Original Paper
Eur Surg Res 2004;36:206–209 DOI: 10.1159/000078854
Erdosteine Improves Oxidative
Damage in a Rat Model of Renal
Ischemia-Reperfusion Injury
A. Gurel
aF. Armutcu
aA. Cihan
bK.V. Numanoglu
cM. Unalacak
dDepartments of aBiochemistry and Clinical Biochemistry, bGeneral Surgery, cPediatric Surgery, and dFamily Medicine, Karaelmas University Faculty of Medicine Zonguldak, Turkey
Received: November 26, 2003
Accepted after revision: February 23, 2004
Dr. Ahmet Gürel
Zonguldak Karaelmas University, Faculty of Medicine Department of Biochemistry, 67600 Kozlu TR–44069 Zonguldak (Turkey)
Tel. +90 372 2610169, Fax +90 372 2610155, E-Mail dragurel@yahoo.com
ABC
Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com
© 2004 S. Karger AG, Basel 0014–312X/04/0364–0206$21.00/0 Accessible online at:
www.karger.com/esr
Key Words
Renal ischemia-reperfusion injuryW ErdosteineW Lipid
peroxidationW Antioxidant enzymesW MyeloperoxidaseW
Xanthine oxidase
Abstract
The aim of the present study was to determine the effects of erdosteine, a new antioxidant and anti-inflammatory agent, on lipid peroxidation, neutrophil infiltration, and antioxidant enzyme activities in a rat model of renal isch-emia-reperfusion (I/R) injury. Twenty-eight rats were di-vided into three groups: sham operation, I/R, and I/R plus erdosteine groups. After the experimental procedure, rats were sacrificed and kidneys were removed and pre-pared for malondialdehyde (MDA) levels, myeloperoxi-dase (MPO), xanthine oximyeloperoxi-dase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. MDA level, MPO and XO activities were significantly increased in the I/R group. On the other hand, SOD and CAT activities were found to be decreased in the I/R group compared to the sham group. Pretreatment with erdosteine significantly diminished tissue MDA level, MPO and XO activities. Our data support a role for erdosteine in attenuation in renal damage after I/R injury of the kidney, in part at least by inhibition of neutrophil sequestration and XO activity.
Copyright © 2004 S. Karger AG, Basel
Introduction
Tissue subjected to a period of ischemia undergoes morphological and functional damage, which increases during the reperfusion phase. Renal ischemia-reperfusion (I/R) injury represents a major complication of renal transplantation, heminephrectomies, and surgical treat-ment of suprarenal aortic aneurysms. Reperfusion of emic kidneys increases the hazardous effect of early isch-emic injury by release of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydro-gen peroxides and accumulation of activated neutrophils. This cascade of events is known as reperfusion injury [1]. In experimental renal ischemia, ROS sources include the electron transport chain, oxidant enzymes such as xan-thine oxidase (XO), and phagocytes. ROS cause lipid peroxidation of cellular membranes, denaturation of pro-tein, polysaccharide depolymerization, and deoxyribonu-cleotide degradation and, hence, disruption of the struc-tural integrity and capacity for cell transport and energy production [2]. The complex, interrelated sequence of events that underlies I/R injury involves priming the endothelium during ischemia to produce both free radi-cals and chemoattractants which, upon reperfusion, se-quester and activate neutrophils, thus amplifying the inju-ry. Like other body compartments, kidneys have enzymes (superoxide dismutase (SOD), catalase (CAT) and
Erdosteine in Kidney I/R Injury Eur Surg Res 2004;36:206–209 207
thione peroxidase (GPX)) and nonenzymic (tocopherols, carotenes, glutathione) antioxidant defenses to cope with this potential damage.
Erdosteine (CAS 84611-23-4) is a mucolytic agent and contains two blocked sulfhydryl groups that are released following its metabolic process. It has been shown that its active metabolites have exhibited free radical scavenging and anti-inflammatory activities [3, 4].
The objective of the present study was to investigate the protective effects of erdosteine on kidney after renal I/R by assessing the malondialdehyde (MDA) level, ities of antioxidant enzymes such as SOD, CAT and activ-ities of ROS producing enzymes such as myeloperoxidase (MPO) and XO in injured renal tissues.
Materials and Methods
Male Wistar albino rats of the same age, weighing between 250 and 300 g, were obtained from Zonguldak Karaelmas University Medical Faculty Experimental Research Center and housed in sepa-rate cages under standard conditions, with a 12/12 h light-dark cycle. The rats were given standard rat chow and water ad libitum.
Test Drug. Erdosteine (Ilsan, Turkey) was administered orally to rats at doses of 10 mg kg–1 b.w. day–1.
Twenty-eight adult male albino Wistar rats were randomly di-vided into three groups: group 1: sham operation (n = 10); group 2: I/R (n = 9), and group 3: I/R plus erdosteine (I/R + E) (n = 9). Rats were anesthetized with ether and right renal arteries were exposed via a midline laparotomy. Following unilateral renal ischemia (30 min) and reperfusion (30 min), right nephrectomy was performed. Ani-mals in the sham operation group underwent a surgical procedure similar to the other groups but the artery was not occluded.
After reperfusion, kidneys were removed, washed twice with cold saline solution, placed into glass bottles, labeled, and stored in a deep freeze (–30° C) until processing (maximum 10 h). Tissues were
homogenized in 4 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) using a glass homogenizer for 2 min at 5,000 rpm, after cutting the organs into small pieces with scissors. The homogenate was then centrifuged at 5,000 g for 60 min to remove debris. Clear upper supernatant fluid was taken and CAT activity and protein concentra-tion were carried out in this stage. The supernatant soluconcentra-tion was extracted with an equal volume of an ethanol/chloroform mixture (5/3, volume per volume (v/v)). After centrifugation at 5,000 g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at +4° C.
MDA Determination. The tissue MDA levels were determined by the method of Draper and Hadley [5] based on the reaction of MDA with thiobarbituric acid (TBA) at 95° C. In the TBA test reaction,
MDA and TBA react to form a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2–3 at 95° C for 15 min. The sample was mixed with 2.5 volumes of 10%
(w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation and the aliquot of supernatant was reacted with an equal volume of 0.67% TBA in a boiling water-bath for 15 min. After cooling, the absorbance was read at 532 nm.
Arbi-trary values obtained were compared with a series of standard solu-tions (1,1,3,3-tetramethoxypropane). Results were expressed as nanomoles per milligram tissue (nmol/mg tissue).
MPO Activity Determination. MPO activity was determined using a 4-aminoantipyrine/phenol solution as the substrate for MPO-mediated oxidation by H2O2 and changes in absorbance at 510 nm
were recorded. One unit of MPO activity is defined as that which degrades 1 Ìmol H2O2 min at 25° C. Data are presented as units per
gram tissue protein [6].
XO Activity Determination. XO activity was assayed spectropho-tometrically at 293 nm and 37° C with xanthine as substrate [7]. The
formation of uric acid from xanthine results in increase in absorben-cy. One unit of activity was defined as 1 Ìmol of uric acid formed per minute at 37° C, pH 7.5, and expressed in units per gram tissue
pro-tein.
SOD Activity Determination. Total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. [8]. The principle of the method is based on the inhibition of NBT reduction by the xanthine-XO system as a superoxide generator. Activity was assessed in the ethanol phase of the lysate after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per milliliter.
CAT Activity. CAT activity was determined according to Aebi’s method [9]. The principle of the method was based on the determina-tion of the rate constant (s–1, k) or the H
2O2 decomposition rate at
240 nm. Results were expressed as k (rate constant) per gram pro-tein.
Protein Assays. Protein assays in the samples were made by the method of Lowry et al. [10].
Statistical Analysis
All statistical analyses were carried out using SPSS statistical soft-ware (SPSS for Windows, Version 11.0). All data were presented in mean (B) standard deviations. Differences in measured parameters among the three groups were analyzed by Kruskal-Wallis test. Dual comparisons between groups that present significant values were evaluated with Mann-Whitney U test. The differences were consid-ered to be significant when the probability was ! 0.05.
Results
The results are shown in table 1. The kidney MDA lev-els were significantly increased in the I/R group compared to the sham operation group. Erdosteine attenuated the increase in the levels of MDA in kidney. Oxidant enzyme activities (MPO and XO) of kidney in the I/R group were significantly increased compared to the sham group. Oxi-dant enzyme activities did not increase in the erdosteine-administered group, and the differences in oxidant en-zyme activities between the erdosteine-treated group and sham group were not significant. There were statistically significant decreases in SOD and CAT activities in the I/R group in comparison with the sham and I/R + E groups.
208 Eur Surg Res 2004;36:206–209 Gurel/Armutcu/Cihan/Numanoglu/ Unalacak
Table 1. MDA level, free radical scavenger enzyme activities as well as CAT activity, and oxidant enzyme activities such as XO in kidney I/R injury
Groups n MDA nmol/mg tissue MPO U/g protein XO U/g protein SOD U/mg protein CAT k/g protein Sham 10 3.24B0.45 0.008B0.002 6.59B1.19 0.273B 0.028 0.444B0.076 I/R 9 4.56B1.09a 0.011B0.002b, c 8.09B1.83b, c 0.233B0.064d 0.330B0.103c, e I/R + E 9 3.41B0.81 0.008B0.002 6.67B1.55 0.281B0.62 0.414B0.097
MDA = Malondialdehyde; MPO = myeloperoxidase; XO = xanthine oxidase; SOD = superoxide dismutase; CAT = catalase; I/R = ischemia/reperfusion; I/R + E = ischemia/reperfusion + erdosteine.
a Statistically significant increase (p ! 0.01) compared to sham and I/R + E groups. b Statistically significant increase (p ! 0.01) compared to sham group.
c Statistically significant difference (p ! 0.05) between I/R and I/R + E groups. d Statistically significant decrease (p ! 0.05) compared to sham and I/R + E groups. e Statistically significant decrease (p ! 0.01) compared to sham group.
Discussion
Renal I/R injury results in decreased glomerular filtra-tion and renal blood flow and increased urine output characterized by natriuresis and impaired concentrating ability. The generation of ROS is a crucial step in the pathogenesis of tissue damage. Thus, consequences of the attack of biomolecules by ROS, such as lipid peroxida-tion, could result, thereby, in altering the structure of bio-logical membranes. Research on animal and clinical stud-ies have shown that there is a close relationship between a lipid peroxidative reaction and secondary pathological changes following reperfusion period [11]. In the present study, the levels of MDA, an end product of lipid peroxi-dation, are significantly increased in the I/R group. Erdos-teine significantly decreased MDA levels of tissues. This anti-lipoperoxidative effect of erdosteine may be ex-plained by its direct free radical scavenger property. Ho-soe et al. [12] demonstrated the selective scavenging
activ-ity of erdosteine and its metabolites for H2O2 and
hypo-chlorous acid. In addition, it has been documented that the S isomer of erdosteine is effective in protecting mice against lethal doses of paraquat which is able to from free radicals when administered intraperitoneally [4]. In some studies, MDA, which is an indicator of lipid peroxidation, was shown to be decreased by erdosteine treatment, and this shows that the increase of lipid peroxidation that is caused by ROS can be prevented by erdosteine treatment [13].
XO is the last enzyme in the pathway of degradation of purine derivatives from nucleic acids. During reperfu-sion, hypoxanthine, which has accumulated during
isch-emia, is metabolized to xanthine by XO. In this process, superoxide anion radical is converted to hydrogen perox-ide or hydroxyl radical. The stimulated generation of oxy-gen radicals is possibly responsible for the disturbance of cell membranes by lipid peroxidation and leads to tissue and organ damage. However, the role of XO in renal isch-emia reperfusion destruction is under debate. Although it was claimed in some studies that ROS produced by XO is important [14], it was claimed not to be important in some other studies [15]. Rhoden et al. [16] showed bio-chemically and histologically that XO inhibitor treatment results in a decrease of lipid peroxidation and improve-ment of renal functions. In our study, XO activity was significantly higher in the I/R group when compared with the sham group, while it was kept at normal level by erdosteine in the reperfusion phase. The mechanism of effect of erdosteine on XO activity is unknown, but we suggest that erdosteine may act as an inhibiting factor in XO activity during the reperfusion phase. This effect of erdosteine may be an important factor in decreased oxi-dative damage in such an animal model.
Post-ischemic tissues are known to accumulate large numbers of inflammatory cells. These cells, particularly neutrophils, are known to be primary mediators of reper-fusion injury. In this study, the tissue-associated MPO activity, which is an index of neutrophil infiltration, was increased in kidney tissues after I/R. MPO plays an important role in production of oxidants by neutrophils, which are potential sources of ROS and are considered to be major effector cells in remote organ damage. Accord-ing to our results, treatAccord-ing rats with erdosteine attenuated the increase in the tissue levels of MPO and MDA caused
Erdosteine in Kidney I/R Injury Eur Surg Res 2004;36:206–209 209
by the I/R condition. Hayashi et al. [17] reported that erdosteine inhibited neutrophil infiltration in mice intra-tracheally treated with LPS. In addition, it has been sug-gested that erdosteine exhibits antioxidant properties by blocking production of ROS in human neutrophils and suppresses the oxidative burst of human polymorphonu-clear leukocytes [3]. These effects may prevent damage to the cell membranes partly caused by oxygen free radicals released from polymorphonuclear leukocytes.
The antioxidation defense system is known to inhibit lipid peroxidation in mammalian tissues by destroying some of the ROS that has an important role in initiation of the lipid peroxidation process. The antioxidant defense system operates through enzymatic and nonenzymatic components. SOD and CAT are antioxidant enzymes par-ticipating in the detoxifying process of superoxide
radi-cals and H2O2 in subsequent reactions. We studied these
two enzymes to investigate the enzymic antioxidant status of rat kidneys after I/R. Some authors have reported that the SOD and CAT activities in kidney gradually de-creased after reoxygenation [18]. This decrease may be related to the consumption of activated enzymes against oxidative stress. In our study, it was observed that SOD and CAT activities were significantly lower in the IR group than the sham group and erdosteine treatment pre-vented a decrease of enzyme activities. The interaction of
erdosteine with antioxidant enzymes has been investi-gated by many other researchers. Yıldırım et al. [19] reported that erdosteine treatment in cisplatin-induced renal tubular damage causes an improvement in SOD, CAT and G SH-Px activities. Besides, Fadillioglu et al. [13] declared in a doxorubicin-induced cardiomyopathy study that erdosteine treatment caused an increase of tis-sue CAT and GSH-Px activities, but no alteration was observed in SOD activity. Improvement of antioxidant enzyme activity in the erdosteine group might be a result of the free radical scavenging effect of this drug. ROS might be cleaned from the environment with the help of free SH groups owned by erdosteine metabolites and enzyme consumption might be prevented this way.
This is the first study in which erdosteine was used to prevent I/R injury in the kidney. This study also demon-strates that prophylactic administration of erdosteine pro-tects kidneys from reperfusion injuries. In conclusion, it is important to inhibit lipid peroxidation to prevent renal I/R injury and we suggest that acute administration of erdosteine would be helpful in clinical practice, e.g. at reconstructive renal surgery and transplantation. Taken together, our data support a role for erdosteine in attenua-tion renal damage after I/R injury of the kidney, at least in part by inhibition of neutrophil sequestration and XO activity.
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