H., Li, L., et al., 2011. Effects of lignans extracted from Eucommia ulmoides and aldose reductase inhibitor epalrestat on hypertensive vascular remodeling. J. Ethnopharmacol. 133 (1), 6–13. Halperin, R.O., Sesso, H.D., Ma, J., Buring, J.E., Stampfer, M.J., Gaziano, J.M., 2006. Dyslipidemia and the risk of incident hypertension in men. Hypertension 47 (1), 45–50. Jing, X., Huang, W.H., Tang, Y.J., Wang, Y.Q., Li, H., Tian, Y.Y., et al., 2015. Eucommia ulmoides Oliv. (Du-Zhong) lignans inhibit Angiotensin II-stimulated proliferation by
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