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NDT Advance Access originally published online on February 20, 2008
Nephrology Dialysis Transplantation 2008 23(8):2586-2592; doi:10.1093/ndt/gfn040
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Association between global leukocyte DNA methylation, renal function, carotid intima-media thickness and plasma homocysteine in patients with stage 2–4 chronic kidney disease

Prabath W. B. Nanayakkara1,2, Jessica C. Kiefte-de Jong3, Coen D. A. Stehouwer4, Frans J. van Ittersum2,5, Margreet R. Olthof3, Rob M. Kok6, Henk J. Blom6, Coen van Guldener7,8, Piet M. ter Wee2,5 and Yvo M. Smulders1,2

1 Department of Internal Medicine 2 Institute for Cardiovascular Research, VU University Medical Center 3 Institute of Health Sciences, Faculty of Earth and Life Sciences, VU University, Amsterdam 4 Department of Internal Medicine, University Hospital Maastricht, Maastricht 5 Department of Nephrology 6 Department of Clinical Chemistry 7 The Institute for Research in Extramural Medicine, VU University Medical Center, Amsterdam 8 Department of Internal Medicine, Amphia Hospital, Breda, The Netherlands

Correspondence and offprint requests to: Prabath W. B. Nanayakkara, Department of Internal Medicine, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel: +31-204444444 (Ext. 986791); Fax: +31-204440505; E-mail: p.nanayakkara{at}vumc.nl



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusion
 Reference
 
Background. Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular disease (CVD). Preliminary evidence suggests a role for global DNA hypomethylation in the pathogenesis of atherosclerotic complications in CKD. The aims of this study in patients with stage 2–4 CKD were (1) to assess the association between renal function and DNA methylation, (2) to assess the association between DNA methylation and two markers of atherosclerosis [common carotid intima-media thickness (CCA-IMT)] and brachial artery endothelium-dependent, flow-mediated dilatation (BA-FMD) and (3) to examine the effect of a multi-step treatment strategy on DNA methylation.

Methods. In the Anti-Oxidant Therapy In Chronic Renal Insufficiency study (ATIC-study), 93 patients with stage 2–4 CKD were included. In a randomized, double-blind, placebo-controlled design, the treatment group received pravastatin to which vitamin E was added after 6 months and homocysteine-lowering B-vitamin therapy after another 6 months. DNA methylation was assessed using tandem mass spectrometry. CCA-IMT and BA-FMD were assessed using B-mode ultrasonagraphy.

Results. At baseline, global DNA methylation was not associated with the estimated glomerular filtration rate (P = 0.32) or with CCA-IMT (P = 0.62) or BA-FMD (P = 0.51). No effect of the treatment strategy including B-vitamin on global DNA methylation was found either in the total study group or within separate strata of homocysteine concentration and renal function.

Conclusion. In patients with stage 2–4 CKD, global DNA methylation is not associated with renal function or with CCA-IMT or BA-FMD. A treatment strategy that includes B-vitamins did not alter global DNA methylation in these patients. These data do not support the role of DNA hypomethylation in CKD-associated vascular disease in patients with stage 2–4 CKD.

Keywords: Atherosclerosis; chronic kidney disease (CKD); common carotid intima-media thickness (CCA-IMT); DNA methylation; homocysteine.



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusion
 Reference
 
Chronic kidney disease (CKD) is associated with an increased incidence of cardiovascular disease (CVD). When the glomerular filtration rate (GFR) decreases below 70 ml/min, the probability of CVD increases markedly [1]. This increase is not fully explained by traditional cardiovascular risk factors.

Recently, impaired one-carbon metabolism (Figure 1) [2–4] has been recognized as one of the possible mechanisms responsible for increased atherogenicity in CKD patients. In patients with end-stage renal disease (ESRD), an elevated homocysteine concentration with impaired transsulfuration and remethylation of homocysteine, and elevated S-adenosylhomocysteine (SAH) levels, has been demonstrated [5,6] (Figure 1). Increased SAH inhibits methyltransferases, leading to impairment of methylation reactions [7]. Global DNA hypomethylation has been demonstrated in dialysis patients [8] and is implicated as an important candidate contributing to CVD in patients with ESRD [9]. However, even prior to ESRD, both CVD incidence and plasma homocysteine concentrations rise sharply in proportion to the loss of renal function [6]. Whether DNA hypomethylation is also a feature of these earlier stages of renal insufficiency is currently unknown.


Figure 1
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  		Fig. 1 One-carbon metabolism. Methionine derived from the diet, protein breakdown or from remethylation of homocysteine forms S-adenosylmethionine (SAM or Ado Met). SAM can donate a methyl group by methyltransferases to the carbon 5' position of cytosine within the cytosine–guanine dinucleotide for DNA hypomethylation. The demethylated product of SAM, S-adenosylhomocysteine (SAH or Ado Hcy), is hydrolyzed further to homocysteine and adenosine (transmethylation). To prevent accumulation of homocysteine, it has to be hydrolyzed into cysteine by cysthationine-β-synthase (CBS; transsulfuration) and leave the human body, or can be remethylated by a methyl group of 5-methyltetrahydrofolate (5, MTHF) back into methionine (SAM: S-adenosylmethionine, SAH: S-adenosylhomocysteine, CBS: cythionine β-synthase, MTHF: methylenetetrahydrofolate, THF: tetrahydrofolate, MTHFR: 5,10-methylenetetrahydrofolate reductase, MS: methionine synthase).

 
Global DNA hypomethylation has been associated with various diseases including atherosclerotic vascular disease [10]. Lower global DNA methylation has been shown in atherosclerotic arteries of animals [11] and humans [12]. Moreover, in a small case-control study, patients with vascular disease had lower leukocyte global DNA methylation compared to healthy controls [13]. However, data in support of an association between DNA hypomethylation and vascular disease in patients with mild-to-moderate kidney disease are not available and potential mechanisms that might explain such an association have not been studied.

In view of these considerations, we designed this study to assess, in a population of stage 2–4 CKD patients, the relationship between (i) renal function and DNA methylation, (ii) DNA methylation and two established surrogate markers of arterial vascular disease: common carotid artery intima-media thickness (CCA-IMT) [14] and brachial artery flow-mediated endothelial-dependant vasodilatation (BA-FMD) [15] and (iii) to assess the effect on DNA methylation of a stepwise treatment strategy which includes B-vitamins.



   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusion
 Reference
 
This study is a part of the Anti-Oxidant Therapy In Chronic Renal Insufficiency study (ATIC-study): a randomized, double-blind, placebo-controlled clinical trial that was performed to investigate the effect of a treatment strategy designed primarily to achieve a stepwise oxidative stress reduction on vascular structure and function in patients with CKD [16]. Between May 2001 and December 2002, CKD patients (n = 700) with a creatinine clearance of 15– 70 ml/min/1.73 m2 (according to the Cockcroft–Gault equation [17]) from seven outpatient clinics in Amsterdam, The Netherlands, were screened for eligibility for participation.

Patients with diabetes mellitus, active vasculitis, nephrotic syndrome, renal transplantation, hypercholesterolaemia (>7 mmol/l) and cholesterol-lowering therapy within the past 3 months or a history of cardiovascular disease were excluded from the study. Out of 118 eligible patients, 93 gave informed consent. Baseline data of these individuals were used to assess the association between global leukocyte DNA methylation on the one hand and renal function, CCA-IMT and BA-FMD on the other hand. Longitudinal data were used to assess the treatment effect on DNA methylation.

Baseline measurements
Data were collected with regard to age, medication and smoking status (having smoked in the past year). A detailed history was obtained to exclude clinically relevant peripheral, cerebral and coronary vascular disease at baseline. Height and weight were measured with the individuals wearing light clothing.

Blood samples were taken after an overnight fast. Global DNA methylation was measured by liquid chromatography-tandem mass spectrometry as described in detail by Kok et al. [18]. In short, DNA was isolated from leukocytes and 1 µg of genomic DNA was hydrolyzed using formic acid. Cytosine (cyt) and 5-methylcytosine (mcyt) were separated using gradient elution reversed phase chromatography with a mobile phase containing 5 mmol/l nonafluoropentanoic acid as ion-paring reagent. Mcyt and cyt were detected by liquid chromatography electro spray ionization tandem mass spectrometry operating in the multiple reaction monitoring mode and quantified using stable isotope dilution. The level of DNA methylation is expressed as the methylcytosine/total-cytosine ratio (mcyt:tcyt).

The intra- and inter-assay coefficient of variation (CV) for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n = 9) and 3.5% (n = 8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n = 6) and 6.5% (n = 14), respectively, for Escherichia coli pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The mean intra- and inter-assay CV for humans (n = 10) was 1.4% and 4.1%, respectively.

Renal function was estimated by the Modification of Diet in Renal Disease (MDRD) study equation (estimated GFR in ml/min/1.73 m2): 170 x [plasma creatinine µmol/l x 0.0113]–0.999 x [age]–0.176 x [plasma urea mmol/l x 2.8]–0.170 x [Albumin g/l x 0.1]+0.318 x [0.762 if female] x [1.18 if black] as described in detail elsewhere [19].

Plasma total (free plus protein-bound) homocysteine was measured with an automated fluorescence polarization immunoassay on an Abbott IMx analyser (Abbott Laboratories, Abbott Park, IL, USA), with an inter-assay CV <4% [20].

Plasma concentrations of CRP were measured with a highly sensitive in-house enzyme-linked immunosorbent assay (ELISA) with rabbit anti-CRP (Dako, Copenhagen, Denmark) as a capturing and tagging antibody, with intra- and inter-assay CVs of 3.8% and 4.7%, respectively.

The 677C>T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene has been suggested to affect global DNA methylation under specific conditions, and was therefore assessed by the polymerase chain reaction [21].

Vascular measurements were taken after acclimatization in a temperature-controlled (25°C) room. The common carotid artery intima-media thickness (CCA-IMT) measurements were performed using a Pie Medical Scanner 350 (Pie Medical, Maastricht, The Netherlands) with a linear array transducer of 7.5 MHz attached to a data registration and processing unit (Wall Track System II) as described elsewhere [16]. The measurement protocol for the brachial artery endothelium-dependent, flow-mediated dilatation (BA-FMD) has also been described in detail elsewhere [16]. Briefly, baseline diameter (mean of three measurements) and peak flow velocity (mean of two measurements) were determined. After the release of the pressure cuff, maximum peak flow velocity was measured within 15 s and diameter was measured at 45, 90, 12, 150 and 300 s. All ultrasound measurements were performed by a single trained operator and assessed at 0, 6, 12 and 18 months.

Intervention
The study design was described in detail elsewhere [16]. After randomization, participants in the active treatment group were treated with pravastatin 40 mg/day for 6 months. Subsequently vitamin E 300 mg/day (450 IU {alpha}-tocopherolacetate) was added for another 6 months, and lastly homocysteine-lowering therapy (folic acid 5 mg/day, pyridoxine 100 mg/day and vitamin B12 1 mg/day all in one tablet) was added for another 12 months. The total duration of the study was 24 months. Patients in the placebo group received matching placebo tablets at the onset and 6 and 12 months later. In order to establish whether this treatment strategy had any effect on DNA-methylation, the mcyt:cyt ratio in leukocyte DNA was reassessed at 6-month intervals during this study period.

Statistical analysis
Statistical analysis was carried out with Stata Statistical Software for Windows, release 7 [22]. Linear regression analysis was performed at baseline to investigate primarily the association between global DNA methylation (mCyt:tCyt ratio) on the one hand and renal function, CCA-IMT and BA-FMD on the other hand. Additional analyses were performed after stratification into tertiles of baseline values of renal function, and homocysteine concentration. Stratified analysis was also performed within MTHFR 677 C>T categories. Subjects with a CRP concentration >10 mg/l during the study were excluded in the analysis because inflammation could influence leukocyte DNA methylation, as was recently suggested [23].

Patients continuing trial participation after the baseline measurements were analysed according to intention to treat. The treatment effect was determined by performing generalized estimating equations (GEE) with the mCyt:tCyt ratio as a dependent variable. The primary independent variable in the GEE model was the treatment strategy (1 = intervention group, 0 = placebo group) adjusted for time and the baseline mCyt:tCyt ratio. To assess the effect at the different time points, time was treated as a categorical variable and represented by dummy variables. The GEE analysis assesses the relationship between the treatment modalities and the mCyt:tCyt ratio by correction for the within subject's dependence as a result of the repeated observations [24]. A p-value <0.05 was considered as significant.



   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusion
 Reference
 
Out of 93 included patients, 15 were excluded because of an elevated CRP level, leaving 78 subjects for analysis. Baseline characteristics of these patients are reported in Table 1. At baseline, the mCyt:tCyt ratio ranged from 3.70 to 5.43% (median: 4.41%). In comparison, the normal range, identified in healthy subjects aged 18–62 years with the same analytical method, was 2.57–4.81% (median: 4.10%) [18].


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  		Table 1 Baseline characteristics of the participants (N = 78)

 
We observed no association between DNA methylation and age (β = –0.003; P = 0.36), sex (β = –0.03; P = 0.70), BMI (β = –0.007; P = 0.34), smoking status (β = –0.015; P = 0.83) and CRP concentration (β = –0.004; P = 0.73). Stratification according to the MTHFR 677C>T genotype did not change these results (data not shown).

Global leukocyte DNA methylation and renal function
There was no association between DNA methylation and renal function in univariate regression analysis (β = –0.002; P = 0.32). No difference in DNA methylation was found between the highest or second tertile of renal function compared to the lowest tertile (Figure 2). Additional analysis within strata of MTHFR 677C>T genotype and tertiles of homocysteine concentrations did not influence these results.


Figure 2
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  		Fig. 2 Global DNA methylation in tertiles of the estimated glomerular filtration rate (N = 78).

 
Global leukocyte DNA methylation and homocysteine concentrations
Global DNA methylation was not associated with homocysteine concentrations in univariate analyses (β = 0.003; P = 0.46). No difference was found in DNA methylation between the highest or second tertile of homocysteine concentration compared to the lowest tertile (Figure 3). We found no significant difference in DNA methylation in subjects with the MTHFR 677 TT and CT genotype compared to MTHFR 677 CC genotype (β = 0.19; P = 0.07 and β = 0.06; P = 0.44, respectively). The lack of association between DNA methylation and homocysteine concentration was also confirmed in the separate strata of the MTHFR 677 C>T genotype.


Figure 3
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  		Fig. 3 Global DNA methylation in tertiles of homocysteine concentration (N = 78).

 
Global leukocyte DNA methylation and carotid intima-media thickness and flow-mediated dilatation of the brachial artery
In univariate analysis, we found no association between DNA methylation and CCA-IMT (β = –0.13; P = 0.62) and BA-FMD (β = 0.007; P = 0.51). Adjustment for sex, age, BMI, smoking status, renal function, CRP and homocysteine concentration weakened this association further (β = –0.004; P = 0.99 and β = 0.003; P = 0.78, respectively).

No difference in DNA methylation was found within strata of CCA-IMT and BA-FMD (Figures 4 and 5). Analysis within strata of renal function, homocysteine and the MTHFR 677 C>T genotype did not change these results.


Figure 4
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  		Fig. 4 Global DNA methylation by tertiles of CCA-IMT (N = 78).

 

Figure 5
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  		Fig. 5 Global DNA methylation by tertiles of BA-FMD (N = 78).

 
Effect of B-vitamin-containing treatment on DNA methylation
Out of 93 included patients, 6 withdrew after the baseline measurements and 87 underwent at least one of the follow-up measurements. Fifteen subjects were excluded because of an elevated CRP level during the study (leaving n = 38 and n = 39 in placebo and treatment group, respectively).

Overall, we found no significant effect of the treatment strategies, including B-vitamins, on DNA methylation (β = –0.001; P = 0.96; Figure 6). No effect of the intervention was found within strata of DNA methylation, renal function and homocysteine at baseline.


Figure 6
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  		Fig. 6 Mean mCyt:tCyt ratio (%) following treatment with placebo, or with pravastatin, vitamin E and B-vitamins (N = 78).

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusion
 Reference
 
The main findings of this study are that in stage 2–4 CKD patients global DNA methylation is (i) not associated with renal function or homocysteine concentration, (ii) not associated with established markers of atherosclerosis (CCA-IMT) and endothelial function (BA-FMD) and (iii) not altered by a treatment strategy which includes B-vitamins.

These results are significant since they contradict several findings regarding global DNA methylation in previous small-scale studies.

DNA methylation, renal function and homocysteine
Some years ago, Ingrosso et al. [8] demonstrated leukocyte DNA hypomethylation in dialysis patients. A later study failed to show a relationship between renal function and DNA methylation both in stage 3–5 CKD patients and in ESRD patients receiving dialysis [23].

Combined with our results, it thus appears that leukocyte DNA hypomethylation is not a feature of mild-to-moderate renal failure. Leukocyte DNA hypomethylation might have occurred in the original Ingrosso study in haemodialysis patients due to leukocyte activation on dialysis membranes, and renal failure per se may not play a significant role. Additional studies are needed to elucidate this further.

High plasma homocysteine level is thought to be associated with DNA hypomethylation. An association between homocysteine and DNA methylation was also demonstrated in haemodialysis patients by Ingrosso et al. [8], but we could not reproduce this finding in stage 2–4 CKD. Our results are in line with a recent report by Stenvinkel et al. in CKD patients [23]. Also in another study, no association was demonstrated between homocysteine concentration and DNA methylation in healthy subjects aged 18–62 years [18]. Our findings thus do not support the concept of DNA hypomethylation being responsible for hyperhomocysteinaemia-associated vascular damage, at least not in stage 2–4 CKD.

Global DNA leukocyte methylation and arterial wall properties
Preliminary evidence suggests that global DNA hypomethylation is involved in atherogenesis [2]. However, most of these studies were carried out in animals using DNA methylation from plaques whereas evidence in humans using leukocyte DNA methylation is marginal. Only a single small case-control study reported lower leukocyte DNA methylation in CVD patients compared to age–sex matched healthy controls [13].

We did not observe an association between leukocyte DNA methylation and two strong surrogate markers of CVD. Although this does not exclude a role for DNA methylation in atherothrombotic disease, it does suggest that intima-media thickening and endothelial vasomotor dysfunction are not features of DNA-hypomethylation-associated arterial vascular disease.

Effect of B-vitamin treatment on global DNA leukocyte methylation
We anticipated that the B-vitamins, in particular folic acid, could increase the level of DNA methylation because we previously demonstrated that these vitamins effectively lowered homocysteine concentration in our population (from 20.16 ± 6.80 to 10.45 ± 4.02 µmol/l, versus no significant difference in the placebo group) [16]. Both an increased remethylation rate and a decrease in homocysteine (which converts intracellularly to the methylation inhibitor SAH; Figure 1) are conceivable mechanisms for such effect of B-vitamins. Ingrosso et al. [8] indeed showed reversal of DNA hypomethylation after folate treatment. However, a recent study showed no association between B-vitamin status and DNA methylation [18]. It is noteworthy that Ingrosso et al. [8] used short-term very high dose folate treatment (15 mg 5-methylenetetrahydrofolate) in patients with confirmed DNA hypomethylation.

Study limitations
An important general limitation of global leukocyte DNA methylation is in the proper interpretation of what is really measured. Firstly, the extent to which variability in global DNA methylation reflects variability in epigenetic regulation of gene expression, or variability in methylation of non-coding, repetitive DNA regions, is unknown. Also, the degree to which leukocyte DNA methylation reflects the level of DNA methylation in other tissues, for example vascular tissue, is undetermined. Finally, we do not know to what extent DNA methylation reflects methylation of other, potentially more relevant molecules, such as proteins, enzymes, lipoproteins, etc.

One can argue that the cut-off point to define inflammation is high (CRP>10 mg/l). Stenvinkel et al. demonstrated a linear association between CRP levels and DNA-methylation only in patients with CRP >10 mg/l, while in patients with lower CRP levels there was no linear association between CRP levels and DNA methylation [23]. Marked inflammation, as evidenced by CRP >10 mg/l, arguably affects leukocyte metabolism and turnover rate, which may in turn affect leukocyte DNA methylation. As we intended to focus on the effect of renal function on methylation, we thus excluded patients with CRP>10 in our analysis. To prevent subtle confounding by low-grade inflammation in this study, adjustment for CRP concentration was performed.

Limitations specific to this study include the relatively small sample size in particular in subgroup analyses of strata of continuous variables and of the MTHFR 677 C>T genotype. As the TT genotype renders individuals more susceptible to DNA hypomethylation, for example in response to low folate status [25], an association between renal function and DNA methylation in MTHFR 677 TT subjects can thus not be excluded. With respect to the intervention part, our study had a power of 80% (at an alpha of 0.05) to detect a 0.13% absolute difference in DNA methylation. We previously found the smallest real difference in DNA methylation using the precise tandem mass spectrometry technique to be 0.11% [18]: a difference we thus would have been able to detect in the present study.

Finally, stage 5 CKD patients were not represented in our study, thus precluding conclusions on DNA methylation in more severe renal failure.



   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
Reference
 
In conclusion, in non-diabetic patients with mild-to-moderate CKD without clinical features of atherosclerosis, global DNA methylation was not associated with renal function, with homocysteine concentration and with arterial wall properties. A treatment strategy that includes B-vitamins did not alter global DNA methylation in these patients. Our study indicates that DNA hypomethylation is not a feature of mild-to-moderate kidney disease, and is not a likely contributor to accelerated atherosclerosis in renal patients.



   Acknowledgments
 
The Dutch kidney foundation (project number C97-1707) and Bristol-Myers Squibb, The Netherlands provided the funding for ATIC study but had no influence on the data analysis or manuscript preparation.

Conflict of interest statement. None declared.



   Reference
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Reference
 

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Received for publication: 11.11.07
Accepted in revised form: 21. 1.08


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