EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Disturbances in metabolism of lipo-proteins and oxidative modification of low-density lipoprotein contribute to cardiovascular disease and development of oxidative stress in patients under renal replacement therapy (hemodialysis and renal transplant). This study was designed to compare oxidized low-density lipoprotein levels and lipid profiles in renal transplant recipients and hemo-dialysis patients.

Materials and Methods: We investigated the concentration of oxidized low-density lipoprotein in hemodialysis (n = 38) and renal transplant (n= 59) patients who had no active inflammatory disease, liver disease, or malignancy, and results were compared to a control group (n = 30).

Results: Renal transplant recipients had hypercholesterolemia, hypertriglyceridemia, and increased oxidized low-density lipoprotein levels (P = .019) compared with the control group. Hemodialysis patients had moderate hypertriglyceridemia (not significant), hypercholesterolemia, decrease in high-density lipoprotein, and increase in oxidized low-density lipoprotein levels (P < .0001) compared with the control group. In the renal transplant group, oxidized low-density lipoprotein level had a negative correlation with the duration after transplant (r = -0.407; P = .026), positive association with cyclosporine level (r = 0.288; P = .04), and negative correlation with high-density lipoprotein level (r = -0.30; P = .05); oxidized low-density lipo-protein/high-density lipoprotein ratio also had a positive correlation with cyclosporine level (r = 0.309; P = .027) and negative correlation with high-density lipoprotein level (r = -0.72; P < .001) in the renal transplant group and high-density lipoprotein in the hemodialysis group (r = -0.87; P < .001). Multiple stepwise regression analyses showed that oxidized low-density lipoprotein only was associated with cyclosporine level (R2 = 0.155; β= 0.393; P = .024).

Conclusions: History of cardiovascular disease is the most important factor associated with end-stage renal disease, and high oxidized low-density lipoprotein level, oxidized low-density lipo-protein/high-density lipoprotein ratio, and high-density lipoprotein level may affect cardiovascular disease.

Key words : Cardiovascular disease, End-stage renal disease, Oxidative stress, Ox-LDL

Chronic kidney disease (CKD) is an important public health problem. Recent epidemiologic evidence shows that 1 million patients with-end stage renal disease (ESRD) globally have undergone kidney replacement therapy.¹ When kidney function decreases for > 3 months, CKD is established. The progressive stage of this condition is ESRD, with inevitable kidney damage and loss of kidney function.^2,3

Cardiovascular disease (CVD) is a major cause of high morbidity and mortality in ESRD patients. Half of all individuals with advanced CKD (patients who require renal replacement therapy such as hemo-dialysis [HD] or renal transplant [RT]) die from a CVD event, and fatality in this population is 10- to 30-times higher than in normal individuals.^4-7 Oxidative stress and lipid abnormalities are among the risk factors for atherosclerosis in patients on HD and after RT.8 Reduced serum levels of high-density lipoprotein (HDL) cholesterol and high levels of triglycerides and low-density lipoprotein (LDL) cholesterol are the primary lipid abnormalities in these patients, but total cholesterol levels are commonly normal or low. Acute rejection is more severe in RT patients who have hyperlipidemia than normal lipid profiles.^4,9,10 Oxidative stress is accumulated in renal failure patients because of the imbalance between the production of reactive oxygen species and insufficient antioxidant defense mechanisms.^11,12 Therefore, oxidized LDL (ox-LDL), a good marker to characterize oxidative stress that mediates coronary artery disease,¹³ is evaluated in our study. Several studies have shown that ox-LDL acts as a chemoattractant for monocytes and macrophages and stimulates inflammatory reactions that gradually result in the expansion of atherosclerotic plaques.^14-17

Materials and Methods

Patients
This study was performed at Shahid Beheshti University of Medical Sciences. The ethics committee of the university approved the study. Recruitment of the patients occurred from January 2013 through August 2013. The study groups were composed of 38 HD patients (22 males and 16 females) and 59 RT recipients (32 males and 27 females). Informed consent was obtained from all patients. The control group consisted of 30 subjects (17 males and 13 females) who were normolipidemic and did not have any disease. Patients with history of active infection within 3 months, malignancy, or chronic liver disease were excluded. In the RT group, inclusion criteria included treatment with conventional triple immunosuppressive drugs (cyclosporine, myco-phenolate mofetil, and prednisolone) and no evidence of acute allograft rejection during the 3 months before recruitment. The causes of renal failure in these patients were diabetic nephropathy, chronic glomerulonephritis, polycystic kidney disease, hypertensive ischemic nephropathy, obstructive nephropathy, and unknown etiology. There was no posttransplant diabetes mellitus in the RT patients; all diabetes mellitus patients in the RT group were diabetic before RT (during HD). The HD group consisted of patients who had regular HD for ≥ 3 months (mean, 43 mo), 3 sessions/wk (4 h/session), by synthetic high-flux membranes (2008B Hemo-dialysis System, Fresenius Medical Care, Bad Homburg, Germany).

Laboratory measurements
All samples were obtained from a peripheral vein after overnight fasting for 12 hours, just prior to the beginning of HD (HD patients) or prior to treatment with immunosuppressive drugs (including cyclo-sporine) (RT patients). After coagulation and centrifugation at 1500 × g at 4°C for 10 minutes, serum aliquots were separated and frozen at -80°C for further assay (maximum, 7 mo). Levels of serum creatinine, albumin, urea, uric acid, calcium, phosphorus, total plasma cholesterol, triglycerides (TG), and HDL cholesterol were measured by enzymatic colorimetric methods with an automated chemical analyzer. The LDL cholesterol was calculated by using the Friedewald formula and very LDL (VLDL) was obtained from dividing TG by 5. Serum total calcium and phosphorus were measured with commercial kits (Pars Azmoon, Tehran, Iran). Plasma ox-LDL concentration was measured with a commercially available enzyme-linked immuno-sorbent assay kit (Cusabio Biotech, Wuhan, China), with detection range between 1.56 and 100 mU/mL; the intra-assay and interassay variations were < 8% and < 10%. The standard curve concentrations used for the enzyme-linked immunosorbent assays were 100 mU/mL, 50 mU/mL, 25 mU/mL, 12.5 mU/mL, 6.25 mU/mL, 3.12 mU/mL, 1.56 mU/mL, and 0 mU/mL. The minimum detectable dose of human ox-LDL was < 0.78 mU/mL. Cyclosporine was measured in RT patients by a radioimmunoassay kit (DIAsource Immuno Assays, Louvain-la-Neuve, Belgium); intra-assay and interassay variation were ≤ 9.2% and 7.3%. The measurement range of cyclosporine (from analytical sensitivity to highest calibrator) was 1.61 to 2500 ng/mL.

A standardized questionnaire was administered by trained staff at a clinic visit to obtain demographic information, lifestyle risk factors (including cigarette smoking and physical activity), self-reported history of CVD, diabetes, hypercholesterolemia, hyper-tension, and medications used. Body height and weight were obtained by trained staff and was used to calculate body mass index (weight/height2 [kg/m2]).

Statistical analyses
All data were expressed as mean ± standard deviation (SD). Depending on data distribution, multiple group comparisons were performed by 1-way analysis of variance for continuous variables, and signiﬁcant differences between HD, RT, and control groups were assessed by Tukey–Kramer test. We calculated sample size with type I error test .05 and type II error test 0.1; the power of the test was 90%.

Intergroup comparisons were performed using chi-square test for categorical variables. The corre-lations between study variables were calculated by Pearson and Spearman rank correlation coefﬁcients. Multiple regression analysis was used to investigate the relation between concentration of ox-LDL as dependent variable and lipid, lipoproteins, albumin, calcium, and cyclosporine as independent variables using stepwise method. Values at < 5% level (P < .05) were considered statistically significant. The analyses were adjusted for age, sex, current cigarette smoking, regular physical activity, and body mass index and were performed with a statistical package (SPSS for Windows, Version 20.0, SPSS Inc., Armonk, NY, USA).

Table 1 and 2 summarize the demographic, clinical, and biochemical characteristics of the HD and RT patients and control group. Body mass index and sex were similar between HD and RT patients and the control group. Dialyzed patients had significantly lower levels of total cholesterol and LDL compared with the control subjects (P = .01) and RT patients (P < .0001) (Table 2). In addition, HD patients had lower HDL (P = .04), moderate hypertriglyceridemia, and increased ox-LDL level (P < .0001) than the control group. In RT patients, TG was signiﬁcantly higher than in the control group (P < .0001). The differences in the levels of creatinine, urea, phosphorus, and calcium between the HD and RT groups compared with the control subjects were inevitable because of the difference in renal function. The RT patients had moderately increased ox-LDL level than the control group (P = .019) (Figure 1). In the RT group, ox-LDL had a negative correlation with the duration after transplant (r= -0.407; P = .026) and HDL level (r= -0.30; P = .05) and positive association with plasma cyclosporine level (r= 0.288; P = .04) (Figure 2); ox-LDL/HDL also showed a positive correlation with cyclosporine level (r= 0.309; P = .027) and negative association with HDL level (r= -0.72; P < .001) in the RT group and with HDL in the HD group (r= -0.87; P < .001). Multiple stepwise regression analyses showed that ox-LDL only was associated with cyclosporine level (R2 = 0.155; β= 0.393; P = .024).

The results of this study showed that serum ox-LDL was higher in HD and RT patients than the control group. Serum ox-LDL correlated with serum cyclosporine level, HDL level, and the duration after transplant in the RT group. In addition, ox-LDL/HDL ratio had a correlation with cyclosporine level in the RT group and HDL in the HD group.

In the present study, we observed that ox-LDL level was higher in HD and RT patients than control subjects. Kimak and associates reported increased ox-LDL in HD patients and moderately increased ox-LDL in RT patients, similar to our results. They excluded any ESRD patients with hypertension and smoking, but we evaluated associations between these factors and ox-LDL.⁸ However our results indicated that ox-LDL level was not affected by sex, age, duration of dialysis, or smoking status in the RT or HD patients.^8,18

In our study, cyclosporine level may affect plasma levels of ox-LDL in the RT group. In the RT group, ox-LDL had a positive association with plasma level of cyclosporine. This was in accordance with other reports that showed that in RT patients, tacrolimus therapy was associated with a better lipid profile and lower in vivo LDL oxidation compared with cyclosporine treatment. It was demonstrated that ox-LDL is a marker of atherosclerosis in RT and HD patients.^19-21 Many factors such as dyslipidemia, hypertension, and diabetes which result in atherosclerosis in ESRD patients were observed in our RT recipients and HD patients. These factors were aggravated by the immunosuppressive drugs that prevent allograft rejection in RT patients. Cyclosporine and ox-LDL are known to cause vasoconstriction, and this may be of possible clinical importance because lipid disorders favoring the formation of ox-LDL are common in patients receiving cyclosporine treatment.^12,22,23

In this study, we showed that HDL concentration was negatively correlated with ox-LDL in RT patients and ox-LDL/HDL was a parameter that inde-pendently predicted ox-LDL in the HD group. This can be an indication of oxidative stress in uremia. The recent observations of Samouilidou and coworkers suggest that increased LDL oxidation may be attributed to decreased activity of enzymes with antioxidant effect on HDL, such as paraoxonase 1.^24,25 Consistent with our results, Pawlak and associates confirmed increased ox-LDL/HDL ratios in dialyzed patients; the inverse association was observed between ox-LDL and HDL levels in the entire group of dialyzed patients.⁴ Toikka and coworkers showed that HDL has antioxidant and anti-inflammatory properties; the antioxidant effect of HDL was shown by inhibition of LDL oxidation in healthy young men.²⁶

We also observed a negative correlation between ox-LDL and longer duration after RT. The LDL level is more susceptible to oxidation after 6 months compare to after 10 months; the cyclosporine blood level was maintained at the same level as in previous months.²⁷

In conclusion, the present study showed that the high concentrations of circulating ox-LDL in atherogenic lipoprotein proﬁles, and decreased HDL cholesterol levels, may impair endothelial function and play an important role in the development of atherosclerosis. In addition, the ox-LDL/HDL ratio was elevated in RT and HD patients and it may have diagnostic importance for CVD. The findings may provide new strategies for therapeutic targets to prevent the development of CVD in patients with ESRD.

Volume: 13 Issue: 6 December 2015

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