Hemodialysis patients have a much higher overall mortality rate than the general population (1,2). Cardiovascular disease, a frequent complication in these patients, is a major cause of the mortality (3). In this setting, important risk factors for cardiovascular disease include hypertension, diabetes mellitus, and increased oxidant stress (4). Hyperhomocysteinemia has recently been identified as a predictor of atherosclerotic complications in the general population (5,6,7,8). It is also frequent among patients with renal failure (9,10). Several recent studies showed that in dialysis patients, hyperhomocysteinemia was a risk factor for cardiovascular complications (10,11,12).
Serum and intracellular levels of homocysteine (Hcy) are regulated by remethylation to methionine or transsulfuration to cysteine (13). The methyl donor in the vitamin B12-dependent remethylation is 5-methyltetrahydrofolate generated from reduction of 5,10-methylenetetrahydrofolate by the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Elevated plasma concentrations of total Hcy (tHcy) result chiefly from genetic defects in the enzymes involved in homocysteine metabolism or from nutritional deficiency of the vitamin cofactors (14). Because renal uptake and metabolism normally account for about 70% of Hcy elimination from plasma (10,15), impaired renal function also results in hyperhomocysteinemia (9,10). Hcy causes endothelial cell dysfunction and injury via production of potent reactive oxygen species during its autooxidation (14,16). This metabolite is thrombogenic, as it increases thromboxane formation, antagonizes nitric oxide, enhances platelet aggregation, and inhibits protein C and thrombomodulin (14,16). Hcy also is a potent mitogen for vascular smooth muscle cells (17,18). Therefore, in end-stage renal disease, elevated plasma Hcy concentrations could contribute to the high prevalence of cardiovascular disease and the increased mortality rate (10,11,12,19). Hyperhomocysteinemia at an earlier stage could also accelerate progression of chronic renal disease.
Recently, a common C to T mutation at nucleotide position 677 (C677T) has been identified in the gene coding for MTHFR, which is involved in the remethylation of Hcy (20). The C677T mutation causes a valine for alanine substitution, which decreases MTHFR activity and tends to elevate plasma concentrations of tHcy in individuals who are homozygous for the mutation (TT genotype) (20). This genotype also has a reported association with coronary heart disease (21,22), but this remains controversial (23). In subjects with normal renal function, the TT genotype causes only a 25% increase in plasma tHcy compared to subjects with other genotypes (23), but in patients with end-stage renal disease undergoing maintenance dialysis, the TT genotype causes a 40 to 100% increase in plasma tHcy (19,24,25) compared to other genotypes who already have two to three times higher concentrations of tHcy than normal subjects.
Considering this synergistic tHcy-increasing effect of renal insufficiency and the TT genotype, the MTHFR mutation could affect the prevalence of vascular disease and associated mortality in patients on maintenance dialysis. In patients with renal insufficiency, more severe hyperhomocysteinemia resulting from the MTHFR mutation may hasten loss of renal function.
In the current study, we investigated independent effects of the MTHFR mutation on serum tHcy concentrations and the prevalence of symptomatic vascular disease in a large population of patients maintained on hemodialysis. We also investigated the effect of the MTHFR mutation on the clinical course of patients with chronic renal failure.
Materials and Methods
During 1997 and 1998, we studied all patients undergoing maintenance hemodialysis at five dialysis centers in the Hokuriku district in Japan (two in Niigata prefecture, one in Toyama prefecture, and two in Fukui prefecture). The cohort comprised 545 patients ages 19.6 to 88.1 yr. Hemodialysis was initiated because of end-stage renal disease resulting from chronic glomerulonephritis (n = 336), diabetic nephropathy (n = 96), polycystic kidney disease (n = 25), Alport's syndrome (n = 2), nephrosclerosis (n = 36), chronic interstitial nephritis (n = 17), toxemia (superimposed preeclampsia) (n = 7), urologic malformation or malignancy (n = 2), miscellaneous nephropathies (n = 8), and renal atrophy of unknown etiology (n = 16). All patients underwent 4-h hemodialysis sessions three times weekly using high-flux membranes. Informed consent for this study was obtained from each subject, and the protocol of this study was approved by the Institute Review Board of Fukui Medical University. A smoking history was obtained that was considered positive if the patient smoked currently or described a previous smoking habit. Hypertension was defined as BP >140/>90 mmHg, or administration of antihypertensive drugs. Nine percent (n = 49) of our dialysis patients received vitamin B12 or vitamin B6 supplements, but none received folate supplements.
Consecutive subjects visiting the affiliated clinics of the Fukui Medical University Hospital for an annual health examination served as healthy control subjects. The age range of these subjects was 16 to 87 yr (n = 676). Their serum creatinine concentrations were <1.2 mg/dl, and they had no clinical signs of coronary artery disease, cerebral vascular disease, or peripheral vascular disease.
Venous blood samples (serum and whole blood) were collected postprandially from all hemodialysis patients, because hemodialysis was usually initiated about 2 h after breakfast or 3 to 4 h after lunch according to the dialysis program at our centers. The serum concentration of total cholesterol was measured by a standard enzymatic method. HDL-cholesterol (HDL-C) was measured in the supernatant after precipitation of other lipoprotein fractions with phosphotungstic acid. Serum tHcy concentrations were measured by automated HPLC with reversed-phase separation and fluorescence detection according to the method of Araki and Sako (26). Serum folate (5-methyltetrahydrofolate) and vitamin B12 concentrations were measured by immunoassay using a commercial analyzer and kit (ACS 180 Immunoassay Analyzer; CIBA Corning, Oberlin, OH). Total pyridoxal was measured by HPLC with fluorescence detection according to the method of Yoshida and coworkers (27). Deficiencies of folate, vitamin B12, and pyridoxal were defined as concentrations of <5.2 nmol/L, <183 pmol/L, and <24 nmol/L, respectively. Whole blood samples for MTHFR genotyping also were obtained from the healthy control subjects. No diurnal variations of serum concentrations of tHcy, folate, total cholesterol, or HDL-C have been reported (28,29).
Assessment of Vascular Disease
Coronary Artery Disease. Coronary artery disease was established from a review of medical records. A previous myocardial infarction was determined by a physician's diagnosis based on chest pain, confirmatory electrocardiographic changes, and enzyme determinations or findings from coronary angiography. Angina pectoris was diagnosed when a stress test was positive and/or coronary artery stenosis (>75% of luminal diameter) was demonstrated by angiography. Patients with a history of myocardial infarction or angina pectoris were classified as having coronary artery disease.
Peripheral Vascular Disease. Peripheral vascular disease was diagnosed by diminished pulses on clinical examination combined with angiography.
Cerebral Vascular Disease. Cerebral vascular disease was defined as a stroke or transient ischemic attack, i.e., functional deficit for at least 24 h and positive findings on computed tomography or magnetic resonance imaging, or an acute functional deficit lasting for 24 h or less, respectively. Patients with cerebral or subarachnoid hemorrhage were excluded.
Determination of MTHFR C/T Genotypes
Genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit (Collectagene; Takara, Ootsu, Shiga, Japan). Identification of the C677T transition in the MTHFR gene was performed by PCR as described previously (20,21).
Data for tHcy, folate, total pyridoxal, and vitamin B12 were transformed to natural logarithms (ln) for analysis. Continuous variables are expressed as mean ± SD. For logarithmically transformed data, the antilogs of the mean and SD are reported. Intergroup differences for continuous variables were assessed by the Mann-Whitney U test or by ANOVA. χ2 analysis was used to assess intergroup differences of noncontinuous variables. Tests to identify trends were used to assess associations between TT genotype frequency and age or duration of dialysis. Multiple linear regression analysis was performed to determine the independent predictors of serum tHcy concentrations. The differences in slopes of the relation between serum tHcy and serum folate by MTHFR genotype among dialysis patients were examined by linear regression with an interaction term. Multivariate logistic regression analysis was to assess the independent contribution of clinical variables including tHcy or MTHFR gene status to vascular disease in dialysis patients. A value of P < 0.05 (two-tailed) was considered statistically significant. All statistical analyses were performed using SPSS statistical software (Statistical Package for the Social Sciences, Inc., Chicago, IL).
Clinical or Laboratory Data in Relation to MTHFR Gene Status in Hemodialysis Patients and Healthy Control Subjects
The characteristics of dialysis patients and control subjects are shown in Table 1. The genotype distribution of MTHFR genotypes in hemodialysis patients (n = 545) was similar to that in control subjects (n = 676), although they were not age-matched (Table 1). The frequencies of T and C alleles were also similar in patients and control subjects (T allele, 40.5% versus 37.9%). Genotype distributions were in the Hardy-Weinberg equilibrium.
Vitamin Concentrations and tHcy Levels in Hemodialysis Patients
Serum concentrations of vitamins and tHcy were measured in a total of 464 hemodialysis patients. Mean levels for folate, pyridoxal, and vitamin B12 in the hemodialysis patients are shown in Table 1. Folate, vitamin B12, and pyridoxal deficiencies were identified in 3.2% (n = 15), 1.3% (n = 6), and 30.7% (n = 141), respectively, of all patients. The mean concentration of tHcy in the patients was 34.5 ± 1.7 μmol/L (Table 1).
Patient Characteristics According to the MTHFR Genotype
Patients with the TT genotype were significantly younger at the time of study and at initiation of dialysis than those with the CT or CC genotype (Table 2). Patients with the TT genotype also had a significantly higher serum concentration of tHcy (about two times) and a significantly lower serum concentration of folate than those with other genotypes (Table 2). No significant differences in other characteristics were observed between the MTHFR genotypes.
Independent Predictors of Serum tHcy Concentration in Hemodialysis Patients
To determine whether the MTHFR mutation independently predicted the serum tHcy concentration in hemodialysis patients, multiple linear regression analyses were performed using a model that included serum levels of folate, pyridoxal, vitamin B12, creatinine, and albumin; gender; dialysis duration; and the MTHFR mutation. Multivariate analysis identified the mutation and serum concentrations of albumin, folate, and vitamin B12 as independent predictors for serum tHcy concentrations (Table 3). By linear regression analysis, the negative slope of the regression lines relating tHcy and folate concentrations differed significantly between the genotypes; the strength of the relation increased with an increasing number of T alleles (P < 0.0001) (Figure 1).
Cardiovascular Disease, Serum tHcy Concentrations, and MTHFR Genotype
Of 545 patients, 105 (19.3%) had at least one of the three types of vascular disease (coronary, cerebrovascular, or peripheral vascular disease). In the patients with vascular disease, mean serum concentration of tHcy was 34.3 ± 1.6 μmol/L versus 34.6 ± 1.7 μmol/L in patients without vascular disease (NS). No significant difference was evident in the prevalence of vascular disease between MTHFR genotypes (Table 2).
When multiple logistic regression analysis was conducted in all patients (n = 545) using model 1, which included age, gender, diabetes mellitus, hypertension, total cholesterol concentrations, HDL-C concentrations, albumin levels, duration of dialysis, MTHFR gene status, and smoking, significant predictors for all prevalent vascular diseases were age, diabetes mellitus, and hypertension, but not MTHFR genotype, and significant predictors for apparent coronary artery disease alone were age and diabetes mellitus. Furthermore, a logistic analysis also was conducted in patients whose tHcy concentration was determined (n = 464) using model 2, which included the serum tHcy concentration in addition to all variables in mode 1 except for MTHFR gene status. By this analysis, age and diabetes mellitus, but not tHcy concentration, were independently associated with all types of vascular disease and with coronary artery disease alone.
Association of the TT Genotype with Age at Study, Age at Initiation of Dialysis, and Dialysis Duration
Because patients with the TT genotype were significantly younger at the time of study and at initiation of dialysis (Table 2), we investigated the distribution of TT genotype according to quartile of age at study or age at initiation of hemodialysis (HD age). This genotype was significantly less common in older patients and males. The prevalence of the TT genotype was not correlated with age in control subjects (Table 4). Patients in the lowest quartile for age at the time of the study had a significantly higher prevalence of the TT genotype than age-matched healthy control subjects considering all subjects and considering male subjects (Table 4). Furthermore, in patients with nonhereditary progressive chronic renal disease (n = 468, including glomerulonephritis, diabetic nephropathy, and nephrosclerosis), the prevalence of the TT genotype was significantly less in patients who were older at initiation of hemodialysis. In contrast, the prevalence did not change with age among control subjects (Table 5). Patients in the lowest quartile of HD age had a significantly higher prevalence of TT genotype than age-matched control subjects for the 468 patients and for males in this patient group (Table 5). Inclusion of patients with hereditary kidney diseases (n = 27) did not alter these results (data not shown).
To investigate whether the TT genotype was affected by the duration of dialysis, the prevalence of the TT genotype was compared between the quartiles of duration of dialysis according to HD age (Table 6). In the middle two quartiles of HD age, the TT genotype prevalence was significantly lower in patients with longer duration of dialysis (Table 6). In these middle quartiles, patients with the TT genotype (n = 47) had a significantly lower age at time of study and a significantly shorter duration of dialysis than those without TT genotypes (n = 224) (55.8 ± 7.3 yr versus 58.4 ± 7.7 yr, P < 0.05, and 5.4 ± 6.3 yr versus 7.7 ± 6.9 yr, P < 0.01, respectively). The three genotypes did not differ with respect to other clinical features including HD age (data not shown). Patients with a tHcy concentration >57 μmol/L representing the arithmetic mean had a significantly shorter duration of dialysis than those with a tHcy concentration <57 μmol/L (4.7 ± 5.3 yr versus 7.4 ± 6.7 yr, P < 0.05).
In this study, we found that the MTHFR genotype independently predicts the serum tHcy concentration of hemodialysis patients. Although we measured tHcy and vitamins postprandially, our findings were consistent with previous smaller surveys of fasting plasma tHcy concentrations in dialysis patients (19,24,25). Other independent predictors for serum tHcy identified in our study were serum concentrations of albumin, folate, and vitamin B12. This finding is in agreement with a previous study of patients on peritoneal dialysis (19). Albumin is the major protein to which homocysteine is covalently bound; folate and vitamin B12 are essential in the vitamin B12-dependent remethylation of homocysteine to methionine (10,13).
We found in a large population of hemodialysis patients that in subjects with the TT genotype, the negative correlation between serum tHcy and serum folate concentrations was more pronounced, as shown previously for the general population (30) and for patients with vascular disease and normal renal function (31). Because the C to T mutation at nucleotide position 677 may be within a folate-binding region of MTHFR (20), the mutant enzyme may have reduced the affinity for methylene tetrahydrofolate. The diminished affinity may decrease the intracellular production of methyl tetrahydrofolate, an essential substrate in the remethylation of homocysteine to methionine. Therefore, patients with the TT genotype may be more susceptible to intracellular deficiency of folate activity and consequent metabolic dysfunction than individuals with other genotypes. Importantly, serum folate levels do not directly reflect intracellular folate activity (32). Consequently, individuals with the TT genotype could have a lower intracellular level of folate than those with other genotypes despite having similar serum folate concentrations. This may partially explain why the negative correlation between serum tHcy and serum folate concentrations was more pronounced in individuals with the TT genotype.
The most notable finding in our current study of dialysis patients was that the prevalence of the TT genotype was less at higher age and for longer duration of dialysis and less than in healthy control subjects. First, patients with the TT genotype were younger at the time of study and at initiation of dialysis than patients with other genotypes. Second, the TT genotype was more prevalent among patients within the youngest age quartile studied than among control subjects of similar age; the prevalence of the TT genotype was less in older patients (Table 4). Third, when the analysis was limited to patients with nonhereditary progressive chronic renal disease, the TT genotype was more prevalent in the patient quartile that was youngest at initiation of hemodialysis compared to age-matched control subjects (Table 5). Finally, among patients with both nonhereditary and hereditary diseases in the middle two age quartiles (38.0 to 50.3 yr and 50.4 to 63.3 yr) at initiation of hemodialysis, the TT genotype was less prevalent in patients with longer duration of dialysis (Table 6). Together, these findings are compatible with the hypothesis that relatively young patients with the TT genotype may be at higher risk to develop end-stage renal failure. On the other hand, patients with the TT genotype who begin dialysis at middle age may have a higher risk of death that increases with dialysis duration.
As found in the present study and previous investigations (19,24,25), dialysis patients with the TT genotype have a greater excess of homocysteine compared to those with other genotypes. Recently, hyperhomocysteinemia has been associated with serious cardiovascular events including death in dialysis patients (11,12,19,33). These findings, therefore, may support a link between the genotype and increased mortality related to dialysis, even though we were unable to connect the TT genotype or tHcy concentrations to vascular disease in dialysis patients. This may be due to several limitations of our study. Because it was cross-sectional, we may have underestimated the prevalence of vascular disease in patients with the TT genotype because of vascular deaths associated with the genotype. Additionally, we did not study subclinical vascular disease.
Hyperhomocysteinemia may aggravate progression in chronic renal disease. Recent studies have associated the mutant C677T MTHFR allele with overt diabetic nephropathy (34) and hyperhomocysteinemia with microalbuminuria (35). Thus, the TT genotype via an elevated Hcy level could induce renal injury and accelerate progression to end-stage renal failure. Such acceleration may partly account for the lower age at initiation of dialysis in patients with the TT genotype, particularly in male patients. The gender-related difference may be related to the action of estrogen to lower Hcy concentrations in premenopausal women (36,37). In other words, a synergistic effect may exist between the TT genotype and male gender (38).
Although our results indicate a possible influence of the MTHFR mutation on the clinical course of patients with renal failure, a large prospective study is needed to test the present observations concerning this mutation. If the present results are confirmed, it is possible that folate therapy for hyperhomocysteinemia in patients with renal failure (28,39) will have to be modified according to MTHFR genotype.
In summary, we identified the MTHFR genotype as an independent predictor of serum tHcy concentration in dialysis patients. In subjects with the TT genotype, the negative correlation between Hcy and folate concentration was more pronounced. The prevalence of the TT genotype was less in patients who were older at initiation of dialysis or who were treated longer on dialysis. We propose the hypothesis that the TT genotype accelerates progression to renal failure and increases mortality.
We thank Drs. Kyoko Ei, Mizue Oda, Isei Ei, Yoshikazu Miyakawa, Kazuko Sugiyama, and Eiko Okada for providing samples and collaborating in this study. We also are indebted to Yuko Mikami for her very skillful technical assistance and to Dr. Akihiko Seo for statistical advice.
American Society of Nephrology
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