Factors Associated with Hyperhomocysteinemia After Renal Transplantation

Abstract

Recent studies show that clinically stable renal transplant recipients have an increased prevalence of hyperhomocysteinemia (hyperHcy), but the mechanism of this disorder has not yet been elucidated. The aim of the present study was to evaluate the factors associated with hyperHcy after a successful renal transplantation. In 106 stable renal transplant recipients, total serum Hcy level (tHcy), folate, total protein, serum creatinine concentration, creatinine clearance, lipid status, body weight (BW), body mass index (BMI), and body fat (BF) were determined. The mean doses of cyclosporine, prednisolone, and azathioprine (mg/kg/day) were recorded. The mean serum tHcy level was significantly higher in renal transplant patients than in healthy controls (22.02 ± 8.02 versus 13.0 ± 3.3 μmol/L; p < 0.001), and the incidence of patients with hyperHcy was 82%. Comparison of the group of 20 patients with tHcy level <15 μmol/L and the group of 86 patients with tHcy level >15 μmol/L revealed that the latter was significantly older, heavier, had been longer on dialysis before renal transplantation, and had older donors and poorer renal graft function. Significant correlation was found between tHcy level and recipient age, dialysis duration, BW, creatinine clearance, serum creatinine, and folate concentration. However, multivariate analysis indicated that creatinine clearance (p = 0.025) and BW (p = 0.03) were the only determinants of elevated total Hcy level in renal transplant recipients. HyperHcy persists after successful kidney transplantation in the majority of renal transplant recipients, and its appearance is primarily associated with creatinine clearance and body weight.

Keywords:

• hyperhomocysteinemia
• kidney transplantation
• creatinine clearance
• body weight

INTRODUCTION

With the increased survival of renal allografts, the long-term survival of renal transplant recipients became mostly influenced by the occurrence of cardiovascular diseases.[1],[2] The exceptionally high prevalence of these diseases in renal transplant recipients is explained by the influence of both traditional atherogenic risk factors and the factors related to chronic renal failure, long-term dialysis, and immunosuppressive therapy. Recent studies suggest that a number of substances, such as homocysteine (Hcy) or cell adhesion molecules, may contribute to the high prevalence of cardiovascular diseases in renal transplant recipients.[3–5] Elevated total plasma Hcy (tHcy) occurs in almost all patients with chronic renal failure, and there is profuse evidence suggesting that the kidney plays a prominent role in Hcy metabolism.[6–9] Patients on maintenance dialysis have two to four times higher tHcy levels than healthy persons. Although a marked decrease of tHcy level could be expected after successful kidney transplantation, the majority of studies have revealed maintenance of hyperHcy in renal transplant recipients despite a significant posttransplant improvement of renal function.[10–12]

This study was undertaken with the objective to determine the incidence of patients with hyperHcy among our renal transplant patients and possible factors associated with its maintenance after successful kidney transplantation.

METHODS

Patients

The study involved 106 renal transplant recipients randomly selected from the population of 276 recipients transplanted and regularly followed for more than 1 yr at the Institute of Urology and Nephrology, Clinical Center of Serbia. The characteristics of the patients are shown in Table 1. Eighty-six patients received a kidney from a related living donor and 20 from a cadaver. The causes of end-stage renal disease (ESRD) were chronic glomerulonephritis (n = 41), chronic pyelonephritis (n = 3), nephrosclerosis (n = 5), polycystic kidney disease (n = 4), congenital urinary tract anomalies (n = 13), other (n = 30), and unknown (n = 10). The immunosuppressive protocol consisted of cyclosporine, prednisolone, and azathioprine. None of the patients received vitamin B or folate supplements. Demographic (age and gender), clinical (dialysis and transplant duration, current immunosuppressive drugs, body weight, and height) and biological parameters (serum levels of folate and total Hcy) were assessed on the same day for each patient.

Table 1 Characteristics of the study population (n = 106)

	Study population
Age (years)	38.85 ± 10.47
Gender (M/F)	66/40
Dialysis duration (months)	32.69 ± 28.27
Donor age (years)	54.88 ± 11.81
Transplant duration (months)	63.43 ± 50.10
Creatinine clearance (mL/min)	56.33 ± 75.75

Data are given as mean ± standard deviation.

The control group consisted of 15 healthy persons, nine males and six females, aged 38.5 ± 6.36 yr.

Informed consent was obtained from all examined persons, and the study was approved by the Ethics Committee of the Clinical Center.

Methods

The total serum Hcy level was measured by high-performance liquid chromatography (HPLC) after reduction of the disulfide bonds by dithiothreitol (normal range: 10–15 μmol/L).

Serum creatinine concentration was determined by an enzymatic method, while creatinine clearance was calculated using the formula of Cockcroft and Gault.[13]

Serum total proteins, cholesterol, and triglycerides were measured enzymatically using automatic methods. Serum folate concentration was determined by ion capture assay (Abbot-AxSYM).

Arterial hypertension was diagnosed when the systolic blood pressure was ≥140 mmHg or diastolic pressure was ≥90 mmHg, or if antihypertensive treatment was prescribed. The mean arterial blood pressure (MAP) was calculated as diastolic blood pressure plus 1/3 of the pulse pressure.

Body mass index (BMI) was calculated according to the following formula: weight (kg)/height² (m²). Body fat (BF%) was estimated from the sum of the thickness of four skinfolds according to the method of Durnin and Wormslay.[14]

Statistics

The one-sample Kolmogorov–Smirnov test was used to check for normal distribution of the variables. The data are presented as mean values with standard deviation (X ± SD). Student's t-test and the Mann–Whitney U test were used for comparing differences between groups. Relationships between variables were estimated using Pearson's rank and Spearmen's correlation tests as appropriate. Independent predictors of total Hcy were identified by multiple regression and logistic regression analyses. The calculations were performed using SPSS.

RESULTS

The mean tHcy serum level was significantly higher in renal transplant recipients than in healthy controls (22.02 ± 8.02 versus 13.0 ± 3.3 μmol/L; p < 0.001). In addition, individual variations of the tHcy level were much greater in transplant patients (9.6–53 μmol/L) than in healthy controls (10–16 μmol/L). The incidence of patients with hyperHcy was 82% (males 90%, females 65%). Males had a significantly higher mean tHcy level than females (23.94 ± 8.5 versus 19.27 ± 6.02 μmol/L; p < 0.05) (Figure 1). BW was significantly higher (76.76 ± 13.77 versus 63.04 ± 11.73 kg; p < 0.001), but BF (22.20 ± 5.49 versus 28.15 ± 5.83%; p < 0.001) significantly lower in males than in females. There was no significant difference in renal function between the genders.

Figure 1. Total Hcy level and serum folate concentration in controls and renal transplant recipients (RTR).

Serum folate concentration was 14.66 ± 9.34 nmol/L (range: 5.6–45.3 nmol/L) in the renal transplant recipients, but 15.8 ± 3.72 nmol/L in the healthy control group. Half of the recipients had serum folate concentrations below the lower limit of the healthy control group. There was no significant difference in serum folate concentration between the genders (Figure 1).

Recipients with hypertension had a significantly higher mean tHcy level compared to those with normal blood pressure (23.4 ± 7.5 versus 20.4 ± 6.4 μmol/L; p < 0.05).

The patients were divided into two groups according to their serum levels of tHcy: group 1 (n = 20) had tHcy in the normal range for the method used (<15 μmol/L), while group 2 (n = 86) had tHcy above the upper normal level (>15 μmol/L). Comparison between the groups revealed that the latter were significantly older and on dialysis longer before transplantation, had older donors and lower creatinine clearance, higher serum creatinine and cholesterol concentrations, and higher BW compared to the former (Table 2).

Table 2 Comparison between the groups formed according to their total serum homocysteine levels

Parameters	Group 1 (tHcy <15 μmol/L)	Group 2 (tHcy >15 μmol/L)
Recipient age (years)	33.45 ± 12.31	39.30 ± 9.22
Donor age (years)	46.90 ± 11.06	56.0 ± 11.15
Dialysis duration (months)	16.12 ± 14.15	29.06 ± 24.57
Transplant duration (months)	80.42 ± 58.84	63.89 ± 47.20
Cyclosporine dose (mg/kg/day)	2.84 ± 1.19	2.67 ± 1.02
Prednisolone dose (mg/kg/day)	0.16 ± 0.06	0.16 ± 0.05
Azathioprine dose (mg/kg/day)	1.37 ± 0.56	1.25 ± 0.38
Systolic blood pressure (mmHg)	136.18 ± 13.87	141.39 ± 13.53
Diastolic blood pressure (mmHg)	83.24 ± 7.28	85.30 ± 7.78
Mean arterial pressure (mmHg)	101.44 ± 9.64	104.04 ± 9.05
Serum creatinine (μmol/L)	133.84 ± 51.18	187.90 ± 77.19
Creatinine clearance (mL/min)	63.59 ± 17.52	54.65 ± 18.91
Serum total protein (g/L)	73.44 ± 5.18	70.89 ± 6.12
Serum cholesterol (g/L)	5.62 ± 1.18	6.31 ± 1.29
Serum triglyceride (g/L)	2.17 ± 1.51	2.30 ± 1.10
Body mass index	23.81 ± 3.50	24.86 ± 3.51
Body fat (%)	26.91 ± 7.46	23.87 ± 5.93
Body weight (kg)	61.41 ± 10.75	73.79 ± 14.44
Total serum homocysteine (μmol/L)	12.87 ± 1.54	24.38 ± 7.32
Serum folate (nmol/L)	12.25 ± 3.18	14.37 ± 9.13

*p < 0.05. Data are given as mean ± standard deviation (student t-test and Mann–Whitney U test).

Univariate analysis showed significant correlations between tHcy level and recipient age, BW, hemodialysis duration, creatinine clearance, serum creatinine, and serum folate concentration (Table 3).

The results of multivariate analysis, presented in Table 4, show that the best-fitted model includes creatinine clearance and BW. Logistic regression analysis used for parameters without normal distribution (hemodialysis duration, systolic and diastolic blood pressure, prednisolone doses, serum creatinine, and serum folate concentration), as well as gender and graft sources (living related donor or cadaver), revealed no significant associations.

Table 3 Correlation coefficients between total homocysteine serum level and recipient age, body weight, HD duration, creatinine clearance, serum creatinine, and folate concentration

Variables	Coefficient of correlation	p of statistical significance
Recipient age	0.210	0.03
Body weight	0.194	0.05
Hemodialysis duration	0.268	0.01
Creatinine clearance	−0.205	0.04
Serum creatinine concentration	0.340	0.001
Serum folate concentration	−0.316	0.01

Table 4 Results of multivariate regression analysis with homocysteine as the dependent variable

Variable	B	Standard error	Beta	t	p	Tolerance	VIF
(Constant)	17.742	4.974		3.567	0.001
Recipient age (years)	3.262E–02	0.093	0.040	0.349	0.728	0.788	1.269
Creatinine clearance (mL/min)	−0.106	0.046	−0.244	−2.277	0.025	0.885	1.130
Body weight (kg)	0.134	0.064	0.240	2.091	0.039	0.771	1.297

DISCUSSION

The significantly higher prevalence of cardiovascular diseases in patients with ESRD than in the general population can be only partly explained by the high prevalence of so-called traditional risk factors in this population. Therefore, attention was directed to some nontraditional risk factors frequently observed in ESRD. High incidence of hyperHcy, one of the nontraditional risk factors, was found in dialysis[9],[15],[16] as well as in adult and pediatric renal graft recipients.[11],[12],[17] This was confirmed in our study of 106 adult renal transplant recipients, among whom 82% had hyperHcy. Previous studies have mainly dealt with the relation between hyperHcy and cardiovascular diseases. An association between hyperHcy and cardiovascular morbidity and mortality was described in the general population,[4],[18] in uremic patients,[9],[16],[19] and in renal transplant patients,[17],[20] although some authors found no connection.[21],[22] Little is known about mechanisms leading to hyperHcy in chronic renal failure, especially after successful kidney transplantation, and that remains an intriguing objective of current studies. Regardless of the mechanisms leading to hyperHcy, we consider that the analysis of factors associated with hyperHcy after a successful renal transplantation could be important. Moreover, it could be proposed that strategies directed to these factors might reduce the incidence of hyperHcy and its detrimental effects on the cardiovascular system.

Our study showed that patients with hyperHcy had significantly lower creatinine clearance and higher serum creatinine levels. Multivariate regression analysis confirmed that creatinine clearance was one of few determinants of elevated total Hcy level in renal transplant recipients. The significant impact of renal function on serum Hcy levels was already described.[6],[8],[23],[24] Recent data suggest that renal uptake and metabolism can account for 70% of the daily total Hcy elimination from plasma by glomerular filtration and metabolism in tubular cells. The tubular cells can either degrade Hcy via the transsulfuration pathway or remethylate Hcy to methionine.[9] However, the total Hcy level after renal transplantation was found to be much higher than in patients with equivalent function of their native kidneys.[8] As every transplantation starts with an inevitable ischemic lesion of the tubular cells, which may be additionally damaged by immune reactions and immunosuppressive drugs, diminished tubular Hcy metabolism in the allograft could contribute to increased total serum Hcy levels in renal transplant recipients. However, controversial results were obtained in the studies investigating the relationship between immunosuppressive agents and Hcy. No significant correlation between the tHcy level and the doses of cyclosporine, prednisolone, and azathioprine was found in our study or in many other studies.[25–27] Others reported a significant positive correlation between the levels of serum Hcy and cyclosporine[28] but a lowering effect of mycophenolic acid on total plasma Hcy.[29]

Multivariate regression analysis revealed that, besides creatinine clearance, only BW was a significant determinant of elevated total Hcy level in our renal transplant recipients. These results are in concert with those obtained by other authors in renal transplant patients and the general population. They underline the role of obesity in the occurrence of hyperHcy.[30],[31] Although multivariate analysis showed that creatinine clearance and BW were the only determinants of elevated tHcy level, comparison of patients with tHcy in the normal range and those with hyperHcy showed that the latter were more frequently males, were significantly heavier and older, and had significantly higher serum cholesterol. These data are not only in agreement with earlier reports,[17],[24],[32],[33] but they also indicate the relation between hyperHcy and traditional cardiovascular risk factors. Our study also revealed that recipients with hypertension, another cardiovascular risk factor, had a significantly higher total Hcy level, although MAP did not correlate significantly with total Hcy levels. Although smoking, an important cardiovascular risk factor, was reported to contribute to the elevated total Hcy level, it was not included in the present analysis, because only three patients had a smoking history. The relation observed between hyperHcy and other cardiovascular risk factors and factors contributing to cardiovascular diseases (dialysis duration) may suggest their combined effect in the development of cardiovascular diseases. However, another question has already been opened, whether hyperHcy had any causative role in the development of cardiovascular diseases or represented a marker for a diseased cardiovascular system.

The unknown mechanism causing hyperHcy and the few factors found to be associated with elevated tHcy in renal transplant patients represent a modest signpost for its prevention and treatment. We and others have shown a significant negative correlation between serum folate and tHcy levels.[10],[16],[34],[35] Moreover, folate and vitamin B were reported to have a lowering effect on total Hcy level in dialysis[36],[37] and renal transplant patients.[37],[36] That suggests that an evaluation of Hcy lowering therapy in prospective control studies might be warranted before the hyperHcy history is elucidated in renal transplant patients.

To conclude, hyperHcy was found in 82% of the kidney transplant recipients regularly followed at our institute. Comparison of patients with tHcy in the normal range and those with hyperHcy showed that the latter were more frequently males, were significantly heavier and older, had been on dialysis for a longer time before renal transplantation, had older donors, and had significantly higher serum cholesterol levels. However, multivariate analysis showed that creatinine clearance and body weight were the only determinants of elevated tHcy in examined renal transplant patients.

ACKNOWLEDGEMENT

This work was supported by grants from The Ministry of Science and Technology, The Republic of Serbia, No. 1919.