EVALUATION OF LIPIDS PEROXIDATION PRODUCTS VS. PROINFLAMMATORY CYTOKINES IN HEMODIALIZED PATIENTS

Abstract

Background: Patients with end-stage renal failure on chronic hemodialysis often demonstrate accelerated development of atherosclerotic changes and cardiovascular complications. In those patients oxidative stress facilitates the intensity of lipid peroxidation process, expressed as increased products of lipid peroxidation (malonaldehyde and 4-hydroxyalkenals). Simultaneously, structures modified by peroxidation and glycation of autoantigenic character are formed. Additionally, increase in proinflammatory cytokines is found in their patients on hemodialysis. The aim of the study was to find out the relations between the products of lipid peroxidation and proinflammatory cytokines in patients on hemodialysis. Methods: Plasma concentrations of malonaldehyde and 4-hydroxyalkenals were estimated by spectrophotometric method, glutathione peroxidase as well as concentration of IL-6, its soluble receptor and TNFα were measured using ELISA kits. Results: Aldehyde concentrations (malonaldehyde and 4-hydroxyalkenals) were found to be five fold higher in comparison with the controls. Concentrations of proinflammatory cytokines: IL-6, TNFα and soluble IL-6 receptor were also higher than in the control group. Patients on hemodialysis showed positive correlation between concentrations of MDA + 4HNE and IL-6 and TNFα. Conclusion: The correlations found between the products of lipid peroxidation and proinflammatory cytokines suggest causative relation between the intensity of peroxidative processes and stimulation of immunological response in hemodialysed patients, which may increase the risk of atherosclerotic changes in those patients.

• Hemodialysis
• Lipid peroxidation products
• Proinflammatory cytokines
• Glutathione peroxidase

INTRODUCTION

Patients with end-stage renal failure on hemodialysis present with accelerated development of atherosclerosis and cardiovascular complications. Well documented is the role of impaired lipid and lipoprotein metabolism in the formation of atherosclerotic changes. Hemodialysed patients have distorted lipoprotein profile, manifested as hyperglyceridemia, lowered concentration of HDL cholesterol and increased concentration of LDL lipoprotein peroxidation products such as malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE) [1-3].

Many modified structures form in the process of LDL lipoprotein peroxidation, which in combination with apolipoprotein B lysine become highly immunogenic [[4]].

Simultaneously, those patients have increased proinflammatory cytokines observed: TNFα, IL-6 and soluble IL-6 receptor [5-6].

The aim of the study was to find out if there was any dependence between the concentrations of lipid peroxidation products expressed as sum of MDA+4-HNE concentrations and proinflammatory cytokines: TNFα, IL-6 and soluble IL-6 receptor (sIL-6R).

SUBJECTS AND METHODS

The study covered 33 patients with chronic renal failure treated by hemodialysis in the nephrological clinic. The disease was caused by: glomerulonephritis (23 cases), renal hypoplasia (3 cases), polycystic kidney disease (2 cases), renal failure due to stasis (2 cases), bilateral nephrostomy (1 case), renal failure in the course of lupus (1 case), diabetes (1 case). The control group consisted of 70 healthy individuals. Mean period of hemodialysis was 120 ± 84 months (24-288). The patients were hemodialysed for 4 hours 3 times a week. At first cuprophane membranes were used for dialysis, and for over a year polisulphonic membranes and bicarbonate fluids have been in use. The blood samples were collected prior to hemodialysis, before heparin was given. None of the patients had shown the symptoms of infection for at least a month nor taken immunosupressive or immunomodulating drugs. The blood was drawn with anticoagulant and immediately centrifuged. Plasma samples were used for further determination of cytokines, lipid peroxidation and glutathione peroxidase. The samples were kept frozen at temp. −70°C till the time of testing.

Lipid peroxidation products, expressed as a total plasma content of malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE), were determined by spectrophotometric method with the use of LPO-568 Bioxytech kits (Oxis International, Inc., USA). The test sensitivity was 0.1 nmol/mL, measurement precision for simultaneous series CV = 7.9%, repeatability CV = 9.2%.

Glutathione peroxidase concentration was measured by ELISA method with the use of Bioxytech pl-GPx EIA kits (Oxis International, Inc., USA); test sensitivity < 2.5 ng/mL.

To determine IL-6 concentration Hbt Human ELISA kit (HyCult Biotechnology b.v., Uden, Netherlands) was used; test sensitivity 10 pg/mL.

TNFα concentration was measured with the use of Predicta TNFα ELISA kit (Genzyme Diagnostic, USA); test sensitivity 3 pg/mL, repeatability for lower values: (37.2 pg/mL) CV = 6.5%, for high values (275.9 pg/mL) CV = 4.3%.

The concentration of sIL-6 was determined by soluble IL-6 receptor (ICN Pharmaceuticals, Inc. Diagnostic Division, USA); test sensitivity 6 ng/mL, repeatability for the values 78 ng/mL, CV = 4.3% and 42 ng/mL, CV = 5.8%.

Statistic analysis of the results were done with the Statistica 5.1 software (Stat Soft Inc.). To evaluate significance of differences of normal distribution Student t-test for independent variables was used. In the cases of no compatibility with normal distribution nonparametric tests were used. The dependence between particular parameters in the studied groups was assessed by Pearson's coefficient.

RESULTS

The obtained concentration values of lipoprotein peroxidation products, glutathione peroxidase, proinflammatory cytokines and soluble IL-6 receptor in patients with chronic kidney failure on hemodialysis are presented in Table 1

Table 1. Mean Concentrations of Lipid Peroxidation Products, Glutathione Peroxidase, Proinflammatory Cytokines, and Soluble IL-6 Receptor in Patients with Chronic Renal Failure on Hemodialysed Patients with Reference to Control Group

		Control Group		Hemodialysed
Parameter	Unit	Mean	SD	Mean	SD	r	p
MDA +4 HNE	μmol/L	2.02	1.48	10.12	3.64	10.633	0.0001
GPX	mg/mL	6.04	2.77	5.97	2.17	0.079	0.9371
TNFα	pg/mL	3.04	2.03	6.02	2.46	−6.108	0.0001
IL-6	pg/mL	1.09	2.51	16.15	13.88	−7.648	0.0001
SIL-6R	ng/mL	29.81	10.59	50.85	36.83	−3.355	0.0013

Hemodialysed patients demonstrated statistically significantly increased concentrations of malonaldehyde and 4-hydroxyaldehyds compared with the controls. Glutathione peroxidase was slightly lowered, however statistically insignificant. The concentrations of TNFα, interleukine 6 and its soluble receptor were statistically significantly increased in comparison with the controls, too.

The study also examined the correlations between the concentrations of aldehydes (MDA+4-HNE) and TNFα, IL-6 and soluble receptor for IL-6 (sIL-6R). In the group of patients on hemodialysis positive correlation between MDA+4-HNE and TNFα r = 0.3969 (p = 0.0270) as well as between MDA+4-HNE and IL-6 r = 0.3892 (p = 0.0387) were found.

Positive correlations between the concentrations of malonaldehyde+4-hydroxyaldehydes and TNFα and Il-6 are illustrated on Fig. 1-2

Figure 1. Relation between MDA+4-HNE and TNFα concentrations in patients on hemodialysis.

Figure 2. Relation between MDA+4-HNE and IL-6 in patients on hemodialysis.

DISCUSSION

The patients with chronic renal failure treated by hemodialysis have increased plasma concentrations of lipid peroxidation products, which suggests intensification of lipid peroxidation processes. Studies by other authors carried out in hemodialysed patients confirm that especially before hemodialysis there is a statistically significant increase in blood plasma concentrations of malonaldehyde and 4-hydroxyalkenals [[3]], [7-8] and a significant increase in the concentrations of thiobarbituric acid reacting substances (TBARS) [[9]].

It was confirmed in our own research. The patients on hemodialysis had the concentration of lipid peroxidation products, expressed as a sum of malonaldehyde and 4-hydroxyalkenals concentrations, up to 5× higher compared with the controls.

The formation of advanced glycation end products (AGEs) may be the factor responsible for the production of free radicals and lipid peroxidation in those patients. AGEs can form in patients with uremia, especially those hemodialysed, even at the normal glucose level. Impaired 3-deoxyglucosone reductase (3-DG) is one of the factors leading to AGE formation. In vivo 3-DG formation does not only come from Amadori product but also fructose and fructose-3-phosphate. In healthy organism, it gets reduced quickly and is eliminated with urine out of the body. However in patients with uremia due to low activity of 3-DG reductase the product is accumulated, which contributes to AGE formation [[10]]. Hemodialysed patients have much increased concentrations of pentosidine and N^ε-carboxymethylolisine (CML) [11-12]. They also have proteins modified by peroxidation, which analogically to AGEs were called advanced oxidation protein products (AOPP) [[13]].

Cellular protective mechanism against adverse effects of free radicals is antioxidation system which includes glutathione peroxidase (GPX). It is supposed decreased activity of antioxidants and increased concentration of free radicals results in intensified oxidative stress and development of cardiovascular complications in hemodialysed patients. In case of chronic renal failure blood plasma glutathione peroxidase activity decreases due to selenium deficit [[14]], presence of uremic inhibitor [15-16] as well as glutathione peroxidase glycation.

Our research evaluating glutathione peroxidase concentration (concentration measured by ELISA method) proved statistically insignificantly lower concentration of that enzyme.

The patients dialysed for chronic renal failure suffer from disfunctions of the immunological system. The majority of immunological processes are shaped by cellular interactions where cytokine network plays a decisive role. Hemodialysed patients had higher concentrations of TNFα, IL-6 compared to the reference group. Differences in concentrations were statistically significant. Other researchers report similar results [[5]], [[17]], also pointing increased cytokine synthesis.

Hemodialysis leads to chronic monocyte stimulation due to contact with incompatible dialysing membrane, complements activation and the presence of endotoxines in dialysing fluid [18-19]. IL-6 has a relatively short half-life time. Afterwards it is eliminated with the urine whereas its soluble receptor stays in the circulation longer. Our research found increased concentration of soluble IL-6 receptor. High receptor concentration may be a trace of more durable activation of the immunological system. Apart from that, its impaired elimination with the urine may also contribute to increased soluble IL-6 receptor concentration.

The basic aim of the research was to find if hemodialysed patients had dependences between parameters, which indirectly inform about lipid peroxidation processes, and activation of immunological response expressed as increased cytokine synthesis and secretion. We started from the assumption that LDL lipoprotein modified by peroxidation and glycation become autoantigenic stimulating immunocompetent cells for cytokine synthesis. In this way immunological activation with subsequent inflammatory process and secondary lipidemia with the growth in peroxidation of lipoprotein fraction could participate together in the initiation and progression of atherosclerotic changes. The research proved hemodialysed patients have positive correlations between the concentrations of lipid peroxidation products and TNFα and IL-6. The above data, especially those referred to the control group, seem to confirm the fact of immunological stimulation following intensification of lipoprotein peroxidation processes, which contributes to the acceleration of artheromatomas development.