Stabilization of Oxidative Stress 1 Year after Kidney Transplantation: Effect of Calcineurin Immunosuppressives

Abstract

Kidney transplantation (KT) is one of the best treatments for patients with chronic renal disease. It leads to improved kidney function, but the oxidative stress (OS) is only partially eliminated after KT. This study evaluated the effect of KT on outcomes, such as (a) specific kidney functions, (b) metabolic parameters, as well as (c) OS-related markers in 70 patients (46 males, 24 females; mean age = 54 ± 11) before and 1 year after KT. Post KT, the patients were divided into two groups: those receiving only cyclosporine A (N = 36) and those receiving only tacrolimus (N = 34). Improved kidney function (creatinine, urea, and glomerular filtration rate) and biochemical and hematological parameters were found 1 year after KT. OS-related markers (total antioxidant capacity, advanced oxidation protein, and lipid peroxidation products) decreased, but glutathione level increased after KT. Alterations in superoxide dismutase and catalase activities were also found. Glutathione peroxidase levels were unchanged. The level of oxidized low-density lipoprotein was surprisingly, not significantly increased. There was no significant difference between calcineurin inhibitors in any of the measured parameters. Improved renal function after KT is linked to reduction in OS but independent of immunosuppressive therapy.

• kidney transplantation
• immunosuppressive therapy
• cyclosporine A
• tacrolimus
• oxidative stress

INTRODUCTION

Chronic renal failure (CRF) is characterized by complex changes in cellular metabolism that affect all organs and systems including the cardiovascular system. Atherosclerosis and cardiovascular disorders are the main causes of mortality and morbidity in uremic patients.¹ The development of these diseases is connected with several risk factors such as endothelial dysfunction, dyslipidemia, hypertension, diabetes, and others. In recent years, oxidative stress (OS) has been postulated to be an important nontraditional risk factor for cardiovascular disorders in patients with CRF.² The increased OS and production of reactive oxygen and nitrogen species are connected with the accumulation of various oxidatively modified molecules with prooxidant properties in the body. These conditions modulate a number of signaling pathways (e.g., synthesis of antioxidants and inflammatory mediators). They also modify the structures and functions of biomolecules and negatively affect the functions of the vasodilator, nitric oxide. The enhanced level of superoxide radical interacts with nitric oxide to form reactive short-lived peroxynitrite that leads to endothelial dysfunction and vasoconstriction.^3,4

Kidney transplantation (KT) is a treatment method for patients suffering from CRF. Successful KT leads to amelioration of renal function and normalization of many metabolic abnormalities. Normalization of antioxidant status and reduction of OS are also expected. However, reports related to OS in KT patients are somewhat controversial.⁵

The calcineurin inhibitors, particularly cyclosporine A (CyA) and tacrolimus (Tac), are indispensable to treatment protocols in the posttransplant period with positive influence on graft survival. However, both immunosuppressives have side effects. One is enhanced OS and a number of studies demonstrate the prooxidant effects of both calcineurin inhibitors,^6,7 although CyA has been described as a more potent OS inducer than Tac.⁸

This study aimed at evaluating changes in antioxidant status characteristics in plasma and erythrocytes of kidney recipients 1 year after KT in connection with renal function. The study of the prospective effects of Tac- or CyA-based immunosuppressive therapy on OS was another objective.

MATERIALS AND METHODS

Study Design

A prospective, randomized, 1-year single-center study was designed to assess the course of changes in OS-related parameters and specific kidney function markers before KT and 1 year after KT. The trial was conducted according to the International Conference on Harmonisation Good Clinical Practice (ICH-GCP) guidelines of the University Hospital, Olomouc, and the study protocol was approved by the local Ethics Committee. All participants signed the informed consent at the outset. The study took place from May 2006 to August 2011 in the Department of Internal Medicine III, University Hospital.

Patients

In the beginning, 96 patients who underwent cadaveric KT were included in the study. All were over 18 years of age. Exclusion criteria included patients undergoing systemic immunosuppressive therapy for reasons other than KT; patients with malignant disease or significant uncontrolled concomitant infections; patients who returned to hemodialysis; and female patients who were pregnant or breastfeeding. Twenty-six patients were excluded during the study because of graft failure with return to regular hemodialysis program, death, and cancer. Seventy patients (46 males, 24 females; mean age = 54 ± 11) completed the study. The causes of end-stage renal disease and consequent of KT were chronic glomerulonephritis (n = 38), chronic tubulointerstitial nephritis (n = 14), polycystic kidney disease (n = 5), diabetic nephropathy (n = 11), vascular nephrosclerosis (n = 1), and Alport syndrome (n = 1) (see Table 1). None of the patients was taking vitamin supplements (folic acid, vitamin C, or vitamin E). None had active viral hepatitis B or C. Patients were treated with calcineurin inhibitors CyA (N = 36) or Tac (N = 34) combined with mycophenolate mofetil and corticosteroids. The initial daily dose of CyA was 3 mg/kg, divided into two doses. The target CyA blood levels were 200–300 ng/mL at month 1 and 100–200 ng/mL at month 12 after KT. The CyA level was measured using fluorescence polarization immunoassay ARCHITECT Cyclosporine Reagent Kit (Abbott Diagnostics, Prague, Czech Republic). The initial daily dose of Tac was 0.1 mg/kg, divided into two doses. The target blood Tac level was 5–15 ng/mL at month 1 and 4–10 ng/mL at month 12 after KT. The Tac level was measured using microparticle enzyme immunoassay ARC Tac Reagent Kit (Abbott Diagnostics). The daily dose of mycophenolate mofetil was 20 mg/kg. Prednisone was progressively tapered to reach a daily dose of 20 mg at day 1, 15 mg at month 3, and 5 mg at month 12 after KT. The dose given to each patient to reach the required serum levels can be considered for standardized as well as the duration of the therapy, which was equal for each subject. No induction immunosuppressive therapy with antithymocyte globulin was administered. Besides immunosuppressives patient after KT received heterogeneous medications such as statins, diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin blockers, β-blockers, calcium antagonists, allopurinol, bisphosphonates, vitamin D, calcitriol, and others.

Table 1.  Characteristics of the study population.

Patients	70
Sex: M/F	46/24
Age of kidney recipients, years	54 ± 11
Immunosuppressive therapy, CyA/Tac	36/34
Hemodialysis/peritoneal dialysis	56/14
Dialysis period (months)	29 ± 38
Etiology of CRF
Chronic glomerulonephritis	38
Chronic tubulointerstitial nephritis	14
Polycystic kidney disease	5
Diabetic nephropathy	11
Vascular nephrosclerosis	1
Alport syndrome	1

Note: CRF, chronic renal failure; CyA, cyclosporine A; Tac, tacrolimus.

Sample Collection and Preparation

Blood samples were obtained 1 day before KT and 1 year after KT. Samples were collected from the patients after overnight fasting from antecubital vein into Vacuette^® (Greiner Bio-One, Kremsmünster, Austria) serum and K₃EDTA tubes (Greiner Bio-One). Basic biochemical and hematological parameters were determined in all samples. Serum, plasma, and isolated erythrocytes were used for the determination of OS parameters.

Clinical Chemistry and Hematology

Biochemical parameters [urea, creatinine, total protein, albumin, C-reactive protein, uric acid, and high-density lipoprotein (HDL)] were determined by commercial kits (Roche, Mannheim, Germany) using a Modular Analytics Evo analyser (Roche). Glomerular filtration rate was estimated using the modification of diet in renal disease formula.

Low-density lipoprotein (LDL) levels were estimated indirectly from measurements of total cholesterol (Chol), triacylglycerol (TAG), and HDL by means of Friedewald’s equation.

Hematological parameters such as erythrocyte number, hemoglobin (Hb), and hematocrit were analyzed using Coulter LH 750 (Beckman Coulter, Fullerton, CA, USA) and Sysmex XE-5000 (Toa Sysmex, Koba, Japan) in hospital laboratories.

OS Parameters

Advanced oxidation protein products (AOPPs) were measured according to the spectrophotometric method described by Witko-Sarsat et al.⁹ The total antioxidant capacity (TAC) was estimated using a TAC assay kit (Randox Laboratories Ltd., Crumlin, UK). The amount of malondialdehyde (MDA) was determined using the thiobarbituric acid reaction method.¹⁰ The level of glutathione (GSH) was assessed according to Sedlak and Lindsay using Ellman’s reagent.¹¹ Superoxide dismutase (SOD) activity was measured using the indirect spectrophotometric method based on the generation of superoxide radicals by a mixture of nitro blue tetrazolium, NADH, and phenazine methosulfate.¹² Glutathione peroxidase (GPX) activity was assayed spectrophotometrically at 340 nm by the modified method of Tappel.¹³ Catalase (CAT) activity was assessed according to Beers and Sizer.¹⁴ The oxidized LDL (oxLDL) concentration was measured from blood plasma using a commercial Oxidized LDL ELISA kit (Mercodia, Uppsala, Sweden) according to the manufacturer’s directions.

Statistical Analysis

All values were expressed as median, the first quartile, and the third quartile or as means ± SD. The data were statistically analyzed using the Wilcoxon signed-rank test with Bonferroni correction of significance levels, Spearman’s rank correlation coefficient, and Mann–Whitney U-test. The data were analyzed for statistically significant differences on the day before (day 0) and after KT (1 year). The SPSS v.15 statistical package (SPSS Inc. Chicago, IL, USA) was used for all analyses. The level of significance was set at 5%.

RESULTS

Biochemical and Hematological Parameters

One year after KT, there were improvements in parameters of kidney functions such as creatinine, urea, and glomerular filtration rate. Glomerular filtration rate significantly increased (p < 0.001) but did not reach normal levels. Lipid metabolism parameters were also changed 1 year after KT. A significant diminution in TAG (p = 0.041) and a near significant elevation in cholesterol were found (p = 0.056). Simultaneously, there was a significant increase in HDL (p < 0.001) confirmed by elevation of apolipoprotein A1 (ApoA1; p = 0.020). Nevertheless, there was a significant augmentation of LDL (p = 0.017) and its characteristic apolipoprotein B (ApoB) constituent (p = 0.071). The improvement in renal function was confirmed by the normalization of phosphate (p < 0.001) and calcium (p = 0.012) metabolism. Further, the stabilization and normalization of kidney transplant function were confirmed by the reduction of anemia detected before KT. In contrast, there was a diminution in total protein level 1 year after KT in comparison with before the transplantation when renal function was insufficient (Table 2).

Table 2.  Markers of clinical chemistry and hematology of the renal transplant recipients.

Parameter	Day before transplantation	1 Year after transplantation	p
Chol (mmol/L)	4.80 (4.22–5.61)	5.13 (4.55–5.92)	0.056^b
TAG (mmol/L)	2.13 (1.21–3.52)	1.81 (1.30–2.50)	0.041^a
HDL (mmol/L)	1.19 (0.91–1.56)	1.43 (1.08–1.88)	<0.001^a
LDL (mmol/L)	2.52 (1.82–3.09)	2.73 (2.31–3.31)	0.017^b
ApoA1 (mmol/L)	1.32 (1.17–1.58)	1.50 (1.31–1.77)	0.020^a
ApoB (mmol/L)	0.82 (0.68–0.94)	0.85 (0.78–1.05)	0.071^a
Urea (mmol/L)	16.0 (9.4–20.7)	8.4 (6.4–10.6)	<0.001^a
Creatinine (mmol/L)	635.0 (482.5–761.0)	124.0 (101.0–143.5)	<0.001^a
Calcium (mmol/L)	2.30 (2.18–2.44)	2.37 (2.29–2.46)	0.012^a
Phosphate (mmol/L)	1.72 (1.37–2.12)	1.07 (0.89–1.22)	<0.001^b
Total protein (g/L)	72.6 (68.0–77.6)	70.3 (64.7–73.6)	<0.001^b
Albumin (g/L)	45.0 (42.0–48.0)	45.0 (42.1–47.8)	0.774^a
Glomerular filtration rate (mL/s)	0.140 (0.110–0.183)	0.890 (0.668–1.085)	<0.001^a
Uric acid (mmol/L)	307.0 (210.0–366.0)	368.0 (296.0–421.0)	<0.005^a
C-reactive protein (mg/L)	3.00 (1.00–6.75)	1.40 (1.00–4.10)	0.181^a
Hb (g/L)	116.0 (108.0–122.5)	139.5 (121.3–149.8)	<0.001^b
Hematocrit	0.340 (0.310–0.378)	0.410 (0.370–0.440)	<0.001^a
Erythrocytes (10^12/L)	3.61 (3.40–3.95)	4.57 (4.21–5.01)	<0.001^a

Notes: Data are expressed as median (25 and 75 percentiles). Chol, total cholesterol; TAG, triacylglycerol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; Hb, hemoglobin.

^aWilcoxon signed-rank test.

^bPaired t-test.

Antioxidant Parameters

The primary focus of this study was the determination of selected parameters of antioxidant status and OS in plasma and erythrocytes 1 year after the transplantation. The TAC of plasma was significantly reduced (p = 0.001). The AOPP level was also reduced in comparison to the level before KT. The amount of oxLDL particles, considered to be a sensitive marker of OS, was surprisingly, not significantly increased (p = 0.160). The erythrocyte level of an important intracellular antioxidant GSH was significantly elevated (p < 0.0001) after KT. The concentration of MDA, a nonspecific product of lipid peroxidation, was significantly decreased 1 year after KT. Furthermore, SOD activity was diminished (p = 0.0001) and CAT activity was increased (p = 0.0004) in comparison with the activities before KT. GPX activity was not significantly changed (Table 3).

Table 3.  Oxidative stress characteristics of the renal transplant recipients.

Parameter	Day before transplantation	1 Year after transplantation	p
AOPPs (mmol/L)	174 (121–296)	141 (111–174)	0.002^a
oxLDL (mmol/L)	55.0 (40.1–77.3)	63.4 (52.7–76.9)	0.160^a
TAC (mmol/L)	1.87 (1.74–2.00)	1.70 (1.54–1.85)	0.001^a
MDA (mmol/g)	0.441 (0.370–0.511)	0.362 (0.285–0.443)	0.0001^a
GSH (mmol/g)	7.76 (5.62–9.90)	11.31 (9.33–12.98)	<0.0001^a
SOD (U/g)	2.57 (2.06–3.15)	2.19 (1.22–2.87)	0.0001^b
CAT (U/g)	75.7 (55.6–88.1)	80.3 (65.5–100.9)	0.0004^a
GPX (U/g)	30.3 (19.7–40.1)	26.7 (18.2–40.1)	0.096^a

Notes: Data are expressed as median (25th and 75th percentiles). AOPPs, advanced oxidation protein products; oxLDL, oxidized LDL; TAC, total antioxidant capacity; MDA, malondialdehyde; GSH, glutathione; GPX, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase.

^aWilcoxon signed-rank test.

^bPaired t-test.

The Effect of Immunosuppressives

There were no differences in the initial levels of biochemical or hematological characteristics between participants treated either by CyA or by Tac. Further, similar trends in normalization of monitored parameters were found after the 1-year treatment by the immunosuppressives (Figure 1, Table 4).

Figure 1.  Modulation of selected biochemical and hematological parameters in patients, 1 year after KT. The data are expressed as median, the first quartile, and the third quartile of percentage of changes in parameters at day 360 and at day 0.

Figure 2.  Modulation of select parameters of antioxidant status and OS in patients 1 year after KT. The results are expressed as median, the first quartile, and the third quartile of percentage of changes in parameters at day 360 and at day 0.

Table 4.  Markers of clinical chemistry and hematology of the renal transplant recipients treated with CyA and Tac.

	Day before transplantation			1 Year after transplantation
Parameter	CyA	Tac	p	CyA	Tac	p
Chol (mmol/L)	4.94 (3.96–5.70)	4.70 (4.33–5.33)	0.605^a	5.42 (4.58–6.18)	5.05 (4.52–5.66)	0.176^b
TAG (mmol/L)	2.29 (1.37–3.73)	1.99 (0.92–3.04)	0.165^a	1.81 (1.34–2.62)	1.83 (1.23–2.12)	0.418^a
HDL (mmol/L)	1.22 (0.90–1.51)	1.19 (0.91–1.64)	0.995^a	1.49 (1.00–2.01)	1.39 (1.13–1.72)	0.705^a
LDL (mmol/L)	2.39 (1.79–3.22)	2.52 (1.90–3.05)	0.625^b	2.86 (2.31–3.56)	2.63 (2.29–3.20)	0.332^b
ApoA1 (mmol/L)	1.34 (1.15–1.53)	1.34 (1.18–1.74)	0.508^a	1.49 (1.26–1.95)	1.51 (1.33–1.67)	0.741^b
ApoB (mmol/L)	0.815 (0.693–0.978)	0.820 (0.660–0.915)	0.339^b	0.860 (0.750–1.058)	0.840 (0.770–0.995)	0.632^a
Urea (mmol/L)	16.40 (9.78–20.75)	15.60 (9.05–20.75)	0.698^b	8.00 (6.33–10.03)	9.10 (6.45–12.40)	0.424^a
Creatinine (mmol/L)	564.0 (466.5–718.3)	667.0 (518.0–794.5)	0.167^b	115.0 (99.3–142.3)	132.0 (109.0–179.5)	0.142^a
Calcium (mmol/L)	2.31 (2.15–2.43)	2.28 (2.19–2.48)	0.908^b	2.41 (2.31–2.46)	2.39 (2.29–2.50)	0.848^a
Phosphate (mmol/L)	1.73 (1.44–2.06)	1.77 (1.36–2.14)	0.834^a	1.11(0.91–1.24)	1.06 (0.87–1.18)	0.243^b
Total protein (g/L)	71.9 (66.9–78.0)	73.7 (69.2–77.1)	0.647^b	71.00 (67.30–75.00)	68.55 (64.13–73.33)	0.249^b
Albumin (g/L)	43.0 (41.0–48.0)	45.0 (42.5–49.0)	0.477^b	44.70 (42.00–47.00)	45.50 (42.33–48.10)	0.391^a
Glomerular filtration rate (mL/s)	0.140 (0.110–0.190)	0.140 (0.110–0.185)	0.893^a	0.920 (0.753–1.095)	0.835 (0.560–1.055)	0.308^b
Uric acid (mmol/L)	313.0 (233.0–388.5)	290.0 (206.5–349.3)	0.154^a	357.0 (266.0–453.0)	380.0 (329.5–428.5)	0.375^b
C-reactive protein (mg/L)	3.00 (1.00–6.00)	3.00 (1.00–7.25)	0.962^a	2.00 (1.00–4.00)	1.05 (1.00–6.65)	0.882^a
Hb (g/L)	115.0 (101.0–125.3)	116.5 (112.3–122.5)	0.328^b	139.0 (121.0–151.0)	140.0 (121.5–148.5)	0.800^b
Hematocrit	0.335 (0.300–0.378)	0.345 (0.320–0.373)	0.194^a	0.420 (0.360–0.450)	0.410 (0.370–0.435)	0.734^b
Erythrocytes (10^12/L)	3.51 (3.08–3.95)	3.68 (3.46–3.96)	0.168^b	4.56 (4.22–5.03)	4.61 (4.18–4.99)	0.865^b

Notes: Data are expressed as median (25 and 75 percentiles). Chol, total cholesterol; TAG, triacylglycerol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; CyA, cyclosporine A; Tac, tacrolimus; Hb, hemoglobin.

^aMann–Whitney U-test.

^bStudent’s t-test.

Table 5.  OS characteristics of the renal transplant recipients treated with CyA and Tac.

	Day before transplantation			1 Year after transplantation
Parameter	CyA	Tac	p	CyA	Tac	p
AOPPs (mmol/L)	195.5 (112.7–388.2)	174.7 (126.8–278.9)	0.543^a	154.2 (117.0–192.4)	137.3 (113.9–168.5)	0.200^a
oxLDL (mmol/L)	56.9 (45.6–79.9)	50.9 (36.2–66.5)	0.107^a	66.8 (53.3–77.8)	60.8 (50.1–75.7)	0.338^b
TAC (mmol/L)	1.89 (1.74–2.00)	1.84 (1.76–2.00)	0.624^a	1.72 (1.52–1.86)	1.66 (1.48–1.85)	0.968^b
MDA (mmol/g)	0.453 (0.365–0.518)	0.431 (0.370–0.494)	0.647^a	0.336 (0.268–0.472)	0.378 (0.307–0.435)	0.299^a
GSH (mmol/g)	7.84 (5.14–10.14)	7.85 (5.84–9.79)	0.778^a	10.74 (8.80–12.36)	11.98 (9.72–13.99)	0.347^b
SOD (U/g)	2.59 (1.91–3.17)	2.58 (2.18–3.12)	0.766^b	1.81 (0.89–2.59)	2.55 (1.45–3.05)	0.060^b
CAT (U/g)	78.6 (55.2–98.5)	70.1 (56.3–85.5)	0.565^a	89.4 (66.6–119.1)	71.5 (64.5–87.3)	0.135^a
GPX (U/g)	29.6 (21.1–39.7)	32.5 (18.3–41.6)	0.916^a	25.1 (17.8–39.4)	28.0 (18.7–43.2)	0.462^a

Notes: Data are expressed as median (25th and 75th percentiles). AOPPs, advanced oxidation protein products; oxLDL, oxidized LDL; TAC, total antioxidant capacity; OS, oxidative stress; CyA, cyclosporine A; Tac, tacrolimus; MDA, malondialdehyde; GSH, glutathione; GPX, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase.

^aMann–Whitney U-test.

^bStudent’s t-test.

The initial antioxidant characteristics showed the homogeneity of categorization in groups treated either by CyA or by Tac. The changes in antioxidant parameters observed 1 year after KT were similar in both groups of patients and the trends of OS parameters modulation were comparable (Figure 2, Table 5).

DISCUSSION

KT is one of the best therapeutic approaches in the treatment of CRF. The transplantation improves the excretion of products of metabolism and exogenous substances including substances with prooxidant properties. This accompanies the normalization of health status and antioxidant–oxidant balance. During the transplantation (ischemia/reperfusion) and also in the posttransplant period (immunosuppressive therapy), the patients suffer from OS.¹⁵ OS is considered a nontraditional risk factor for atherosclerosis and cardiovascular complications that indirectly affect the life span of the graft and mortality and morbidity of patients after KT.^2,3 This trial aimed at investigating the long-term effects of transplantation on renal functions and OS-related parameters.

The results showed that the restoration of kidney effectiveness was accompanied by an increase in glomerular filtration rate and a decrease in urea and creatinine levels in plasma. The improvement in renal excretion capacity of low molecular weight substances was associated with reduction of plasma TAC. In patients suffering from chronic renal diseases, the TAC value does not correspond with the degree of OS. The TAC value increases when low molecular weight substances are accumulated in plasma due to insufficient excretion capacity of the kidney.¹⁶ However, the improvement in antioxidant status and the decrease in OS were confirmed by reduced AOPP levels in plasma, as well as increased GSH concentration and diminished MDA levels in erythrocytes. A partial improvement in renal function and redox status following KT that we observed is in agreement with Aveles et al. These authors found an increased thiol content and a diminution in carbonyl level in plasma, 9 months after transplantation.¹⁷ Previously, Cofan et al.⁸ reported that the concentration of oxLDL decreased after the replacement of immunosuppressive drugs CyA and Tac. Thus, we assumed that the normalization of kidney function is accompanied by the reduction of oxLDL, which is a sensitive, long-term marker of OS and a risk factor for atherosclerosis.^18–20 Surprisingly, 1 year after KT, oxLDL was not diminished. Rather it increased, but not significantly. However, our results are in agreement with a recent study by Kimak et al.²¹ who also showed a moderate increase in oxLDL and changes in other lipid parameters in transplant patients. Besides modulation of lipid parameters, a decrease in paraoxonase 1 (POX1) activity in CRF patients was observed. POX1 is an HDL-associated hydrolase that protects other lipoprotein particles against oxidative modification. The mild increase in oxLDL after KT could be associated with incomplete restoration of POX1 activity and HDL function.^21,22

Our results also demonstrated that the level of AOPPs, one of the end products of OS, is a reliable and sensitive marker for estimating the effectiveness of therapy and the degree of OS in patients suffering from chronic kidney disease. Its determination seems to be better than oxLDL levels for monitoring the real situation in the body.

The main function of the selected antioxidant enzymes, particularly SOD, GPX, and CAT is detoxification of reactive oxygen species and other reactive prooxidants. However, there is evidence that antioxidant enzyme activity is lowered in patients suffering from CRF.²³ Reports further indicate that the renal transplantation is not connected with the normalization of the activity of these enzymes which usually remain lower than those of healthy individuals.^7,17,24 In this study, we found changes in the erythrocyte activity of SOD and CAT following KT that was probably linked to the partial elimination of OS. On the other hand, no influence of KT on GPX activity was found. Our results are not in agreement with Vural et al.⁷ who found an increased GPX and SOD activities in erythrocytes 1 month after KT. On the other hand, McGrath et al.²⁵ showed a marked decrease in SOD activity and no significant changes in GPX activity 6 months after the transplantation compared with subjects without any history of renal disease. Thus, antioxidant enzymes seem to be sensitive markers of persistent antioxidant–oxidant imbalance in kidney transplant recipients.

The calcineurin-inhibiting drugs CyA and Tac are indispensable parts of the therapeutic protocols in transplant patients. However, there are a number of studies indicating that their nephrotoxicity affects long-term graft survival. These drugs have other adverse effects including alteration to endothelial mitochondria, accumulation of nitrotyrosine, and endothelin-induced hypertension.²⁶ Even though the prooxidant activities of Tac and CyA have been intensively studied, a large number of contradictory results have been published. Some authors consider CyA to be a more potent prooxidant than Tac,²⁷ but other studies have not confirmed these findings.⁷ Our results showed that there were no significant differences between effects of CyA and Tac on OS-related parameters after transplantation. This indicates that OS in KT patients may be independent of the immunosuppressive therapeutic regiment used.

The transplant patients are usually treated not only with immunosuppressives but also with combination of therapeutics (statins, diuretics, ACE inhibitors, angiotensin receptor blockers, β-blockers, α-antagonists, allopurinol, bisphosphonates, etc.), which are used for the prevention and/or the treatment of hypertension, dyslipidemia, and hyperuricemia to diminish the risk of graft failure. Most of the drugs have adverse side effects.²⁸ The influence of majority of these medicaments on antioxidant status is marginally known. Statins were found to ameliorate vasculature in patients with kidney diseases and transplant patients by pleiotropic effect explained by the modulation of mainly lipid metabolism, but also inflammatory and OS pathways and endothelial function.^29,30 ACE inhibitors were found to reduce OS in transplant patients.^29,31 Allopurinol, inhibitor of uric acid and purine bases synthesis, is used for the treatment of hyperuricemia. Uric acid is an important antioxidant of body fluids, but its elevated level is connected with inflammation, prooxidant condition, modulation of endothelial and smooth muscle cells, increased risk of cardiovascular events, and chronic allograft nephrotoxicity.³² Allopurinol decreases the level of C-reactive protein, improves glomerular filtration rate, and slows down the progression of renal disease.³³ Bisphosphonates, further component of transplant patient’s medication are applied to improve bone mineral density. Experimental studies have shown that they have antiatherogenic action but without changes in lipid or cholesterol profiles; however, these experimental observations have not been confirmed in clinical trials.³⁴

In conclusion, the successful KT reduced but did not normalize the oxidative imbalance in the organism, perhaps due to the effect not only of immunosuppressive therapy, but also of the influence of another medications and renal dysfunction 1 year after KT. Monitoring of OS-related parameters together with inflammatory markers and renal excretion function are very important in the prognosis of graft function and can be effectively carried out early in the treatment.

ACKNOWLEDGMENT

This work was supported by grants MSM 6198959216 and MZ CR (NS/9964-4).

Declaration of interest: The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper.