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Clinical Transplantation

OXIDATIVE STRESS AND LIPID ABNORMALITIES IN RENAL TRANSPLANT RECIPIENTS WITH OR WITHOUT CHRONIC REJECTION1

Cristol, Jean-Paul2; Vela, Carlos3; Maggi, Marie-France2; Descomps, Bernard2; Mourad, Georges3,4

Author Information

Biochemistry Department and the Transplantation Unit, Nephrology Department, University Hospital Lapeyronie, 34295 Montpellier 05, France

1This work was supported by a grant from the Dèlègation á la Recherche Clinique, Montpellier University Hospital.

2Biochemistry Department.

3Transplantation Unit, Nephrology Department.

4Address correspondence to: Georges Mourad, M.D., Department of Nephrology, University Hospital Lapeyronie, 34295 Montpellier 05, France.

Received 8 September 1997.

Accepted 4 February 1998.

  • Free

Abstract

*Abbreviations: Apo A, apolipoprotein AI; Apo B, apolipoprotein B; CR, chronic rejection; CsA, cyclosporine; GPx, glutathione peroxidase; GSH, glutathione; HDL, high-density lipoprotein; HPLC, high-performance liquid chromatography; LDL, low-density lipoprotein; Lp(a), lipoprotein a; MDA, malonyldialdehyde; PUFA, polyunsaturated fatty acid; RBC, red blood cell; SOD, superoxide dismutase; TC, total cholesterol; TG, triglyceride; Vit E, α-tocopherol.

Chronic rejection (CR*) is the most important cause of graft loss after the first posttransplantation year. Clinically, CR presents itself as a gradual deterioration in renal function, usually associated with proteinuria and hypertension (1-4). Allograft arteriosclerosis is considered the most important underlying histopathological lesion; it is characterized by thickening of the intimal layer of the arterioles, with progressive luminal occlusion. In the early stages, endothelial lesions associated with an accumulation of lymphocytes and monocytes in the broadened subendothelial space are frequently observed. Monocytes occasionally may be transformed into foam cells(5). At the later stages, mononuclear cells are replaced by proliferating cells, including smooth muscle cells and fibroblasts, attracted from the media. This infiltration results in accumulation of various components of the extracellular matrix, including collagen (1-5). The resulting histological picture of CR with endothelial lesions and vascular intimal hyperplasia resembles the early arteriosclerotic lesion, and the term"transplant arteriosclerosis" has been proposed. After the Banff classification (2) for kidney transplant pathology grades, the Fourth Alexis Carrel Conference (3) made the recommendation that the histological diagnosis of CR not be made unless the arteriosclerotic changes are present in the core biopsy. These vascular lesions are not specific for renal allografts because they are often observed in allogeneic transplantation of heart, liver, and lung. The high prevalence of lipid abnormalities after renal transplantation, the well-established role of lipid disturbances in the genesis of arteriosclerosis, and finally the histological similarities between CR and arteriosclerosis suggest that dyslipidemia plays a role in the pathogenesis of CR (6-9). In addition, it has been reported that reactive oxygen species and oxidation of low-density lipoproteins (LDL) play a central role in the development of arteriosclerosis (10,11) by enhancing the recruitment of inflammatory cells and the subsequent microvascular dysfunction. The aim of the present study was to determine whether lipid abnormalities and oxidative stress are associated with the allograft vasculopathy observed during renal allograft chronic rejection.

MATERIALS AND METHODS

Study Design

A cross-sectional study was performed to determine lipid parameters and oxidative metabolism following renal transplantation. Blood samples were collected in fasting patients during their regular outpatient visits. Recipients with cancer, active infection, acute rejection for less than 6 months, and diabetes mellitus were excluded. No patients were given vitamin supplementation or hypocholesterolemic drugs. Seventy-seven patients who had received transplants more than 1 year earlier and who were receiving cyclosporine (CsA) and steroids as maintenance immunosuppression were included. The protocol was approved by Montpellier University's ethical committee, and all patients gave written informed consent. Results were compared with data collected from a cohort of 38 healthy volunteers (control group, 51±9 years old).

CR was defined as a gradual increase in serum creatinine levels with proteinuria and/or hypertension, and was confirmed by the presence of fibroproliferative vasculopathy on graft biopsy. The population of transplant recipients was thus divided into two groups, according to the presence or absence of histologically proven CR. The clinical characteristics of the patients are listed in Table 1.

T1-7
Table 1:
Patient characteristics

Lipid Metabolism

Plasma triglycerides (TG) and total cholesterol (TC) levels were measured by routine enzymatic methods (Boehringer Mannheim, Meyland, France). Plasma high-density lipoprotein (HDL) cholesterol was assayed in the supernatant after precipitation of apolipoprotein B (Apo B)-containing lipoproteins by magnesium phosphotungstate. Plasma apolipoproteins A1 (Apo A), Apo B, and lipoprotein (a) (Lp(a)) were analyzed by immunonephelometric assay (Behring Diagnostic S.A., Rueil Malmaison, France).

Oxidative Stress

Oxidative stress was monitored by determining: (i) the end product of lipid peroxidation, malonyldialdehyde (MDA), and the degradation of plasma and erythrocyte polyunsaturated fatty acids (PUFA); (ii) the nonenzymatic antioxidant system: plasma and red blood cell (RBC) α-tocopherol (Vit E) and RBC glutathione (GSH); (iii) the enzymatic antioxidant system: plasma glutathione peroxidase (GPx) and superoxide dismutase activity (SOD).

MDA determination. The concentrations of MDA were evaluated from the reaction, resulting in the formation of thiobarbituric acid-reactive substances according to Yagi's method (12). Briefly, lipids and proteins were precipitated using 10% phosphotungstic acid and N/12 sulfuric acid. The sediment was resuspended in distilled water and thiobarbituric acid was added. The reaction mixture was heated at 95°C for 60 min; thiobarbituric acid-reacting substances were extracted with butanol. After centrifugation, the butanol layer was taken for fluorometric measurements, which were made at 515-nm excitation and 553-nm emission (Merck F1000, Merck, Paris, France).

RBC GSH measurement. The glutathione content in red blood cells was measured by colorimetric assay (Bioxytech GSH-400; OXIS International S.A., Bonneuil-sur-Marnes, France). Whole blood was centrifuged immediately, and the erythrocyte pellet was lysed in a 6% solution of metaphosphoric acid at 4°C. After centrifugation, the upper layer was collected and kept at-20°C until use. Sample was added to 4-chloro-1-methyl-7-trifluomethyl-quinolinium methylsulfate; the derived thioether was then transformed into a chromophoric thione by the addition of NaOH 30% (maximal adsorbence wavelength: 400 nm). Results were expressed as mmol/mg Hb.

Plasma glutathione peroxidase measurement. Plasma glutathione peroxidase (GPx) was measured by enzyme-linked immunoassay (Bioxytech pl-GPx-EIA; OXIS). One hundred microliters of heparinized plasma were incubated with 100 µl of biotinylated anti-plasma GPx solution for 1 hr at room temperature in wells previously coated with polyclonal GPx antibodies. After five rinses, 100 µl of streptavidin-phosphatase solution were incubated for 1 hr at room temperature. The enzymatic reaction was stopped with 50 µl of NaOH 1 M containing 0.1 M EDTA, and the adsorbence at 405 nm was measured.

SOD activity measurement. The activity of SOD was assessed by spectrophotometry (Bioxytech SOD-525; OXIS). The erythrocyte pellet was lysed in distillated water and was stored at -20°C until use. The erythrocyte lysate was mixed with a chromogen reagent(5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo [c]fluorene) and the chromophore product was measured by spectrophotometry (525-nm wavelength) after SOD-induced oxidation.

Determination of plasma Vit E and RBC Vit E. Plasma Vit E and RBC Vit E were measured by high-performance liquid chromatography (HPLC) according to the method previously described by Cachia et al.(13), using material from Waters chromatography(Millipore Waters, Les Ullis, France).

Vitamin E was extracted from plasma (100 µl) with 600 µl of ethanol/ascorbic acid 10% solution (5:1; v/v) and 5 µg of tocopherol acetate as internal standard. After centrifugation, the supernatant was separated and extracted with hexane (1.5 ml). After a new step of centrifugation, the supernatant was collected and evaporated to dryness. The dry residue was redissolved in 100 µl of methanol run through a Novopack 150×3.9-mm column (4-mm particle size, reverse phase). The elution fluid was monitored at 292 nm (Millipore Waters Lambda max 480). Pure methanol was used as the mobile phase. To correct vitamin E levels to lipoprotein profiles, plasma Vit E was expressed in µmol/mmol of (triglycerides + cholesterol).

The red blood cells were washed three times with 0.9% sodium chloride. Fifty microliters of red blood cell pellet were mixed with 50 µl of conservation buffer (50 mM Tris buffer, 5 mM ascorbate, and 150 mM sodium chloride) and 2 ml of detergent sodium dodecyl sulfate (280 mg/L in water). After vortex-mixing for 2 min, samples were kept at -80°C until they were used. RBC Vit E was extracted from this solution with 250 µl of hexane/ethanol/ascorbic acid 10% (25:1:1) and 250 ng of δ-tocopherol as internal standard. The dried extract was redissolved in a minimum volume of methanol and identified by HPLC analysis. The HPLC apparatus was equipped with an mBondapak 300×3.9-mm column (10-mm particle size, C18 reverse phase) and an electrochemical detector (Precision instrument 201, Waters Chromatography). A mixture of methanol water (98:2; v/v) containing 3 mg/L lithium perchlorate and 1 ml/L acetic acid was used as mobile phase.

Determination of the PUFAs in erythrocytes. Lipid extraction and transesterification: Five hundred microliters of washed erythrocyte pellet were submitted to osmotic hemolysis in 2 ml of distilled water. Five hundred microliters of hemolysate was added to 10 ml of chloroform/methanol(2:1; v/v) and treated according to Folch (25). After a vigorous mixing, 1 ml of KCl 0.15 M (Prolabo) was added. The organic infranatant was separated after centrifugation (20 min at 2000 rev/min). After evaporation of the extract in a stream of nitrogen, the removal was resolubilized in a minimum volume of chloroform/methanol (1:1; v/v), and 3 ml of methanol/sulfuric acid (19:1; v/v) was added. The mixture was refluxed at 80°C for 30 min in a ground glass-stoppered tube. After cooling, the solution was neutralized with NaHCO3. The fatty acids were extracted with hexane (3 ml) in the presence of distilled water (1 ml). After centrifugation, the organic phase was separated and evaporated. The sample was redissolved in a minimum volume of hexane.

Gas chromatography: The PUFAs were fractionated in a fused silica-capillary column (50 m × 0.32 mm internal diameter) coated with 100% cyanopropyl-siloxane 88 phase from Chrompack (Les Ullis, France) in a Fisons Instruments 8000 series gas chromatographer with an on-column injector(AS 800 Autosampler, Fisons Instruments, Rodaro, Italy). The conditions were as follows (14): ionization detector, 250°C; injector, 230°C; and oven program, 10°C/min from 100 to 170°C, then 2°C/min from 170 to 200°C. Hydrogen was used as the carrier gas(flow rate of 1 ml/min). The response of a flame ionization detector (FID-80; Fisons Instruments) was monitored with chrom-card hardware running in an IPC computer (IBM computer) connected to an ESC-P2 Epson printer.

Statistical Analysis

Results were expressed as mean ± SD. To assess the effect of renal transplantation on oxidative stress, the whole group of transplant patients was compared with the control group using Student's t test. The analysis of the effect of chronic rejection on lipid abnormalities and oxidative stress was performed using analysis of variance with three-group comparison (the control group, group I, and group II; analysis of variance with correction for multiple comparisons by a Scheffé F test, StatviewSE 1.03A program, Power PC Macintosh). Since the Lp(a) distribution was not normal, Lp(a) was taken as a qualitative variable, and the percentage of patients in each group having more than 300 mg/L of Lp(a) was compared using a chi-square test. A P-value <0.05 was considered significant.

RESULTS

Lipid Abnormalities

As shown in Table 2, total cholesterol, triglycerides, and Apo B were significantly elevated in groups I and II when compared with controls. Similar values for HDL cholesterol, Apo A, and Lp(a) were observed in transplant patients and controls. There was no significant difference between group I and II patients in any lipid parameter.

T2-7
Table 2:
Lipid metabolism in transplant patients and healthy volunteers

Oxidative Stress

Transplant recipients versus controls. When comparing the whole group of transplant patients (groups I and II) with controls, we observed a significant increase in MDA (1.74±0.35 vs. 1.24±0.29 nmol/ml; P<0.01) associated with a significant decrease in RBC PUFAs, particularly in (n-6) and (n-3) end products (arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid); in contrast, the plasma PUFA levels remained unchanged (Table 3). Impairment of the antioxidative defenses was observed in transplant patients as RBC Vit E and SOD decreased from 1.08±0.31 to 0.72±0.40 mg/ml of RBC pellet and 1.32±0.31 to 1.11±0.25 U/mg Hb, respectively (P<0.01), and GPx increased from 6.63±2.05 to 11.41±5.1 mg/L (P<0.01). Absolute values of plasma Vit E were increased in transplant recipients (16.11±1.10 vs. 13.67±1.14 mg/L); however, after adjustment for the sum of cholesterol and triglycerides, normalized vitamin E levels were similar in both groups (4.79±0.13 vs. 4.6±0.3 µmol/mmol). Finally, RBC GSH levels were unchanged in transplant recipients (5.05±1.2 vs. 4.48±0.91 mmol/mg Hb).

T3-7
Table 3:
Fatty acid composition of plasma and erythrocyte phospholipidsa

Controls versus group I and group II transplant patients. Comparison between controls and groups I and II transplant recipients showed that oxidative stress was significantly different in the three groups: it was higher in group I versus controls and in group II versus controls and group I(MDA: 1.24±0.29 nmol/ml in controls, 1.62±031 nmol/ml in group I, and 1.87±0.43 nmol/L in group II) (Fig. 1). Moreover, as shown in Figure 2, the decrease in antioxidative defenses was more pronounced in chronic rejection patients as RBC Vit E and SOD levels were significantly decreased only in group II compared with controls (0.61±0.38 vs. 1.08±0.31 mg/ml of RBC pellet, P<001, and 1.08±0.20 vs. 1.32±0.31 U/mg Hb, P<0.01, respectively). RBC Vit E and SOD levels were lower in group II patients when compared with group I; however, these differences did not reach statistical significance. There was no correlation between oxidative stress markers or defense mechanisms and creatinine levels or proteinuria. GPx, GSH, and plasma Vit E normalized by lipids were similar in both groups (Table 4).

F1-7
Figure 1:
MDA values in the control group, in group I (transplant recipients without chronic rejection), and group II (transplant recipients with chronic rejection).
F2-7
Figure 2:
RBC Vit E and SOD in controls, group I(transplant recipients without chronic rejection), and group II (transplant recipients with chronic rejection).
T4-7
Table 4:
Defense mechanisms against oxidative stress in patients and healthy volunteers

DISCUSSION

Our study demonstrates clear disturbances of the oxidative metabolism in renal transplant recipients associated with lipid abnormalities. The combination of an atherogenic lipid profile with impaired antioxidant defense mechanisms could conspire to the development or progression of the allograft arteriosclerosis. Interestingly enough, MDA used as a lipid peroxidation marker was significantly increased, and the enzymatic and nonenzymatic antioxidant defenses were markedly impaired in those patients suffering from chronic rejection.

Lipid abnormalities, including increased TC, TG, and Apo B, have frequently been reported in renal transplantation. This well-documented lipid atherogenic profile has previously been implicated in the high frequency of cardiovascular events and mortality observed in these patients(15-17). Posttransplant dyslipidemia, observed even in patients with normal renal function, may probably be related to the immunosuppressive drugs, particularly steroids (18-20) and CsA (21,22). Indeed, withdrawal of steroids from patients maintained on CsA results in a significant reduction in TC and TG concentrations (18,20). CsA also contributes to lipid disorders (increase in TG and diminution in HDL cholesterol), but the effect of the withdrawal of CsA from patients maintained on azathioprine and steroids has not been fully assessed (16). Preliminary data in heart and liver transplants (23,24) as well as in kidney transplants (25) suggest that the incidence of hyperlipidemia and hypercholesterolemia is substantially lower with tacrolimus than with CsA.

There is increasing evidence that hyperlipidemia could be involved in the progression of renal diseases (26), and some authors suggested that lipid disturbances observed in renal transplant recipients could participate in the pathogenesis or progression of chronic rejection. Several groups reported that chronic renal vascular rejection was associated with significantly higher TC and TG observed either before or after transplantation (6,27); in contrast, Guijarro et al.(7) observed an increase only in TG levels. Our study clearly showed a difference between transplant patients and controls, but it failed to confirm the difference in lipid parameters observed by others in patients with or without chronic vascular rejection (Table 2). Taken together, these data suggest that lipid abnormalities are probably a facilitating factor for CR but when isolated are not able to initiate CR. The simultaneous presence of an oxidative stress, a well-established factor for atherosclerosis, may be essential for lipid toxicity.

Concerning oxidative stress, we observed in transplant patients a significant increase in MDA, an end product of oxidative stress, as well as a decrease in RBC PUFAs, the main target of reactive oxygen species(28). In agreement with these results, RBC defense mechanisms are impaired, as demonstrated by the significant decrease in RBC Vit E and SOD activity. By contrast, plasma Vit E and plasma GPx were normal or increased (Table 4). It is well known that plasma Vit E levels are strongly correlated with cholesterol and triglyceride levels and require standardization. Adjustment for the sum of cholesterol and triglycerides can reduce the influence of hyperlipidemia (29,30). In transplant recipients, levels of normalized vitamin E are similar to those of controls, which suggests that the increase in absolute plasma Vit E was linked to the posttransplant-induced hyperlipidemia. On the other hand, in vitro studies and animal models have shown that GPx could be induced by reactive oxygen species and particularly by H202(31-33). Thus, the observation of increased GPx levels could be an adaptative mechanism.

Oxidative stress was commonly observed in hemodialysis patients(34-38), and it could be anticipated that successful renal transplantation would restore the balance between reactive oxygen species production and defense mechanisms. In fact, persistence of oxidative stress after successful transplantation was reported by several authors (37,39,40). Long-term immunological stimulation by the allograft and maintenance treatment with CsA may enhance reactive oxygen species production. In vitro, CsA has been shown to increase lipid peroxidation in microsome systems (41,42). Moreover, in a model of unilateral nephrectomy in the rat, CsA treatment was associated with an increase in cortical MDA and conjugated diene (43). In clinical transplantation, oxidative stress markers were elevated in heart allograft transplant recipients receiving CsA (44). Enhanced susceptibility to in vitro copper-induced LDL oxidation has been reported in CsA-treated renal transplant recipients (45), and conversion from CsA to azathioprine restored normal susceptibility of LDL to copper-induced oxidation (46). In addition to this in vitro-enhanced LDL oxidation, Ghanem et al. (47) have reported an increase in titer and concentration of autoantibodies against MDA-modified LDL, suggesting the presence of "in vivo" LDL oxidation in transplant recipients.

Our study shows that oxidative stress markers are significantly increased in patients with chronic rejection when compared with those with normal renal function (Fig. 1), despite both groups receiving CsA at similar doses and levels (Table 2). Furthermore, RBC Vit E and SOD levels were dramatically decreased in CR patients when compared with controls, although they were not statistically different between patients with or without CR (Fig. 2). In our opinion, two factors may have negatively influenced the sensitivity of our analysis concerning antioxidant defenses in group II versus I: the cross-sectional design (some patients with more aggressive forms of CR were excluded because they had already lost their grafts) and the low number of patients included. The simultaneous presence of hyperlipidemia and enhanced lipid peroxidation suggests that oxidative stress may play a role in the development and/or progression of the vascular lesions observed in CR. A causal relationship between increased lipid peroxidation and accelerated arteriosclerosis was observed in heart transplant recipients, but this relation is not definitively demonstrated because most, if not all, heart transplant recipients develop coronary artery disease within the first year after transplantation (44). A ballooning lesion of the arterial wall is generally considered a relevant model for transplant arteriosclerosis (48). In such models, early vitamin E supplementation largely prevents the progression of vascular lesions(49,50). On the other hand, the observed markers of oxidative stress may be a consequence of the decrease in renal function due to chronic rejection, as oxidative stress was described in chronic renal failure patients before dialysis (51), as well as in experimental glomerulonephritis (52). However, we did not observe any correlation between MDA levels, SOD activity, or RBC Vit E and serum creatinine levels or the degree of proteinuria.

In summary, in renal transplant recipients, particularly those suffering from CR, we observed the simultaneous presence of dyslipidemia and oxidative stress, two well-known risk factors for arteriosclerosis. It could be speculated that oxidative stress enhances the toxicity of lipids and thus plays a role in the initiation or progression of CR. Oxidative stress and lipid disorders certainly act additively with other factors in the progression of CR. Indeed, the occurrence of oxidized LDL in the arterial wall of the renal allograft could be involved in the generation of foam cells or could enhance the mitogenic activity of other growth factors for smooth muscle cells as platelet-derived growth factor or basic fibroblast growth factor (53). Prospective monitoring of oxidative stress and further studies with either hypocholesterolemic drugs or exogenous antioxidant compounds are needed to clarify this important issue and are under investigation in our renal transplant population.

Acknowledgments. The authors thank Dr. Angel Argiles for useful discussion of the manuscript.

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