Ankara Ecz. Fak. Derg. J. Fac.

Pharm, Ankara
37 (2) 91 - 100, 2008 37 (2) 91 - 100, 2008

EFFECT OF HEMODIALYSIS ON OXIDATIVE STRESS IN PATIENTS

WITH CHRONIC RENAL FAILURE

KRONİK RENAL YETMEZLİKLİ HASTALARDA HEMODİYALİZİN OKSİDATİF

STRES ÜZERİNDEKİ ETKİSİ

Aysun HACIŞEVKİ
Gazi University, Faculty of Pharmacy, Department of Biochemistry,
06330, Etiler-Ankara, TURKEY.

ABSTRACT

Oxidative stress is at play in the progression of chronic renal failure (CRF) and in the genesis of
atherosclerosis.The aim of the present study was to evaluate the factors that might influence the oxidative-
antioxidative balance in patients on hemodialysis. The study group was consisted of 64 hemodialysis
patients due to CRF. Twenty-two healthy subjects constituted a control group. We measured changes in
serum superoxide dismutase and glutathione peroxidase activity, and malondialdehyde levels in chronic
renal failure patients and compared with healthy control groups. Superoxide dismutase and glutathione
peroxidase activity, and malondialdehyde levels were assayed with spectrophotometric methods. Superoxide
dismutase activity of CRF patients group were higher than those of control group (p<0.001). Glutathione
peroxidase activity of CRF patients group were lower than those of control group (p<0.001).
Malondialdehyde levels in hemodialysis patients were higher than those of control group (p<0.01). Several
studies of SOD activity in chronic renal failure patients have found conflicting results. We propose that the
increased SOD activity could be a protective mechanism for the cells due to the hyperproduction of free
radicals in chronic renal failure. Decreased serum antioxidant activity in CRF patients on hemodialysis may
contribute to the increased oxidative damage and in the development of renal complications. This study
indicates the existence and increased production of oxidative stress resulting from hemodialysis and
disturbance in antioxidant enzyme system. Our results supports that an increase in oxidative stress may be
considered as one of the major risk factors in chronic renal failure patients.

Key words: SOD, GPx, Malondialdehyde, Chronic renal failure, Antioxidant enzymes
92 Aysun HACIŞEVKİ

ÖZET

Oksidatif stres kronik renal yetmezlik gelişiminde ve ateroskleroz gelişiminde rol oynamaktadır. Bu
çalışmanın amacı hemodiyaliz hastalarında oksidatif-antioksidatif balansı etkileyebilen faktörleri
değerlendirmektir. Çalışmaya 64 hemodiyaliz hastası, kontrol grubu olarak ise 22 sağlıklı kişi dahil
edilmiştir. Kronik renal yetmezlikli hastalarda serum süperoksit dismutaz, glutatyon peroksidaz aktiviteleri
ve malondialdehid düzeylerindeki değişiklikler ölçüldü ve sağlıklı kontrol grubu ile karşılaştırıldı. Bu
parametreler spektrofotometrik method kullanılarak tayin edildi. Hasta grubunun serum süperoksit dismutaz
aktivitesi kontrol grubundan daha yüksek bulunmuştur (p<0.001), glutatyon peroksidaz aktivitesi ise daha
düşüktür (p<0.001). Kronik renal yetmezlikli hastaların malondialdehid düzeyleri kontrol grubundan daha
yüksektir (p<0.01). Kronik renal yetmezlikli hastalarda süperoksit dismutaz aktivitesine yönelik
çalışmalarda çelişkili sonuçlar gözlenmektedir. Bu hastalarda artmış süperoksit dismutaz aktivitesini, artmış
serbest radikal üretimine bağlı koruyucu bir mekanizma olarak değerlendirebiliriz. Çalışmamız, kronik renal
yetmezlikli hastalarda majör risk faktörlerinden biri olarak düşünülen oksidatif stresteki artışı
desteklemektedir.

Anahtar kelimeler: SOD, GPx, Malondialdehid, Kronik renal yetmezlik, Antioksidan enzimler

INTRODUCTION

Reactive oxygen species (ROS) play a key role in the pathophysiological pathways of a wide
variety of clinical and experimental renal diseases (1-3). These ROS, including the superoxide
anion, the hydroxyl radical, hypochlorous acid, and peroxynitrite may be generated by activated
neutrophils, monocytes, and messangial cells during metabolic processes (4). ROS have been
shown to be primary mediators in glomerulonephritis, and are factors in the regulation of
glomerular permeability to proteins, development of morphologic lesions, and alteration of
glomerular hemodynamics (ie, reductions of glomerular blood flow and glomerular filtration rate)
(5).

Oxidative stress has been defined as a loss of balance between the production of free radical
or reactive oxygen species and protective antioxidant systems. A profound imbalance between
oxidants and antioxidants has been suggested in haemodialysis patients. In normal conditions, both
systems are in a steady state in such a way that an increase in oxidative products is followed by a
greater production by antioxidant systems. This balance always favors the antioxidant arm so that
there is a safety zone (6,7).
Ankara Ecz. Fak. Derg., 37 (2) 91 - 100, 2008 93

There is increasing evidence about the presence of oxidative stress in chronic renal failure
patients, and particularly in those submitted to hemodialysis therapy. This seems to be due to
multiple factors including an increase in the production of agents from oxidative metabolism
(oxygen- derived substances generated by activated leucocytes, transition metal compounds, and
other toxins of different molecular weight), and a decrease in antioxidant defenses. Oxidative stress
generated in a physiologic or pathologic way and harms the cellular constituents including
membrane lipids, proteins, and DNA (6). Free radical-mediated changes are thought to be involved
with atherosclerosis in patients with chronic renal failure. Oxidative damage due to reactive oxygen
species has been reported to increase in uremic patients and has thus been suggested to be a
possible factor contributing to the pathogenesis of atherosclerosis in chronic renal failure. There
may be several potential sources of increased free radical production in chronic renal failure (8).
The aim of the present study was to evaluate the factors that might influence the oxidative-
antioxidative balance in patients on hemodialysis.

MATERIALS AND METHODS

CRF patients (34 females and 30 males) at Başkent University, Faculty of Medicine were
enrolled in the study. The study group consisted of 64 hemodialysis patients due to chronic renal
failure. The study was approved by the Başkent University Ethics Committee of Faculty of
Medicine. The weekly duration of dialysis was 12 h in 3-4 h sessions. A control group consisted of
22 healthy persons, with no clinical symptoms of any disease and with the markers of renal
function in the norm. All blood samples were collected from the ulnar vein, in the morning before a
dialysis session. Following the collection of blood, serum was seperated immediately. Samples
were centrifuged and the supernatant is removed and stored -70 ºC until the analysis.

We measured changes in superoxide dismutase and glutathione peroxidase activity, and

malondialdehyde levels in chronic renal failure patients and compared with healthy control groups.
The activities of both superoxide dismutase and glutathione peroxidase were determined with
Cayman commercial kits. The levels of thiobarbituric acid reactive substances (TBARS) were also
determined spectrophotometrically in terms of malondialdehyde (9).

All data are presented as means ±SE and were statistically analyzed using a SPSS 10 for
Windows. Mann Whitney U test were applied to determine the significance of biochemical
parameters among the groups. P value of <0.05 was considered as significant.
94 Aysun HACIŞEVKİ

RESULTS AND DISCUSSION

The general characteristics of hemodialysis (HD) patients and healthy subjects such as age,
quetelet index, sex distribution are presented in Table 1.

Table 1. The demographic and clinical characteristics in HD patients and in controls.

Parameters Healthy subjects HD Patients

Number of patients 22 64
Sex (Male/Female) 9/13 30/34
Age (year) 46.27±2.87 47.28±1.68
2
BMI (kg/m ) 23.97±0.61 22.69±0.55

Data are means±SE.

There are a great number of metabolic derangements in the course of CRF which become
intensified in end-stage of renal disease, when dialysis is required (10). Impairments of enzymatic
system have been reported such as glutathione peroxidase (GSH-Px) or superoxide dismutase
(SOD), deficiency of selenium, zinc, copper, vitamins A, C, and E, and also diminished glutathione
concentration in chronic renal failure (11). Oxidative stress is another accompaniment of CRF.
Gerardi et al (10) and Marnett (12) have found increased serum concentrations of lipid peroxidation
products malondialdehyde and 4-hydroxynonenal in hemodialysis patients. Mezzano et al (13) have
revealed increased concentration of thiobarbituric acid reactive substances , which is a product of
lipid peroxidation, and a higher level of advanced oxidation protein products in patients with
uraemia. Nguyen-Khoa et al (14) have shown, in addition to high levels of thiobarbituric acid
reactive substances and advanced oxidation protein products, a decreased activity of superoxide
dismutase and glutathione peroxidase (11).

Biochemical parameters and the measured parameters are being presented in Table 2.
Serum malondialdehyde levels, the biomarker of lipid peroxidation, were significantly increased in
HD patients compared to healthy subjects (p<0.01) (Fig.1).
Ankara Ecz. Fak. Derg., 37 (2) 91 - 100, 2008 95

Table 2. The demographic and clinical characteristics in serum of HD patients and controls.

Parameters Healthy subjects HD Patients

(n=22) (n=64)

Superoxide dismutase (U/ml) 5.56±0.84 9.88±0.69+

Glutathione peroxidase (nmol/min/ml) 121.85±5.82 56.69±1.85+
Malondialdehyde (µmol/L) 2.41±0.009 3.68±0.08*
Urea (mg/dL) 13.77±1.40 75.33±2.12*
Creatinine (mg/dL) 0.98±0.09 10.36±0.31*

Data are means±SE.

*
Significantly different from controls (p<0.01), + Significantly different from controls (p<0.001)

Oxidative stress is defined as an imbalance between oxidant production and antioxidant

protection. Oxygen free radicals induce damage to tissue in many clinical conditions. Also, it is
well known that, in the diseases in which excessive immunoinflammatory responses are present,
formation of free radicals are accelerated (6,7). Oxidative damage induced by reactive oxygen
species (ROS) is thought to contribute to the development of macrophage foam cells in the walls of
arteries, which may lead to lethal complications of chronic renal failure (CRF) patients such as
cardiovascular disease (15,16). CRF is a disease caused by damage to renal parenchyma by chronic
pathologic processes leading to decreased glomerular filtration rate (GFR). Causes of CRF are
multiple, with the predominance of primary glomerulonephritis (26.4 %), diabetic nephropathy
(19.2 %), tubulointerstitial nephritis (16.5 %), hypertensive nephropathy (8.9 %), and polycystic
kidney disease (8.9 %) (11).
There is increasing evidence about the presence of oxidative stress in chronic renal failure
patients, and particularly in those submitted to hemodialysis therapy. This seems to be due to
multiple factors including an increase in the production of agents from oxidative metabolism and a
decrease in antioxidant defenses. Besides, the use of low biocompatible membranes and purity of
dialysis water has an influence on oxidative stress (6,17-19).

When free radicals overwhelm the antioxidant barrier, they become available for interacting
with phospholipidic structures producing lipid peroxidation. One of the major components of this
reaction is the production of malondialdehyde, which identification would allow us having an idea
96 Aysun HACIŞEVKİ

of the magnitude of lipid peroxidation. The results found in our patients are in agreement with
those of many other authors regarding increased lipid peroxidation (6,20-21).

Malondialdehyde*
140
+ Superoxide dismutase+
Glutathione peroxidase+
120

100

80
+
60

40

20
*+ *+
0
HD Patients Controls
Fig 1. Serum malondialdehyde levels and antioxidant enzyme
activities in HD patients and controls.* Significantly different from
controls p<0.01.+ significantly different from controls p<0.001.

Serum superoxide dismutase levels were significantly increased in CRF patients compared to
healthy subjects (p<0.001) (Fig.1). The plasma glutathione peroxidase levels of patient group have
shown a significant decrease when compared with those of control group (p<0.001) (Fig.1). In
addition, no relationship was found between measured parameters and clinical parameters in both
cases and controls.

Several studies have shown decreased activity of glutathione peroxidase in patients with
renal disorders and chronic renal failure (6-7,22-26). Glutathione peroxidase protects cells from
oxidative damage by catalyzing the reduction of both organic and hydrogen peroxides, using
glutathione as a reducing agent. GPx activity is an important test to assess the oxidative damage in
patients with kidney diseases. Kidney proximal tubular cells are the main source of GPx activity in
the plasma. The progression of renal disorders is accompanied by a decrease in GPx activity (22).
Blood 'Se' levels are frequently reported to be lower in hemodialyzed patients (26). The integrity of
GPX requires adequate intake of 'Se' (27) and its deficiency causes low activity of GPX and
reduction in GPX-protein (apoenzyme) synthesis (28). During oxidative stress, inactivation of GPX
may occur, and on the other hand superoxide anion itself can inhibit peroxidase function (29). The
previous studies indicate an impairment of antioxidant systems and augmentation of oxidants
during haemodialysis sessions (30-31).
Ankara Ecz. Fak. Derg., 37 (2) 91 - 100, 2008 97

The superoxide dismutase enzyme is the front line of defense against reactive oxygen
species-mediated injury (32). Several studies of SOD activity in chronic renal failure patients have
found conflicting results (25-26,33-35). We propose that the increased SOD activities could be a
protective mechanism for the cells due to the hyperproduction of free radicals in chronic renal
failure. The increase in superoxide dismutase activity may originate from excess production of O2.-
in macrophage. The lowering of plasma antioxidant activity in CRF patients on hemodialysis may
contribute to the increased oxidative damage and in the development of renal complications.
During haemodialysis, uraemic toxicity, malnutrition and the progressive worsening of clinical
condition can lead to oxidative stress caused by an hyperproduction of oxidants including ROS and
uraemic toxins with pro-oxidant function, and
defective antioxidant protection. Losses of antioxidants via dialysis and the use of low
biocompatible membranes are the factors that may be responsible for the imbalance between
oxidative and antioxidative mechanisms in HD patients. All these factors contribute to the higher
oxidative stres in haemodialysis patients included in our study.

This study indicates the existence and increased production of an oxidizing stress resulting
from hemodialysis and disturbance in antioxidant enzyme system. We found enhanced oxidative
stress in all patient groups due to an increase in lipid peroxidation and reduced activities of
glutathione peroxidase. Our results supports that an increase in oxidative stress may be considered
to be as one of the major risk factors in chronic renal failure disease.

Acknowledgements: This study was supported by the Research Fund of Gazi University
(02/2006-04).

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Received: 03.09.2009

Accepted: 03.10.2009