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A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with chronic kidney disease
Atherosclerosis 231 (2013) 141e144
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with chronic kidney disease Giorgio Sesti a, *, Gaia Chiara Mannino a, Carlo De Lorenzo a, Annalisa Greco a, Angela Sciacqua a, Maria A. Marini b, Francesco Andreozzi a, Francesco Perticone a a b
Department of Medical and Surgical Sciences, University Magna Græcia of Catanzaro, Via Europa-Località Germaneto, Catanzaro 88100, Italy Department of Systems Medicine, University of Rome-Tor Vergata, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2013 Received in revised form 9 August 2013 Accepted 31 August 2013 Available online 14 September 2013
Objective: Studies reported a relationship between elevated asymmetric dimethylarginine (ADMA) concentrations and adverse renal outcomes. There is evidence that the rs9267551 variant in the DDAH2 gene has a functional impact with the C allele having a higher transcriptional activity resulting in increased expression of DDAH2 in endothelial cells and lower plasma ADMA levels in C allele carriers. Methods: To address whether this variant is associated with chronic kidney disease (CDK), 2852 White European were studied. CKD was defined as estimated glomerular filtration rate (eGFR) <60 ml/min/ 1.73 m2. Results: The proportion of subjects with CKD was significantly lower in C allele carriers than in GG genotype carriers (OR 0.49, 95%CI 0.25e0.97; P ¼ 0.03). In a logistic regression model adjusted for age, gender, BMI, blood pressure, total and HDL cholesterol, triglyceride, and fasting plasma glucose, C allele carriers have a lower risk of CKD compared with GG genotype carriers (OR 0.38, 95%CI 0.18e0.78; P ¼ 0.008). This association was maintained after addition to the logistic regression model of other confounders including glucose tolerance status, presence of dyslipidemia, anti-hypertensive and antidiabetic drugs (OR 0.35, 95%CI 0.15e0.80; P ¼ 0.01). Conclusion: The rs9267551 functional variant of the DDAH2 gene is associated with CKD with carriers of the C allele having a lower risk of renal dysfunction independently from several confounders. Because ADMA predicted progression of renal disease, it is possible that, in GG carriers, ADMA may accumulate at the renal level causing endothelial dysfunction as a consequence of reduced nitric oxide availability and potentiating micro-vascular damage. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Asymmetric dimethylarginine Chronic kidney disease Dimethylarginine dimethylaminohydrolase
Chronic kidney disease (CKD) is a global health problem due to its adverse outcomes, including cardiovascular events and all-cause mortality [1,2]. Decreased estimated glomerular filtration rate (eGFR), the primary measure used to define CKD (eGFR <60 ml/ min/1.73 m2), and cardiovascular disease share common atherosclerotic risk factors including hyperglycemia, hypertension, dyslipidemia, smoking, overweight/obesity, and insulin resistance [3e 10]. Endothelial dysfunction is considered an early alteration in the development and progression of atherosclerosis, and is already present at early stages of renal dysfunction [11e13]. In addition, results of longitudinal studies have demonstrated that endothelial dysfunction is associated with a decline in eGFR independently of * Corresponding author. Tel.: þ39 (0) 961 3647204; fax: þ39 (0) 961 3647192. E-mail address: [email protected] (G. Sesti). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.08.041
traditional cardiovascular risk factors and antihypertensive treatment [14]. Endothelial dysfunction is a wide term that entails diminished production and/or availability of nitric oxide (NO). Inhibition of NO synthesis by endogenous inhibitors of the endothelial NO synthase (eNOS) may have a main role in inducing endothelial dysfunction [15e17]. Although initial studies found increased plasma ADMA concentrations in patients with end-stage renal disease (ESRD) [18,19], subsequent studies have reported a relationship between elevated ADMA concentrations and adverse renal outcomes over time, which could imply a causal role for elevated ADMA levels in the progressive decline of kidney function [20,21]. ADMA is generated by proteins methylation by the enzyme protein arginine N-methyltransferases (PRMTs), and is degraded by the dimethylarginine dimethylaminohydrolase (DDAH) [22,23]. There are two isoforms
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of DDAH, with DDAH1 found in tissues expressing neuronal NOS, and DDAH2 highly expressed in the endothelium [24]. We have recently shown that the rs9267551 variant in the DDAH2 gene has a functional impact with the C allele having a higher transcriptional activity resulting in increased expression of DDAH2 in primary human endothelial cells naturally carrying the C allele. In addition, subjects carrying the C allele exhibit lower levels of circulating ADMA, and higher insulin sensitivity [25]. Interestingly, in genomewide association studies (GWAS) carried out by the Diabetes Genetics Replication And Meta-analysis (DIAGRAMþ), the rs9267551 variant in the DDAH2 gene was nominally associated with type 2 diabetes (P ¼ 3 105) with the G diabetogenic risk allele conferring an odds ratio (OR) of 1.12 (95%CI 1.06e1.19) (Andrew Morris and Mark McCarthy personal communication for the DIAGRAMþ). In view of the important role of ADMA in regulating endotheliumdependent vasodilation and, thereby, renal function, we hypothesized that the rs9267551 polymorphism in the DDAH2 gene may be associated with CKD. To this aim, we investigated the association of rs9267551 polymorphism with CKD in a cohort of subjects of European ancestry. 1. Materials and methods
The protocol was approved by the Institutional Ethics Committees and informed written consent was obtained from participants. All the investigations were performed in accordance with the principles of the Declaration of Helsinki. 1.2. Analytical determinations Serum creatinine was measured in the routine laboratory by an automated technique based on a Creatinine Jaffè compensated Method for serum and plasma (Roche Diagnostics) method implemented in an autoanalyzer. The laboratory references ranges for this assay are 0.7e1.2 mg/dl for males, 0.5e1.0 mg/dl for females. Glucose, triglyceride, total and HDL-C concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). 1.3. Calculations Estimated glomerular filtration rate (eGFR) was calculated by using the CKD-EPI equation [28]: eGFR ¼ 141 min(Scr/k, 1)a max(Scr/k, 1)1.209 0.993Age 1.018 [if female], where Scr is serum creatinine, k is 0.7 for females and 0.9 for males, a is 0.329 for females and 0.411 for males, min indicates the minimum of Scr/k or 1, and max indicates the maximum of (Scr/k or 1).
1.1. Study subjects 1.4. DNA analysis The study group consisted of 2852 adult individuals of European ancestry consecutively recruited at the Department of Systems Medicine of the University of Rome-Tor Vergata and at the Department of Medical and Surgical Sciences of the University “Magna Graecia” of Catanzaro [26]. Recruited subjects participated to a campaign for assessment of cardio-metabolic risk factors. Recruitment mechanisms include word-of-mouth, fliers, and newspaper advertisements. The inclusion criteria were: age 23 years, and presence of one or more cardio-metabolic risk factors including elevated fasting glucose levels, hypertension, dyslipidemia, overweight/obesity, and family history for diabetes. Subjects were excluded if they had end-stage renal disease (ESRD), chronic gastrointestinal diseases, chronic pancreatitis, history of any malignant disease, history of alcohol or drug abuse, positivity for antibodies to hepatitis C virus (HCV) or hepatitis B surface antigen (HBsAg), and hepatic failure. After 12-h overnight fasting, subjects underwent anthropometrical evaluation and a venous blood sample was drawn for laboratory determinations. Body mass index (BMI) was calculated as body weight (kilograms) divided by the square of height (meters). Waist (at the midpoint between the lateral iliac crest and lowest rib) and hip circumference (at the level of the trochanter major) were measured to the nearest 0.5 cm. Readings of clinic blood pressure (BP) were obtained in the left arm of the supine patients, after 5 min of quiet rest, with a sphygmomanometer. Normal fasting glucose (NFG) was defined as fasting plasma glucose <100 mg/dl, impaired fasting glucose (IFG) as fasting plasma glucose 100 and <126 mg/dl, and type 2 diabetes mellitus (T2DM) as fasting plasma glucose 126 mg/dl or current treatment with anti-diabetic drugs. Atherogenic dyslipidemia and hypertension were defined according to the criteria utilized for definition of the metabolic syndrome released in 2009 by a joint statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity [27]. Thus, atherogenic dyslipidemia was defined as triglyceride 150 mg/dl, HDL <40 mg/dl in men and <50 mg/dl in women or drug treatment. Hypertension was defined as systolic blood pressure 130 and/or diastolic 85 mmHg or antihypertensive drug treatment in the subjects with a history of hypertension.
DNA was isolated from whole blood using commercial DNA isolation kit (Promega, Madison, WI). Screening of the rs9267551 polymorphism was performed using a TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA). TaqMan genotyping reaction was amplified on a GeneAmp PCR system 2700 and fluorescence was detected using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA). Genotyping quality was tested by including 3 HapMap samples in each 96-well plate. The agreement rate with the HapMap database genotypes was >99%. 1.5. Statistical analysis The results for continuous variables are given as means SD. Unpaired Student’s t test was used to compare differences of continuous variables between two groups, and the c2-test for noncontinuous variables. The HardyeWeinberg equilibrium between the genotypes was evaluated by c2 test. There was no deviation from the Hardy Weinberg equilibrium in genotype distributions (P ¼ 0.003) using the same threshold of statistical significance utilized by the DIAGRAM þ investigators in the replication studies of the GWAS (defined as a P > 0.001) [29,30]. A multivariate logistic regression analysis was used to determine the association between the rs9267551 polymorphism and CKD. The case-control study has 80% power to detect an association conferring a 0.60-fold reduced risk of CKD for protective C allele according to a dominant model. All tests were two-sided, and a P value <0.05 was considered statistically significant. All analyses were performed using the SPSS software program Version 16.0 for Windows. 2. Results Clinical characteristics of study subjects according to the rs9267551 polymorphism are shown in Table 1. Because of the small number of CC individuals (n ¼ 13) and the a priori hypothesis based on the dominant effect observed in our previous functional studies [25], GC and CC individuals were pooled and analyzed together as C carriers, according to a dominant genetic model. The rs9267551 polymorphism did not show any significant association
G. Sesti et al. / Atherosclerosis 231 (2013) 141e144
143
Table 1 Clinical features of 2852 study subjects according to the rs9267551 polymorphism of DDAH2. Variables
GG
GC
CC
P (G/G vs. G/C þ C/C)
n (male/female) Age (yr) BMI (kg/m2) Waist circumference (cm) SBP (mmHg) DBP (mmHg) Fasting glucose (mg/dl) Total cholesterol (mg/dl) HDL (mg/dl) Triglycerides (mg/dl) Creatinine (mg/dl) eGFR (ml/min/1.73 m2) eGFR <60 ml/min/1.73 m2 [no. (%)] Glucose tolerance status (NFG, IFG, T2DM) [no. (%)]
2601 (1316/1285) 52 13 30.0 6.1 100 14 135 18 82 11 111 47 200 40 49 14 137 80 0.85 0.28 98 28 183 (7.0%) 1377/471/753 (52.9/18.1/28.9) 45.0% 2043 (78.5) 1324 (50.9) 1437 (55.2)
238 (127/111) 54 14 29.2 5.2 99 11 135 18 82 10 115 51 201 40 49 14 141 79 0.83 0.21 99 31 9 (3.7%) 126/37/75 (52.9/15.5/31.5) 39.4% 188 (78.9) 118 (49.5) 135 (56.7)
13 (3/10) 52 11 33.8 6.8 107 15 138 19 83 11 110 53 206 25 52 16 121 44 0.75 0.16 102 24 0 9/1/3 (69.2/7.6/23.0) 44.4% 11 (84.6) 7 (53.8) 7 (53.8)
0.72 0.15 0.16 0.21 0.53 0.75 0.25 0.72 0.77 0.55 0.38 0.88 0.03 0.46
208 (27.6) 545 (72.4)
14 (18.7) 61 (81.3)
3 (100) 0
Family history of type 2 diabetes (%) Hypertension [no. (%)] Anti-hypertensive treatment [no. (%)] Dyslipidemia [no. (%)] Antidiabetic treatment [no. (% of diabetic subjects)] Diet Oral hypoglycemic agents
0.09 0.99 0.88 0.86 0.2
Data are means SD. Differences of continuous variables between two groups were compared using unpaired Student’s t. Categorical variables were compared by c2 test. BMI: body mass index; SBP ¼ systolic blood pressure; DBP ¼ diastolic blood pressure; eGFR ¼ estimated glomerular filtration rate; NFG ¼ normal fasting glucose, IFG ¼ impaired fasting glucose; T2DM ¼ type 2 diabetes mellitus. Dyslipidemia: triglyceride 150 mg/dl, HDL <40 mg/dl in men and <50 mg/dl in women or drug treatment; Hypertension: systolic blood pressure 130 and/or diastolic 85 mmHg or antihypertensive drug treatment.
with age, gender, adiposity, blood pressure, fasting glucose levels, lipid profile, serum creatinine levels and eGFR (Table 1). No differences between genotypes were observed in the proportion of subjects having glucose abnormalities, hypertension, dyslipidemia or family history of type 2 diabetes (Table 1). Likewise, no differences between genotypes were observed in the proportion of subjects on treatment with anti-hypertensive or antidiabetic drugs (Table 1). The proportion of subjects with CKD was significantly lower in subjects carrying the C allele as compared with carriers of the GG genotype (OR 0.49, 95%CI 0.25e0.97; P ¼ 0.03). In a logistic regression model adjusted for age, and gender, carriers of the C allele have a lower risk to have CKD as compared with subjects carrying the GG genotype (OR 0.38, 95%CI 0.18e0.78; P ¼ 0.008). This association was maintained when subjects carrying the CC genotype were excluded (OR 0.42, 95%CI 0.21e0.86; P ¼ 0.01). The association was also maintained when BMI, blood pressure, total and HDL cholesterol, triglyceride, and fasting plasma glucose levels were additionally included in the model (OR 0.34, 95%CI 0.14e0.83; P ¼ 0.01). This association remained significant also after addition to the logistic regression model of other confounders including glucose tolerance status (NFG, IFG, T2DM), presence of dyslipidemia, anti-hypertensive and antidiabetic drugs in addition to age, gender and BMI (OR 0.35, 95%CI 0.15e0.80; P ¼ 0.01). 3. Discussion There is evidence showing that plasma ADMA levels are inversely related to renal function, as measured by the eGFR [20,21]. Moreover, it has been shown that ADMA is an independent predictor of progression to end stage renal disease (ESRD) and death in patients with CKD even after adjustment for traditional and nontraditional risk factors [20,21]. DDAH, the enzyme that hydrolyzes ADMA, has an important role in regulation of its levels since it has been estimated that approximately 83% of ADMA generated daily is metabolized by DDAH, whereas only a negligible amount is excreted unchanged by the kidneys [31]. We have recently shown that the rs9267551 polymorphism in the DDAH2 gene has functional impact with the C allele being associated with
enhanced expression of DDAH2 in primary human endothelial cells and lower plasma ADMA levels [25]. In the light of the important role of ADMA in regulating renal function, we investigated the association of rs9267551 polymorphism with CKD in a cohort of subjects of European ancestry. We found that the rs9267551 polymorphism is significantly associated with CKD with carriers of the C allele having a reduced risk of renal dysfunction as compared with in subjects carrying the GG genotype. This association remained significant even after adjustments for a series of cardiovascular risk factors including blood pressure, lipids, and glucose tolerance status as well as anti-hypertensive and antidiabetic treatments. Since it has been reported that ADMA is inversely related to eGFR and that it predicted progression of renal disease to ESRD, it is possible that in subjects carrying the GG genotype ADMA may accumulate at the renal level causing endothelial dysfunction as a consequence of reduced NO availability and potentiating micro-vascular damage. In support of this hypothesis, it has been shown that ADMA has pronounced renal hemodynamic effects [32]. Continuous ADMA infusion caused a decrease in renal perfusion and an increase in reno-vascular resistance at doses that failed to affect blood pressure, renin-angiotensin and sympathetic system [32]. The relatively large sample size, the inclusion of both sexes, the homogeneity of the sample with detailed characterization of cardio-metabolic variables, and the exclusion of confounding conditions such as chronic gastrointestinal diseases, and history of any malignant disease are major strengths of the present investigation. Nevertheless, the study has some limitations. A first limitation relates to the fact that serum creatinine levels and estimated GFR were used to identify kidney function. Although gold standard methods to measure GFR (isotope clearance measurements) may offer a more sensitive estimate of kidney function, they are expensive and time-consuming techniques which are not feasible in large-scale studies. Moreover, the number of subjects with CKD observed in the study was limited, yet our sample size was powerful enough for establishing an independent association between the rs9267551 polymorphism in the DDAH2 gene and CKD. Additionally, this study is an observational investigation based on
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outpatients observed at a referral university hospital, representing individuals at risk for cardio-metabolic disease, and, therefore, may not be extendible to the general population. Furthermore, the present findings are only based on subjects of European ancestry, and should not be generalized to other ethnic populations. Finally, because of the cross-sectional design of the study, the present results reveal only an association with prevalent and not incident kidney failure. Clearly, prospective studies carried out in different ethnic populations may provide a response to these questions. Disclosure statement The authors have nothing to disclose. References [1] Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 2004;351:1296e305. [2] Perticone F, Sciacqua A, Maio R, et al. Renal function predicts cardiovascular outcomes in southern Italian postmenopausal women. Eur J Cardiovasc Prev Rehabil 2009;16:481e6. [3] Fox CS, Larson MG, Leip EP, Culleton B, Wilson PW, Levy D. Predictors of newonset kidney disease in a community-based population. J Am Med Assoc 2004;291:844e50. [4] Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. Am J Kidney Dis 2003;41:1e12. [5] Chen J, Muntner P, Hamm LL, et al. The metabolic syndrome and chronic kidney disease in U.S. adults. Ann Intern Med 2004;140:167e74. [6] Nerpin E, Riserus U, Ingelsson E, et al. Insulin sensitivity measured with euglycemic clamp is independently associated with glomerular filtration rate in a community-based cohort. Diabetes Care 2008;31:1550e5. [7] Zoppini G, Targher G, Chonchol M, Perrone F, Lippi G, Muggeo M. Higher HDL cholesterol levels are associated with a lower incidence of chronic kidney disease in patients with type 2 diabetes. Nutr Metab Cardiovasc Dis 2009;19:580e6. [8] Sarafidis PA, Ruilope LM. Insulin resistance, hyperinsulinemia, and renal injury: mechanisms and implications. Am J Nephrol 2006;26:232e44. [9] Succurro E, Arturi F, Lugarà M, et al. One-hour postload plasma glucose levels are associated with kidney dysfunction. Clin J Am Soc Nephrol 2010;5:1922e7. [10] Sesti G, Succurro E, Arturi F, et al. IGF-1 levels link estimated glomerular filtration rate to insulin resistance in obesity: a study in obese, but metabolically healthy, subjects and obese, insulin-resistant subjects. Nutr Metab Cardiovasc Dis 2011;21:485e91. [11] Bolton CH, Downs LG, Victory JGG, et al. Endothelial dysfunction in chronic renal failure: roles of lipoprotein oxidation and pro-inflammatory cytokines. Nephrol Dial Transplant 2001;16:1189e97. [12] Stam F, van Guldener C, Schalkwijk CG, ter Wee PM, Donker AJM, Stehouwer CDA. Impaired renal function is associated with markers of endothelial dysfunction and increased inflammatory activity. Nephrol Dial Transplant 2003;18:892e8. [13] Perticone F, Maio F, Tripepi G, Zoccali C. Endothelial dysfunction and mild renal insufficiency in essential hypertension. Circulation 2004;110:821e5. [14] Perticone F, Maio R, Perticone M, et al. Endothelial dysfunction and subsequent decline in glomerular filtration rate in hypertensive patients. Circulation 2010;122:379e84.
[15] Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999;43:542e8. [16] Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 2004;24:1023e30. [17] Perticone F, Sciacqua A, Maio R, et al. Asymmetric dimethylarginine, L-arginine, and endothelial dysfunction in essential hypertension. J Am Coll Cardiol 2005;46:518e23. [18] Vallance P, Leone A, Calver A, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572e5. [19] Zoccali C, Bode-Böger SM, Mallamaci F, et al. Plasma concentration of asymmetricaldimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 2001;358:2113e7. [20] Ravani P, Tripepi G, Malberti F, et al. Asymmetrical dimethylarginine predicts progression to dialysis and death in patients with chronic kidney disease: a competing risks modeling approach. J Am Soc Nephrol 2005;16:2449e55. [21] Fliser D, Kronenberg F, Kielstein JT, et al. Asymmetric dimethylarginine and progression of chronic kidney disease: the mild to moderate kidney disease study. J Am Soc Nephrol 2005;16:2456e61. [22] Ogawa T, Kimono M, Watanabe H, Sasaoka K. Metabolism of NG, NGdimethylarginine and NG, N0 G-dimethylarginine in rats. Arch Biochem Biophys 1987;252:526e37. [23] Ogawa T, Kimono M, Watanabe H, Sasaoka K. Purification and properties of a new enzyme NG, NG-dimethylarginine dimethylaminohydrolase from rat kidney. J Biol Chem 1989;264:10205e9. [24] Leiper JM, Santa Maria J, Chubb A, et al. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distribution and homology to microbial arginine deiminases. Biochem J 1999;343:209e14. [25] Andreozzi F, Presta I, Mannino GC, et al. A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with insulin sensitivity. PLoS One 2012;7:e36224. [26] Scarpelli D, Cardellini M, Andreozzi F, et al. Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes mellitus in Caucasian Italian subjects. Diabetes 2006;55: 1529e33. [27] Alberti KG, Eckel RH, Grundy SM, et al., International Diabetes Federation Task Force on Epidemiology and Prevention., National Heart, Lung, and Blood Institute., American Heart Association., World Heart Federation., International Atherosclerosis Society., International Association for the Study of Obesity. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009;120:1640e5. [28] Levey AS, Stevens LA, Schmid CH, et al., CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration). A new equation to estimate glomerular filtration rate. Ann Intern Med 2009;150:604e12. [29] Voight BF, Scott LJ, Steinthorsdottir V, et al., MAGIC investigators., GIANT Consortium. Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet 2010;42:579e89. [30] Dupuis J, Langenberg C, Prokopenko I, et al. New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat Genet 2010;42:105e16. [31] Achan V, Broadhead M, Malaki M, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23:1455e9. [32] Kielstein JT, Simmel S, Bode-Boger SM, et al. Subpressor dose asymmetric dimethylarginine modulates renal function in humans through nitric oxide synthase inhibition. Kidney Blood Press Res 2004;27:143e7.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with chronic kidney disease Giorgio Sesti a, *, Gaia Chiara Mannino a, Carlo De Lorenzo a, Annalisa Greco a, Angela Sciacqua a, Maria A. Marini b, Francesco Andreozzi a, Francesco Perticone a a b
Department of Medical and Surgical Sciences, University Magna Græcia of Catanzaro, Via Europa-Località Germaneto, Catanzaro 88100, Italy Department of Systems Medicine, University of Rome-Tor Vergata, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2013 Received in revised form 9 August 2013 Accepted 31 August 2013 Available online 14 September 2013
Objective: Studies reported a relationship between elevated asymmetric dimethylarginine (ADMA) concentrations and adverse renal outcomes. There is evidence that the rs9267551 variant in the DDAH2 gene has a functional impact with the C allele having a higher transcriptional activity resulting in increased expression of DDAH2 in endothelial cells and lower plasma ADMA levels in C allele carriers. Methods: To address whether this variant is associated with chronic kidney disease (CDK), 2852 White European were studied. CKD was defined as estimated glomerular filtration rate (eGFR) <60 ml/min/ 1.73 m2. Results: The proportion of subjects with CKD was significantly lower in C allele carriers than in GG genotype carriers (OR 0.49, 95%CI 0.25e0.97; P ¼ 0.03). In a logistic regression model adjusted for age, gender, BMI, blood pressure, total and HDL cholesterol, triglyceride, and fasting plasma glucose, C allele carriers have a lower risk of CKD compared with GG genotype carriers (OR 0.38, 95%CI 0.18e0.78; P ¼ 0.008). This association was maintained after addition to the logistic regression model of other confounders including glucose tolerance status, presence of dyslipidemia, anti-hypertensive and antidiabetic drugs (OR 0.35, 95%CI 0.15e0.80; P ¼ 0.01). Conclusion: The rs9267551 functional variant of the DDAH2 gene is associated with CKD with carriers of the C allele having a lower risk of renal dysfunction independently from several confounders. Because ADMA predicted progression of renal disease, it is possible that, in GG carriers, ADMA may accumulate at the renal level causing endothelial dysfunction as a consequence of reduced nitric oxide availability and potentiating micro-vascular damage. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Asymmetric dimethylarginine Chronic kidney disease Dimethylarginine dimethylaminohydrolase
Chronic kidney disease (CKD) is a global health problem due to its adverse outcomes, including cardiovascular events and all-cause mortality [1,2]. Decreased estimated glomerular filtration rate (eGFR), the primary measure used to define CKD (eGFR <60 ml/ min/1.73 m2), and cardiovascular disease share common atherosclerotic risk factors including hyperglycemia, hypertension, dyslipidemia, smoking, overweight/obesity, and insulin resistance [3e 10]. Endothelial dysfunction is considered an early alteration in the development and progression of atherosclerosis, and is already present at early stages of renal dysfunction [11e13]. In addition, results of longitudinal studies have demonstrated that endothelial dysfunction is associated with a decline in eGFR independently of * Corresponding author. Tel.: þ39 (0) 961 3647204; fax: þ39 (0) 961 3647192. E-mail address: [email protected] (G. Sesti). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.08.041
traditional cardiovascular risk factors and antihypertensive treatment [14]. Endothelial dysfunction is a wide term that entails diminished production and/or availability of nitric oxide (NO). Inhibition of NO synthesis by endogenous inhibitors of the endothelial NO synthase (eNOS) may have a main role in inducing endothelial dysfunction [15e17]. Although initial studies found increased plasma ADMA concentrations in patients with end-stage renal disease (ESRD) [18,19], subsequent studies have reported a relationship between elevated ADMA concentrations and adverse renal outcomes over time, which could imply a causal role for elevated ADMA levels in the progressive decline of kidney function [20,21]. ADMA is generated by proteins methylation by the enzyme protein arginine N-methyltransferases (PRMTs), and is degraded by the dimethylarginine dimethylaminohydrolase (DDAH) [22,23]. There are two isoforms
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of DDAH, with DDAH1 found in tissues expressing neuronal NOS, and DDAH2 highly expressed in the endothelium [24]. We have recently shown that the rs9267551 variant in the DDAH2 gene has a functional impact with the C allele having a higher transcriptional activity resulting in increased expression of DDAH2 in primary human endothelial cells naturally carrying the C allele. In addition, subjects carrying the C allele exhibit lower levels of circulating ADMA, and higher insulin sensitivity [25]. Interestingly, in genomewide association studies (GWAS) carried out by the Diabetes Genetics Replication And Meta-analysis (DIAGRAMþ), the rs9267551 variant in the DDAH2 gene was nominally associated with type 2 diabetes (P ¼ 3 105) with the G diabetogenic risk allele conferring an odds ratio (OR) of 1.12 (95%CI 1.06e1.19) (Andrew Morris and Mark McCarthy personal communication for the DIAGRAMþ). In view of the important role of ADMA in regulating endotheliumdependent vasodilation and, thereby, renal function, we hypothesized that the rs9267551 polymorphism in the DDAH2 gene may be associated with CKD. To this aim, we investigated the association of rs9267551 polymorphism with CKD in a cohort of subjects of European ancestry. 1. Materials and methods
The protocol was approved by the Institutional Ethics Committees and informed written consent was obtained from participants. All the investigations were performed in accordance with the principles of the Declaration of Helsinki. 1.2. Analytical determinations Serum creatinine was measured in the routine laboratory by an automated technique based on a Creatinine Jaffè compensated Method for serum and plasma (Roche Diagnostics) method implemented in an autoanalyzer. The laboratory references ranges for this assay are 0.7e1.2 mg/dl for males, 0.5e1.0 mg/dl for females. Glucose, triglyceride, total and HDL-C concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). 1.3. Calculations Estimated glomerular filtration rate (eGFR) was calculated by using the CKD-EPI equation [28]: eGFR ¼ 141 min(Scr/k, 1)a max(Scr/k, 1)1.209 0.993Age 1.018 [if female], where Scr is serum creatinine, k is 0.7 for females and 0.9 for males, a is 0.329 for females and 0.411 for males, min indicates the minimum of Scr/k or 1, and max indicates the maximum of (Scr/k or 1).
1.1. Study subjects 1.4. DNA analysis The study group consisted of 2852 adult individuals of European ancestry consecutively recruited at the Department of Systems Medicine of the University of Rome-Tor Vergata and at the Department of Medical and Surgical Sciences of the University “Magna Graecia” of Catanzaro [26]. Recruited subjects participated to a campaign for assessment of cardio-metabolic risk factors. Recruitment mechanisms include word-of-mouth, fliers, and newspaper advertisements. The inclusion criteria were: age 23 years, and presence of one or more cardio-metabolic risk factors including elevated fasting glucose levels, hypertension, dyslipidemia, overweight/obesity, and family history for diabetes. Subjects were excluded if they had end-stage renal disease (ESRD), chronic gastrointestinal diseases, chronic pancreatitis, history of any malignant disease, history of alcohol or drug abuse, positivity for antibodies to hepatitis C virus (HCV) or hepatitis B surface antigen (HBsAg), and hepatic failure. After 12-h overnight fasting, subjects underwent anthropometrical evaluation and a venous blood sample was drawn for laboratory determinations. Body mass index (BMI) was calculated as body weight (kilograms) divided by the square of height (meters). Waist (at the midpoint between the lateral iliac crest and lowest rib) and hip circumference (at the level of the trochanter major) were measured to the nearest 0.5 cm. Readings of clinic blood pressure (BP) were obtained in the left arm of the supine patients, after 5 min of quiet rest, with a sphygmomanometer. Normal fasting glucose (NFG) was defined as fasting plasma glucose <100 mg/dl, impaired fasting glucose (IFG) as fasting plasma glucose 100 and <126 mg/dl, and type 2 diabetes mellitus (T2DM) as fasting plasma glucose 126 mg/dl or current treatment with anti-diabetic drugs. Atherogenic dyslipidemia and hypertension were defined according to the criteria utilized for definition of the metabolic syndrome released in 2009 by a joint statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity [27]. Thus, atherogenic dyslipidemia was defined as triglyceride 150 mg/dl, HDL <40 mg/dl in men and <50 mg/dl in women or drug treatment. Hypertension was defined as systolic blood pressure 130 and/or diastolic 85 mmHg or antihypertensive drug treatment in the subjects with a history of hypertension.
DNA was isolated from whole blood using commercial DNA isolation kit (Promega, Madison, WI). Screening of the rs9267551 polymorphism was performed using a TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA). TaqMan genotyping reaction was amplified on a GeneAmp PCR system 2700 and fluorescence was detected using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA). Genotyping quality was tested by including 3 HapMap samples in each 96-well plate. The agreement rate with the HapMap database genotypes was >99%. 1.5. Statistical analysis The results for continuous variables are given as means SD. Unpaired Student’s t test was used to compare differences of continuous variables between two groups, and the c2-test for noncontinuous variables. The HardyeWeinberg equilibrium between the genotypes was evaluated by c2 test. There was no deviation from the Hardy Weinberg equilibrium in genotype distributions (P ¼ 0.003) using the same threshold of statistical significance utilized by the DIAGRAM þ investigators in the replication studies of the GWAS (defined as a P > 0.001) [29,30]. A multivariate logistic regression analysis was used to determine the association between the rs9267551 polymorphism and CKD. The case-control study has 80% power to detect an association conferring a 0.60-fold reduced risk of CKD for protective C allele according to a dominant model. All tests were two-sided, and a P value <0.05 was considered statistically significant. All analyses were performed using the SPSS software program Version 16.0 for Windows. 2. Results Clinical characteristics of study subjects according to the rs9267551 polymorphism are shown in Table 1. Because of the small number of CC individuals (n ¼ 13) and the a priori hypothesis based on the dominant effect observed in our previous functional studies [25], GC and CC individuals were pooled and analyzed together as C carriers, according to a dominant genetic model. The rs9267551 polymorphism did not show any significant association
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Table 1 Clinical features of 2852 study subjects according to the rs9267551 polymorphism of DDAH2. Variables
GG
GC
CC
P (G/G vs. G/C þ C/C)
n (male/female) Age (yr) BMI (kg/m2) Waist circumference (cm) SBP (mmHg) DBP (mmHg) Fasting glucose (mg/dl) Total cholesterol (mg/dl) HDL (mg/dl) Triglycerides (mg/dl) Creatinine (mg/dl) eGFR (ml/min/1.73 m2) eGFR <60 ml/min/1.73 m2 [no. (%)] Glucose tolerance status (NFG, IFG, T2DM) [no. (%)]
2601 (1316/1285) 52 13 30.0 6.1 100 14 135 18 82 11 111 47 200 40 49 14 137 80 0.85 0.28 98 28 183 (7.0%) 1377/471/753 (52.9/18.1/28.9) 45.0% 2043 (78.5) 1324 (50.9) 1437 (55.2)
238 (127/111) 54 14 29.2 5.2 99 11 135 18 82 10 115 51 201 40 49 14 141 79 0.83 0.21 99 31 9 (3.7%) 126/37/75 (52.9/15.5/31.5) 39.4% 188 (78.9) 118 (49.5) 135 (56.7)
13 (3/10) 52 11 33.8 6.8 107 15 138 19 83 11 110 53 206 25 52 16 121 44 0.75 0.16 102 24 0 9/1/3 (69.2/7.6/23.0) 44.4% 11 (84.6) 7 (53.8) 7 (53.8)
0.72 0.15 0.16 0.21 0.53 0.75 0.25 0.72 0.77 0.55 0.38 0.88 0.03 0.46
208 (27.6) 545 (72.4)
14 (18.7) 61 (81.3)
3 (100) 0
Family history of type 2 diabetes (%) Hypertension [no. (%)] Anti-hypertensive treatment [no. (%)] Dyslipidemia [no. (%)] Antidiabetic treatment [no. (% of diabetic subjects)] Diet Oral hypoglycemic agents
0.09 0.99 0.88 0.86 0.2
Data are means SD. Differences of continuous variables between two groups were compared using unpaired Student’s t. Categorical variables were compared by c2 test. BMI: body mass index; SBP ¼ systolic blood pressure; DBP ¼ diastolic blood pressure; eGFR ¼ estimated glomerular filtration rate; NFG ¼ normal fasting glucose, IFG ¼ impaired fasting glucose; T2DM ¼ type 2 diabetes mellitus. Dyslipidemia: triglyceride 150 mg/dl, HDL <40 mg/dl in men and <50 mg/dl in women or drug treatment; Hypertension: systolic blood pressure 130 and/or diastolic 85 mmHg or antihypertensive drug treatment.
with age, gender, adiposity, blood pressure, fasting glucose levels, lipid profile, serum creatinine levels and eGFR (Table 1). No differences between genotypes were observed in the proportion of subjects having glucose abnormalities, hypertension, dyslipidemia or family history of type 2 diabetes (Table 1). Likewise, no differences between genotypes were observed in the proportion of subjects on treatment with anti-hypertensive or antidiabetic drugs (Table 1). The proportion of subjects with CKD was significantly lower in subjects carrying the C allele as compared with carriers of the GG genotype (OR 0.49, 95%CI 0.25e0.97; P ¼ 0.03). In a logistic regression model adjusted for age, and gender, carriers of the C allele have a lower risk to have CKD as compared with subjects carrying the GG genotype (OR 0.38, 95%CI 0.18e0.78; P ¼ 0.008). This association was maintained when subjects carrying the CC genotype were excluded (OR 0.42, 95%CI 0.21e0.86; P ¼ 0.01). The association was also maintained when BMI, blood pressure, total and HDL cholesterol, triglyceride, and fasting plasma glucose levels were additionally included in the model (OR 0.34, 95%CI 0.14e0.83; P ¼ 0.01). This association remained significant also after addition to the logistic regression model of other confounders including glucose tolerance status (NFG, IFG, T2DM), presence of dyslipidemia, anti-hypertensive and antidiabetic drugs in addition to age, gender and BMI (OR 0.35, 95%CI 0.15e0.80; P ¼ 0.01). 3. Discussion There is evidence showing that plasma ADMA levels are inversely related to renal function, as measured by the eGFR [20,21]. Moreover, it has been shown that ADMA is an independent predictor of progression to end stage renal disease (ESRD) and death in patients with CKD even after adjustment for traditional and nontraditional risk factors [20,21]. DDAH, the enzyme that hydrolyzes ADMA, has an important role in regulation of its levels since it has been estimated that approximately 83% of ADMA generated daily is metabolized by DDAH, whereas only a negligible amount is excreted unchanged by the kidneys [31]. We have recently shown that the rs9267551 polymorphism in the DDAH2 gene has functional impact with the C allele being associated with
enhanced expression of DDAH2 in primary human endothelial cells and lower plasma ADMA levels [25]. In the light of the important role of ADMA in regulating renal function, we investigated the association of rs9267551 polymorphism with CKD in a cohort of subjects of European ancestry. We found that the rs9267551 polymorphism is significantly associated with CKD with carriers of the C allele having a reduced risk of renal dysfunction as compared with in subjects carrying the GG genotype. This association remained significant even after adjustments for a series of cardiovascular risk factors including blood pressure, lipids, and glucose tolerance status as well as anti-hypertensive and antidiabetic treatments. Since it has been reported that ADMA is inversely related to eGFR and that it predicted progression of renal disease to ESRD, it is possible that in subjects carrying the GG genotype ADMA may accumulate at the renal level causing endothelial dysfunction as a consequence of reduced NO availability and potentiating micro-vascular damage. In support of this hypothesis, it has been shown that ADMA has pronounced renal hemodynamic effects [32]. Continuous ADMA infusion caused a decrease in renal perfusion and an increase in reno-vascular resistance at doses that failed to affect blood pressure, renin-angiotensin and sympathetic system [32]. The relatively large sample size, the inclusion of both sexes, the homogeneity of the sample with detailed characterization of cardio-metabolic variables, and the exclusion of confounding conditions such as chronic gastrointestinal diseases, and history of any malignant disease are major strengths of the present investigation. Nevertheless, the study has some limitations. A first limitation relates to the fact that serum creatinine levels and estimated GFR were used to identify kidney function. Although gold standard methods to measure GFR (isotope clearance measurements) may offer a more sensitive estimate of kidney function, they are expensive and time-consuming techniques which are not feasible in large-scale studies. Moreover, the number of subjects with CKD observed in the study was limited, yet our sample size was powerful enough for establishing an independent association between the rs9267551 polymorphism in the DDAH2 gene and CKD. Additionally, this study is an observational investigation based on
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outpatients observed at a referral university hospital, representing individuals at risk for cardio-metabolic disease, and, therefore, may not be extendible to the general population. Furthermore, the present findings are only based on subjects of European ancestry, and should not be generalized to other ethnic populations. Finally, because of the cross-sectional design of the study, the present results reveal only an association with prevalent and not incident kidney failure. Clearly, prospective studies carried out in different ethnic populations may provide a response to these questions. Disclosure statement The authors have nothing to disclose. References [1] Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 2004;351:1296e305. [2] Perticone F, Sciacqua A, Maio R, et al. Renal function predicts cardiovascular outcomes in southern Italian postmenopausal women. Eur J Cardiovasc Prev Rehabil 2009;16:481e6. [3] Fox CS, Larson MG, Leip EP, Culleton B, Wilson PW, Levy D. Predictors of newonset kidney disease in a community-based population. J Am Med Assoc 2004;291:844e50. [4] Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. Am J Kidney Dis 2003;41:1e12. [5] Chen J, Muntner P, Hamm LL, et al. The metabolic syndrome and chronic kidney disease in U.S. adults. Ann Intern Med 2004;140:167e74. [6] Nerpin E, Riserus U, Ingelsson E, et al. Insulin sensitivity measured with euglycemic clamp is independently associated with glomerular filtration rate in a community-based cohort. Diabetes Care 2008;31:1550e5. [7] Zoppini G, Targher G, Chonchol M, Perrone F, Lippi G, Muggeo M. Higher HDL cholesterol levels are associated with a lower incidence of chronic kidney disease in patients with type 2 diabetes. Nutr Metab Cardiovasc Dis 2009;19:580e6. [8] Sarafidis PA, Ruilope LM. Insulin resistance, hyperinsulinemia, and renal injury: mechanisms and implications. Am J Nephrol 2006;26:232e44. [9] Succurro E, Arturi F, Lugarà M, et al. One-hour postload plasma glucose levels are associated with kidney dysfunction. Clin J Am Soc Nephrol 2010;5:1922e7. [10] Sesti G, Succurro E, Arturi F, et al. IGF-1 levels link estimated glomerular filtration rate to insulin resistance in obesity: a study in obese, but metabolically healthy, subjects and obese, insulin-resistant subjects. Nutr Metab Cardiovasc Dis 2011;21:485e91. [11] Bolton CH, Downs LG, Victory JGG, et al. Endothelial dysfunction in chronic renal failure: roles of lipoprotein oxidation and pro-inflammatory cytokines. Nephrol Dial Transplant 2001;16:1189e97. [12] Stam F, van Guldener C, Schalkwijk CG, ter Wee PM, Donker AJM, Stehouwer CDA. Impaired renal function is associated with markers of endothelial dysfunction and increased inflammatory activity. Nephrol Dial Transplant 2003;18:892e8. [13] Perticone F, Maio F, Tripepi G, Zoccali C. Endothelial dysfunction and mild renal insufficiency in essential hypertension. Circulation 2004;110:821e5. [14] Perticone F, Maio R, Perticone M, et al. Endothelial dysfunction and subsequent decline in glomerular filtration rate in hypertensive patients. Circulation 2010;122:379e84.
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