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The effect of etoricoxib on kidney ischemia–reperfusion injury in rats: A biochemical and immunohistochemical assessment
International Immunopharmacology 23 (2014) 179–185
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The effect of etoricoxib on kidney ischemia–reperfusion injury in rats: A biochemical and immunohistochemical assessment Bahadir Suleyman a,⁎, Abdulmecit Albayrak b,1, Nezahat Kurt c,2, Elif Demirci d,3, Cemal Gundogdu d,3, Mehmet Aksoy e,4 a
Department of Pharmacology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey Department of Pharmacology, Faculty of Medicine, Ataturk University, Erzurum, Turkey Department of Biochemistry, Faculty of Medicine, Ataturk University, Erzurum, Turkey d Department of Pathology, Faculty of Medicine, Ataturk University, Erzurum, Turkey e Department of Anesthesiology and Reanimation, Faculty of Medicine, Ataturk University, Erzurum, Turkey b c
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 10 June 2014 Accepted 23 June 2014 Available online 25 July 2014 Keywords: Etoricoxib Oxidant–antioxidant parameters Ischemia–reperfusion Bcl-2 Rat
a b s t r a c t The purpose of this study was to investigate the effect of etoricoxib on oxidative injury induced with ischemia– reperfusion (I/R) in rat kidney tissue in terms of biochemistry and immunohistochemistry. Male Albino Wistar rats were divided into renal I/R (RIR), 50 mg/kg etoricoxib + RIR (ETO-50), 100 mg/kg etoricoxib + RIR (ETO-100) and sham operation (SG) groups. Animals in the ETO-50 and ETO-100 groups were given etoricoxib by the oral route at dosages of 50 and 100 mg/kg, respectively. The RIR and SG groups were given distilled water as solvent. One hour after drug administration, 1 h of ischemia and 3 h of reperfusion were applied to the left kidneys of all rats (apart from SG) under 25 mg/kg thiopental sodium anesthesia. At the end of that time, kidneys were extracted and biochemical and immunohistochemical analyses were performed. Etoricoxib reduced, in a dose-dependent manner, levels of MDA, MPO and COX-2 that normally rise with I/R in rat kidney tissues. Etorixicob did not alter COX-1 activity at 50 and 100 mg/kg doses, but significantly prevented loss of tGSH in tissues with I/R. In addition, Bcl-2′ gene expression inhibited with I/R was prevented in renal tubular and glomerular cells. Furthermore, etoricoxib significantly decreased the caspase-3 gene expression which increased with I/R. Etoricoxib significantly prevented I/R injury in a dose-dependent manner. The results of this study show that etoricoxib treatment could decrease kidney injury during IR. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Renal ischemia is seen in partial nephrectomy, organ transplantation and various urological vascular surgical procedures [1,2]. Ischemic tissue being deprived of oxygen leads to increased loss of energy in cells, the accumulation of toxic metabolites, cell function compromise and cell death [3]. The severity of ischemic injury rises in parallel to the duration of ischemia [4]. Ischemic tissues are therefore reperfused. However, the plentiful molecular oxygen (O2) reaching the ischemic tissue through arterial blood in reperfusion leads to the formation
⁎ Corresponding author. Tel.: +90 4642123009/3407; fax: +90 4642123015. E-mail addresses: [email protected] (B. Suleyman), [email protected] (A. Albayrak), [email protected] (N. Kurt), [email protected] (E. Demirci), [email protected] (C. Gundogdu), [email protected] (M. Aksoy). 1 Tel.: +90 4422316566. 2 Tel.: +90 4422316612. 3 Tel.: +90 4422316569. 4 Tel.: +90 4422317928.
http://dx.doi.org/10.1016/j.intimp.2014.06.042 1567-5769/© 2014 Elsevier B.V. All rights reserved.
of excessive free oxygen radicals, a decrease in antioxidant defense mechanisms and oxidative stress [5]. Inflammation is important as well as free oxygen radicals in the formation and continuation and determining the severity of I/R injury [6]. Inflammation in I/R injury begins with an increase in cytosolic calcium concentrations in the ischemic period. Rising calcium levels induce phospholipase A2 activity in the cells and increase the synthesis of arachidonic acid from phospholipids. Cytosolic calcium also accelerates the production of prostaglandin from arachidonic acid by inducing cyclooxygenase (COX) activity [7]. Experimental studies on mice have shown that COX deficiency significantly increases I/R injury [8]. The COX enzyme has different isoforms. The COX-1 and COX-2 isoforms are the most investigated, and their functions have been examined in most detail. COX-1 is a structural enzyme responsible for cytoprotective activity in tissues [9]. COX-2 is induced in damaged tissues and leads to inflammation by increasing proinflammatory prostaglandins from arachidonic acid [10]. This information from the literature indicates that antioxidant and anti-inflammatory activity can be useful both before and after reperfusion of ischemic tissue. Studies show that COX-2 inhibitors protect
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tissue from I/R injury [11]. However, the use of classical non-steroidal anti-inflammatory agents that do not exhibit selectivity for COX-2 leads to severe side-effects associated with COX-1 inhibition [12]. The etoricoxib used in the experiment is a COX-2 selective inhibitor with anti-inflammatory, antipyretic and analgesic effects [13,14]. Etoricoxib has been reported to significantly prevent cerebral ischemia-related oxidative stress [15]. These data suggest that etoricoxib is an antiinflammatory, analgesic, antipyretic and antioxidant drug, which does not lead to COX-1 inhibition-related side effects (bleeding, gastric, intestinal and renal injury), and may be beneficial in the treatment of I/R injury. Our scan of the literature revealed no information concerning any protective effect of etoricoxib in renal damage induced in rats with I/R. The purpose of this study was therefore to perform a biochemical and immunohistochemical investigation of the effect of etoricoxib on damage induced with I/R in the rat kidney.
2. Materials and methods 2.1. Experimental animals Twenty-four male Albino Wistar rats weighing 230–240 g obtained from the Atatürk University Medical Experimental Practice and Research Center were used. Before the experiment, animals were housed and fed in groups at normal room temperature (22 °C). The “Atatürk University Animal Experiments Ethical Committee” (AÜHADYEK) confirmed that all stages of the study were in accordance with ethical norms through written approval No. 42190979-01-02/ 2237 dated 22 May, 2013.
2.2. Chemical substances
2.4. Biochemical procedures Supernatants were obtained from homogenates prepared from kidney tissues and MDA and tGSH levels and MPO enzyme activity in supernatants were determined using appropriate methods. 2.4.1. Sample preparation Tissue weighing 0.2 g was removed from each kidney. pH = 6 potassium phosphate buffer containing 0.5% HDTMAB (0.5% hegza dodecyltrimethyl ammonium bromide) for MPO assay in tissues, 1.15% potassium chloride solution for MDA and pH = 7.5 phosphate buffer for tGSH were made up to 2 mL and homogenized in ice. These were than centrifuged at 10,000 rpm for 15 min at + 4 °C, and the supernatant obtained was used as the analysis specimen. 2.4.1.1. Malondialdehyde (MDA) assay. The concentrations of tissue lipid peroxidation were determined by estimating MDA using the thiobarbituric acid test. In brief, the rat livers were promptly excised and rinsed with cold saline. To minimize the possibility of interference of hemoglobin with free radicals, blood adhering to the tissue was carefully removed. The tissue was weighed, and homogenized in 10 mL of 100 g/L KCl. The homogenate (0.5 mL) was added to a solution containing 0.2 mL of 80 g/L sodium lauryl sulphate, 1.5 mL of 200 g/L acetic acid, 1.5 mL of 8 g/L 2-thiobarbiturate and 0.3 mL distilled water. The mixture was incubated at 98 °C for 1 h. Upon cooling, 5 mL of n-bu-tanol: pyridine (15: l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4000 rpm. The absorbance of the supernatant was measured at 532 mn [16]. 2.4.1.2. Myeloperoxidase (MPO) activity assay. For MPO activity assay, we used oxidation reaction with H2O2 mediated by MPO in 4-amino antipyrin/phenol solution as substrate [17].
The etoricoxib used in the experiment was obtained from Merck Sharp & Dohme, England, and the thiopental sodium from İE Ulagay, Turkey.
2.4.1.3. Total glutathione (tGSH) assay. The DTNB [5,5′-dithiobis (2nitrobenzoic acid)] used for measurement is a disulfide chromogen and is easily reduced by sulfhydryl group compounds. The resulting yellow color is measured spectrophotometrically at 412 nm [18].
2.3. Experimental procedure
2.4.1.4. Superoxide dismutase (SOD) analysis. Measurements were performed according to Sun et al. [19] When xanthine is converted into uric acid by xanthine oxidase, SOD forms. If nitro blue tetrazolium (NBT) is added to this reaction, SOD reacts with NBT and a purple colored-formazan dye occurs. The samples were weighed and homogenized in 2 mL of 20 mM phosphate buffer containing 10 mM EDTA at pH 7.8. The samples were centrifuged at 6000 rpm for 10 min and then the supernatants were used as assay samples. The measurement mixture, containing 2450 μL measurement mixture (0.3 mM xanthine, 0.6 mM EDTA, 150 μM NBT, 0.4 M Na2CO3, 1 g/L bovine serum albumin), 500 μL supernatant and 50 μL xanthine oxidase (167 U/L), was vortexed, and then it was incubated for 10 min. At the end of the reaction, formazan is produced. The absorbance of the purplecolored formazan was measured at 560 nm. As more of the enzyme is present, there is less least O2·− radical that reacts with NBT.
Experimental animals were divided into four groups—renal ischemia– reperfusion control (RIR), 50 mg/kg etoricoxib + renal ischemia– reperfusion (ETO-50), 100 mg/kg etoricoxib + renal ischemia– reperfusion (ETO-100) and a healthy group (SG) scheduled for a sham operation.
2.3.1. Surgical and pharmacological procedures Ischemia and post-ischemia reperfusion procedures were performed under sterile conditions with 25 mg/kg intraperitoneal (i.p.) thiopental sodium anesthesia. One hour before thiopental sodium anesthesia, etoricoxib was administered orally by pipette in doses of 50 mg/kg to the ETO-50 group and 100 mg/kg to the ETO-100 group. The RIR and SG rats were given distilled water as a solvent by the same route. During anesthesia, the left kidneys of all rats were accessed and extracted from the dorsal region. Subsequently (apart from the SG group) ischemia was induced for 1 h by attaching clips to the renal artery and veins. At the end of this period reperfusion was established for 3 h in the RIR ETO-50 and ETO-100 groups (by removing the clips). At the end of that period, RIR, ETO50 and ETO-100 group rats were sacrificed under high-dose anesthesia and the kidneys subjected to I/R were extracted. Biochemical and immunohistochemical examinations were performed on the extracted kidneys. Biochemical and immunohistochemical results from the ETO-50 and ETO-100 groups were compared with those from the RIR and SG groups.
2.4.1.5. Catalase (CAT) analysis. Catalase activity was determined by measuring the rate of decay of H2O2 absorbance at 240 nm. CAT activity was expressed as k/g protein [20]. 2.4.1.6. COX activity analysis. A COX activity kit measures COX POX activity. The reaction principle is based on the colorimetric measurement of oxidized N,N,N,N′-tetramethyl-p-phenylenediamine (TMPD) formation at 590 nm. 2.4.1.7. Experimental protocol. Into the COX standard well, specimen and specimen blank wells were placed 150 μL analysis buffer and 10 μL hem solution. Subsequently, 10 μL from the standard, specimen and inactive
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specimens was added to the wells; 140 μL analysis buffer, 10 μL hem solution, 10 μL specimen and 10 μL SC-560 solution were added to the inhibitor wells. After rinsing for a few seconds in a plate rinser, these were left to incubate for 5 min at 25 °C. Following incubation, first 20 μL colorimetric substrate and then 20 μL arachidonic acid were added to all wells. The plate was mixed in a mixer for a few seconds; after incubation for 5 min at Plate 25 C′ absorbances were read at a wavelength of 590 nm. Total COX activity and COX-1, COX-2 activities were calculated using the formula given below. Once total COX activity had been calculated for every specimen, the COX activities of specimens treated with SC-560 were calculated. These results represented specimens' COX-2 activities. COX-1 activities were calculated by subtracting COX-2 activities from total COX activities. The amount of enzyme oxidizing 1 nmol of TMPD in 1 min at 25 °C was taken as 1 enzyme unit and was given as enzyme activities (unit/wet g tissue) in specimens.
Total COX Activity ¼
ΔA590=5 min 0:21 mLðTVÞ 2 0:00826 Ë cM−1 0:01 mLðSVÞ
TH: total volumeNH: specimen volume*2 mol TMPD is used for the reduction of PGG2 to PGH2. The results were therefore divided into two. 2.5. Immunohistochemical procedures For immunohistochemical procedures, 4-μm-thick sections from rat kidney materials kept in 10% formalin were placed on a positivelycharged slide. Examinations were performed under an Olympus BX51 microscope, and interstitium, glomerular and tubular structures were evaluated separately using the grading system given below on the basis of Bcl-2 staining patterns. Extension and intensity were considered separately in this evaluation. Extension shows the area in which stain is present, while intensity shows the degree of staining. At grading for extension, grade I refers to less than 10% of cells being stained, grade II indicates 10–50% staining and grade III indicates that more than 50% are stained. At grading for intensity, grade I indicates mild staining, grade II moderate staining and grade III intense staining. 2.6. Statistical analysis Results from the experiments were expressed as “mean ± standard error” (x ± SEM). Significance of differences between groups was determined using one-way ANOVA, followed by Fisher's post-hoc LSD
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(least significant differences). All statistical procedures were performed on “IBM SPSS Statistics Version 20” statistical software, and significance was set at p b 0.05. 3. Results 3.1. Biochemical results 3.1.1. MDA, MPO, tGSH, SOD, CAT, COX-1 and COX-2 analysis results As seen in Fig. 1, the MDA amount is 8.3 ± 0.5 in kidney tissue of rats which received 50 mg/kg of etoricoxib; it was 3.1 ± 0.4 μmol/g protein in the dose of 100 mg/kg. Etoricoxib developed I/R in both doses, and significantly reduced the MDA elevation in kidney tissue compared to the RIR control group (P b 0.0001). The MPO activity in kidney tissue of rats which received 100 mg/kg etoricoxib (5.5 ± 0.7) was found to be lower than that of rats which received 50 mg/kg etoricoxib (12.3 ± 1.3) However, the statistical significance level of both doses was the same (P b 0.0001) (Fig. 1). While the tGSH activity was more significantly higher in the kidney tissue of rats which received 100 mg/kg of etoricoxib (12.6 ± 1.2) (P b 0.0001), the significance level was P b 0.05 in the dose of 50 mg/kg (6.3 ± 0.7) (Fig. 1). The SOD (21.8 ± 2) and CAT (18.8 ± 2.7) activities in 100 mg/kg of etoricoxib were calculated to be higher and more significant (P b 0.0001) (Fig. 1). As seen in Fig. 2, the COX-1 activity was found to be almost the same in the kidney tissues of the RIR, ETO-50, ETO-100 and the SG groups. The difference in the COX-1 activity between all these groups was not statistically significant. However, etoricoxib suppressed the COX-2 activity significantly in doses of 50 mg/kg (4.5 ± 0.7) and 100 mg/kg (2.3 ± 0.5) compared to the RIR group (P b 0.0001) (Fig. 2). 3.2. Immunohistochemical results 3.2.1. Bcl-2 results As seen in Fig. 3a, the staining area and the staining density in the tubular cells of the RIR group were determined as grade I. The staining area and the staining density in glomerular cells of the RIR group were also determined as grade I (Fig. 3b). This condition indicates that the staining area of the tubular and glomerular cells was below 10% and the staining density was low (Bcl-2 x 200). However, the staining area of the tubular cells in the ETO-50 group was grade III and the density was grade I (Fig. 3c). This indicates that more than 50% of the tubular cell area of the ETO-50 group was stained mildly (Bcl-2 x 200). The
Fig. 1. The effect of etoricoxib on MDA, MPO, tGSH, SOD and CAT levels in kidney tissue after induction of I/R. The ETO-50, ETO-100 and SG groups were compared with the RIR group and p b 0.05 was regarded as significant. Results are expressed as mean ± standard error (N = 6).
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as grade III and the density was determined as grade II (Fig. 3f). These findings reveal that more than 50% of the tubular and glomerular cell area of the ETO-100 group were stained moderately (Bcl-2 x 200). While the staining area of the tubular cells of the SG group was above 50%, the staining intensity was found to be intense (Fig. 3g). The extensiveness and the density of Bcl-2 in glomerular cells of the SG group were the same as those of the tubular cells (Fig. 3h). This microscopic appearance indicates that the staining area and the density of tubular and glomerular cells of the SG group were grade III (Bcl-2 x 200).
Fig. 2. COX-1 and COX-2 activities in RIR, ETO-50, ETO-100 and SG group rat kidney tissue. Groups were compared with RIR and p b 0.05 was regarded as significant. Results were expressed as mean ± standard error. (N = 6).
staining area of the glomerular cells in the ETO-50 group was observed as grade III and the density was grade II (Fig. 3d). This indicates that more than 50% of the glomerular cells were stained moderately (Bcl-2 x 200). While the staining area in the tubular cells of the ETO-100 group was grade III and the density was grade II (Fig. 3e), the staining area in the glomerular cells was determined
3.2.2. Caspase-3 results As observed in Fig. 4a, the staining extensiveness and density were determined as grade III in the tubular cells; however, while the staining extensiveness was grade III in the glomerular cells, the density was grade II (Fig. 4b, caspase 3x200) in the RIR group. The staining extensiveness and the density of renal tubular cells of the ETO-50 group (grade II) decreased compared to that of the RIR group (Fig. 4c, caspase 3x200). While the extensiveness was grade II in the glomerular cells of 50 mg/kg etoroxib- receiving group, the density was grade I (Fig. 4d, caspase 3x200). Etoricoxib 100 mg/kg decreased the staining density in the tubular cells (grade I) compared to the 50 mg/kg-receiving group; however, the extensiveness was the same in both groups (grade II); the extensiveness and the density were the same (grade I) in the glomerular cells of the 100 mg/kg etoricoxib-receiving group (Fig. 4e, caspase 3x200). The extensiveness and the density in the tubular and glomerular cells of the SG group were found to be the same as those of the ETO-100 group (Fig. 4f, caspase 3x200).
Fig. 3. (3a) Extension and intensity of Bcl-2 in RIR group renal tubular cells. (3b) Extension and intensity of Bcl-2 in RIR group renal glomerular cells. (3c) Extension and intensity of Bcl-2 in ETO-50 group renal tubular cells. (3d) Extension and degree of staining of Bcl-2 in ETO-50 group renal glomerular cells. (3e) Extension and degree of staining of Bcl-2 in ETO-100 renal tubular cells. (3f) Extension and intensity of Bcl-2 in ETO-100 renal glomerular cells. (3g) Extension and intensity of Bcl-2 in SG renal tubular cells. (3h) Extension and degree of staining of Bcl-2 in SG renal glomerular cells.
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Fig. 4. (4a) Extension and intensity of caspase-3 in RIR group renal tubular cells. (4b) Extension and intensity of caspase-3 in RIR group renal glomerular cells. (4c) Extension and intensity of caspase-3 in ETO-50 group renal tubular cells. (4d) Extension and intensity of caspase-3 in ETO-50 group renal glomerular cells. (4e) Extension and degree of staining of Bcl-2 in ETO-100 renal tubular (arrow) and glomerular cells (star). (4f) Extension and intensity of Bcl-2 in SG renal tubular (arrow) and glomerular cells (star).
4. Discussion This study investigated the effect of etoricoxib on damage induced with I/R in the rat kidney in biochemical and immunohistochemical terms. Our results show that the I/R process leads to marked oxidative injury in kidney tissue. Studies of the damage mechanism in I/R have shown that large quantities of free oxygen radicals are produced with reperfusion of ischemic tissue [21]. The most harmful effect caused in cells by free oxygen radicals is lipid peroxidation [22]. This reaction starts with lipid radical formation and concludes with the formation of the more toxic MDA [23]. In our study, the level of MDA in kidney tissue subjected to I/R was significantly higher in the ETO-50 group compared to the ETO-100 and SG rat groups. The decreased MDA amount in the injured kidney tissue which received etoricoxib suggests that 50 and 100 mg/kg doses of etoricoxib are effective against IR-related oxidative stress. A large number of studies have shown that MDA concentrations rise in tissue damaged by I/R [24,25]. Altuner D et al. reported that the I/R procedure causes oxidative damage in kidney tissue [26]. This shows that our experimental results are compatible with those from the literature. MPO activity was also higher in kidney tissue in which I/R was induced than in the SG and etoricoxib groups. MPO is known as a parameter of polymorph nuclear leukocyte (PNL) infiltration and activation [27]. Experimental studies on animals have shown an association between degree of PNL activation and degree of reperfusion injury [28]. 50 and 100 mg/kg doses of etoricoxib have statistically significantly decreased the MPO activity which had increased with IR in kidney tissue. However, there was a higher decrease in the MPO activity at the dose of 100 mg/kg compared to 50 mg/kg. This result suggests that the protective effect of etoricoxib against
IR injury could be associated with the inhibition of PNL activation. One of the most important endogenous antioxidants in defense against oxidative damage is tGSH. Elevated GSH is an indicator of cell function and vitality. A fall in GSH levels is regarded as a damage marker [29]. Studies have shown that glutathione levels decrease in the kidney during the I/R period and that this decrease arise from intense oxidative stress throughout I/R [30]. The results of this study indicate that oxidative IR injury caused by the decreased tGSH amount is preserved with etoricoxib and that the antioxidant effect of etoricoxib and its protective effects on tGSH are more prominent in high dose of etoricoxib. Tok A et al. revealed that the I/R process leads to a decrease in tGSH levels in the rat kidney and causes severe oxidative damage, and that this damage can be reduced with antioxidant activity [31]. Furthermore, etoricoxib protected the SOD activity to a higher extent with 100 mg/kg compared to 50 mg/kg in kidney tissue with IR. As known, SOD is an endogenous antioxidant and its activity decreases in damaged tissues [32]. Yang S et al. also reported that SOD activity decreased in kidney tissue with IR [24]. CAT is another antioxidant, which decreased in IR-induced renal tissue. CAT levels were found to be statistically significantly higher in groups which received 50 mg/kg and 100 mg/kg etoricoxib compared to the RIR group. Etoricoxib was seen to protect the CAT level statistically significantly better at the dose of 100 mg/kg in the tissue subjected to oxidative stress. It was reported that IR decreased the CAT level in kidney tissue subjected to IR-induced oxidative stress, and antioxidant treatment protected the kidney tissue from IR injury [33]. While the oxidant/antioxidant balance changed in favor of oxidants in the RIR group, this balance was in favor of antioxidants in the ETO-50 and ETO-100 groups. The oxidant/antioxidant balance is maintained with superiority of antioxidants in healthy tissues.
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Various aggressive factors which may lead to tissue injury impair the oxidant/antioxidant balance in favor of oxidants [34]. COX-2 activity in the RIR group in our study was also significantly lower than that in the ETO-50, ETO-100 and SG group kidney tissues. COX-1 is involved in the synthesis of cytoprotective prostaglandins [24]. COX-2 is activated with pro-inflammatory agents [35]. COX-2 production is lower in healthy tissues, but COX-2 activity exhibits a marked increase in inflamed tissues [36]. In our study, the COX-2 activity was determined to be lower than COX-1 activity in the kidney tissues of the healthy group, and the COX-2 activity was found to be higher in the RIR group. While etoricoxib in doses of 50 mg/kg and 100 mg/kg significantly prevented the COX-2 activity which increases with IR, it did not change COX-1 activity. Due to the fact that etoricoxib does not affect the COX-1 activity, it does not cause side effects such as renal failure, peptic ulcer and bleeding. As known, non-steroidal antiinflammatory drugs (NSAIDs) are effective in the treatment of acute postoperative pain. However, the side effects of NSAIDs, namely gastric irritation, peptic ulcer, renal failure, platelet inhibition and hemorrhage limit their use [9]. In previous studies too, amiphostin was reported to provide a renoprotective effect through inhibiting the COX-2 activity which increases in renal IR injury [37]. Senbel AM et al. reported that the COX-2 selective antagonist celecoxib protected the kidney tissue from COX-2 activation-related oxidative injury [38]. This information from the literature confirms the accuracy of our results. Again, in this study, Bcl-2 gene expression, an immunohistochemical parameter, was also weak in kidney tubular and glomerular tissue in the RIR group, in which MDA, MPO and COX-2 levels were high and tGSH levels were low. Etoricoxib also increased Bcl-2 gene expression in a dose-dependent manner. The Bcl-2 gene is an oncoprotein that plays an anti-apoptotic role. Bcl-2 inhibits apoptosis, and significantly reduces cell death initiated by various stimuli [39,40]. Ito T et al. also reported that Bcl-2 reduced cell death caused by free radicals [41]. These data indicate that Bcl-2s play a role in the protective mechanism of action of etoricoxib. Fennell DA et al. revealed that this protective effect of Bcl-2 was caused by preventing the release into cytoplasm of cytochrome-C and apoptosis inducing factor (AIF) from mitochondria [42]. The decreased caspase-3 gene expression in the tubular and glomerular cells of the kidneys in the ETO-50 and ETO-100 groups and the increased caspase 3 gene expression in the RIR group compared with the SG group support that etoricoxib suppresses apoptosis by inducing the Bcl-2 gene expression. This is because cytochrome C, which is released to cytoplasm from the mitochondria under stress, initiates the cascade, which activates procaspase-3 with cofactor of dATP, and chromosomal DNA is degraded and cell phagocytosis is provided [43]. That is, the degree of induction of caspase-3 may increase with cytochrome-C′s passing into the cytoplasm from the mitochondria. In addition, substances which inhibit cytochrome-C translocation have been reported to suppress the caspase-3 activation and increase the rate of Bcl-2 [44]. However, there are also studies indicating that some of the factors which release the cytochrome-C enzyme from the mitochondria into cytoplasm are in fact caspase-3 [45].
5. Conclusion In conclusion, renal IR has been understood to be a complex pathological process, which begins with the tissue being deprived of oxygen, continues with oxidative stress, progresses with inflammation, and leads to apoptosis. Etoricoxib is observed to suppress renal IR injury by causing antioxidant, anti-inflammatory and antiapoptotic effects. The protective effect of etoricoxib was found to be more potent at the dose of 100 mg/kg compared to 50 mg/kg. This study may be a reference for investigating the protective effect of other COX-2 selective inhibitors in IR and different tissue injury models. This study also suggests that etoricoxib, which is an antiinflammatory, analgesic, antipyetic and antioxidant drug that does
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The effect of etoricoxib on kidney ischemia–reperfusion injury in rats: A biochemical and immunohistochemical assessment Bahadir Suleyman a,⁎, Abdulmecit Albayrak b,1, Nezahat Kurt c,2, Elif Demirci d,3, Cemal Gundogdu d,3, Mehmet Aksoy e,4 a
Department of Pharmacology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey Department of Pharmacology, Faculty of Medicine, Ataturk University, Erzurum, Turkey Department of Biochemistry, Faculty of Medicine, Ataturk University, Erzurum, Turkey d Department of Pathology, Faculty of Medicine, Ataturk University, Erzurum, Turkey e Department of Anesthesiology and Reanimation, Faculty of Medicine, Ataturk University, Erzurum, Turkey b c
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 10 June 2014 Accepted 23 June 2014 Available online 25 July 2014 Keywords: Etoricoxib Oxidant–antioxidant parameters Ischemia–reperfusion Bcl-2 Rat
a b s t r a c t The purpose of this study was to investigate the effect of etoricoxib on oxidative injury induced with ischemia– reperfusion (I/R) in rat kidney tissue in terms of biochemistry and immunohistochemistry. Male Albino Wistar rats were divided into renal I/R (RIR), 50 mg/kg etoricoxib + RIR (ETO-50), 100 mg/kg etoricoxib + RIR (ETO-100) and sham operation (SG) groups. Animals in the ETO-50 and ETO-100 groups were given etoricoxib by the oral route at dosages of 50 and 100 mg/kg, respectively. The RIR and SG groups were given distilled water as solvent. One hour after drug administration, 1 h of ischemia and 3 h of reperfusion were applied to the left kidneys of all rats (apart from SG) under 25 mg/kg thiopental sodium anesthesia. At the end of that time, kidneys were extracted and biochemical and immunohistochemical analyses were performed. Etoricoxib reduced, in a dose-dependent manner, levels of MDA, MPO and COX-2 that normally rise with I/R in rat kidney tissues. Etorixicob did not alter COX-1 activity at 50 and 100 mg/kg doses, but significantly prevented loss of tGSH in tissues with I/R. In addition, Bcl-2′ gene expression inhibited with I/R was prevented in renal tubular and glomerular cells. Furthermore, etoricoxib significantly decreased the caspase-3 gene expression which increased with I/R. Etoricoxib significantly prevented I/R injury in a dose-dependent manner. The results of this study show that etoricoxib treatment could decrease kidney injury during IR. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Renal ischemia is seen in partial nephrectomy, organ transplantation and various urological vascular surgical procedures [1,2]. Ischemic tissue being deprived of oxygen leads to increased loss of energy in cells, the accumulation of toxic metabolites, cell function compromise and cell death [3]. The severity of ischemic injury rises in parallel to the duration of ischemia [4]. Ischemic tissues are therefore reperfused. However, the plentiful molecular oxygen (O2) reaching the ischemic tissue through arterial blood in reperfusion leads to the formation
⁎ Corresponding author. Tel.: +90 4642123009/3407; fax: +90 4642123015. E-mail addresses: [email protected] (B. Suleyman), [email protected] (A. Albayrak), [email protected] (N. Kurt), [email protected] (E. Demirci), [email protected] (C. Gundogdu), [email protected] (M. Aksoy). 1 Tel.: +90 4422316566. 2 Tel.: +90 4422316612. 3 Tel.: +90 4422316569. 4 Tel.: +90 4422317928.
http://dx.doi.org/10.1016/j.intimp.2014.06.042 1567-5769/© 2014 Elsevier B.V. All rights reserved.
of excessive free oxygen radicals, a decrease in antioxidant defense mechanisms and oxidative stress [5]. Inflammation is important as well as free oxygen radicals in the formation and continuation and determining the severity of I/R injury [6]. Inflammation in I/R injury begins with an increase in cytosolic calcium concentrations in the ischemic period. Rising calcium levels induce phospholipase A2 activity in the cells and increase the synthesis of arachidonic acid from phospholipids. Cytosolic calcium also accelerates the production of prostaglandin from arachidonic acid by inducing cyclooxygenase (COX) activity [7]. Experimental studies on mice have shown that COX deficiency significantly increases I/R injury [8]. The COX enzyme has different isoforms. The COX-1 and COX-2 isoforms are the most investigated, and their functions have been examined in most detail. COX-1 is a structural enzyme responsible for cytoprotective activity in tissues [9]. COX-2 is induced in damaged tissues and leads to inflammation by increasing proinflammatory prostaglandins from arachidonic acid [10]. This information from the literature indicates that antioxidant and anti-inflammatory activity can be useful both before and after reperfusion of ischemic tissue. Studies show that COX-2 inhibitors protect
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tissue from I/R injury [11]. However, the use of classical non-steroidal anti-inflammatory agents that do not exhibit selectivity for COX-2 leads to severe side-effects associated with COX-1 inhibition [12]. The etoricoxib used in the experiment is a COX-2 selective inhibitor with anti-inflammatory, antipyretic and analgesic effects [13,14]. Etoricoxib has been reported to significantly prevent cerebral ischemia-related oxidative stress [15]. These data suggest that etoricoxib is an antiinflammatory, analgesic, antipyretic and antioxidant drug, which does not lead to COX-1 inhibition-related side effects (bleeding, gastric, intestinal and renal injury), and may be beneficial in the treatment of I/R injury. Our scan of the literature revealed no information concerning any protective effect of etoricoxib in renal damage induced in rats with I/R. The purpose of this study was therefore to perform a biochemical and immunohistochemical investigation of the effect of etoricoxib on damage induced with I/R in the rat kidney.
2. Materials and methods 2.1. Experimental animals Twenty-four male Albino Wistar rats weighing 230–240 g obtained from the Atatürk University Medical Experimental Practice and Research Center were used. Before the experiment, animals were housed and fed in groups at normal room temperature (22 °C). The “Atatürk University Animal Experiments Ethical Committee” (AÜHADYEK) confirmed that all stages of the study were in accordance with ethical norms through written approval No. 42190979-01-02/ 2237 dated 22 May, 2013.
2.2. Chemical substances
2.4. Biochemical procedures Supernatants were obtained from homogenates prepared from kidney tissues and MDA and tGSH levels and MPO enzyme activity in supernatants were determined using appropriate methods. 2.4.1. Sample preparation Tissue weighing 0.2 g was removed from each kidney. pH = 6 potassium phosphate buffer containing 0.5% HDTMAB (0.5% hegza dodecyltrimethyl ammonium bromide) for MPO assay in tissues, 1.15% potassium chloride solution for MDA and pH = 7.5 phosphate buffer for tGSH were made up to 2 mL and homogenized in ice. These were than centrifuged at 10,000 rpm for 15 min at + 4 °C, and the supernatant obtained was used as the analysis specimen. 2.4.1.1. Malondialdehyde (MDA) assay. The concentrations of tissue lipid peroxidation were determined by estimating MDA using the thiobarbituric acid test. In brief, the rat livers were promptly excised and rinsed with cold saline. To minimize the possibility of interference of hemoglobin with free radicals, blood adhering to the tissue was carefully removed. The tissue was weighed, and homogenized in 10 mL of 100 g/L KCl. The homogenate (0.5 mL) was added to a solution containing 0.2 mL of 80 g/L sodium lauryl sulphate, 1.5 mL of 200 g/L acetic acid, 1.5 mL of 8 g/L 2-thiobarbiturate and 0.3 mL distilled water. The mixture was incubated at 98 °C for 1 h. Upon cooling, 5 mL of n-bu-tanol: pyridine (15: l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4000 rpm. The absorbance of the supernatant was measured at 532 mn [16]. 2.4.1.2. Myeloperoxidase (MPO) activity assay. For MPO activity assay, we used oxidation reaction with H2O2 mediated by MPO in 4-amino antipyrin/phenol solution as substrate [17].
The etoricoxib used in the experiment was obtained from Merck Sharp & Dohme, England, and the thiopental sodium from İE Ulagay, Turkey.
2.4.1.3. Total glutathione (tGSH) assay. The DTNB [5,5′-dithiobis (2nitrobenzoic acid)] used for measurement is a disulfide chromogen and is easily reduced by sulfhydryl group compounds. The resulting yellow color is measured spectrophotometrically at 412 nm [18].
2.3. Experimental procedure
2.4.1.4. Superoxide dismutase (SOD) analysis. Measurements were performed according to Sun et al. [19] When xanthine is converted into uric acid by xanthine oxidase, SOD forms. If nitro blue tetrazolium (NBT) is added to this reaction, SOD reacts with NBT and a purple colored-formazan dye occurs. The samples were weighed and homogenized in 2 mL of 20 mM phosphate buffer containing 10 mM EDTA at pH 7.8. The samples were centrifuged at 6000 rpm for 10 min and then the supernatants were used as assay samples. The measurement mixture, containing 2450 μL measurement mixture (0.3 mM xanthine, 0.6 mM EDTA, 150 μM NBT, 0.4 M Na2CO3, 1 g/L bovine serum albumin), 500 μL supernatant and 50 μL xanthine oxidase (167 U/L), was vortexed, and then it was incubated for 10 min. At the end of the reaction, formazan is produced. The absorbance of the purplecolored formazan was measured at 560 nm. As more of the enzyme is present, there is less least O2·− radical that reacts with NBT.
Experimental animals were divided into four groups—renal ischemia– reperfusion control (RIR), 50 mg/kg etoricoxib + renal ischemia– reperfusion (ETO-50), 100 mg/kg etoricoxib + renal ischemia– reperfusion (ETO-100) and a healthy group (SG) scheduled for a sham operation.
2.3.1. Surgical and pharmacological procedures Ischemia and post-ischemia reperfusion procedures were performed under sterile conditions with 25 mg/kg intraperitoneal (i.p.) thiopental sodium anesthesia. One hour before thiopental sodium anesthesia, etoricoxib was administered orally by pipette in doses of 50 mg/kg to the ETO-50 group and 100 mg/kg to the ETO-100 group. The RIR and SG rats were given distilled water as a solvent by the same route. During anesthesia, the left kidneys of all rats were accessed and extracted from the dorsal region. Subsequently (apart from the SG group) ischemia was induced for 1 h by attaching clips to the renal artery and veins. At the end of this period reperfusion was established for 3 h in the RIR ETO-50 and ETO-100 groups (by removing the clips). At the end of that period, RIR, ETO50 and ETO-100 group rats were sacrificed under high-dose anesthesia and the kidneys subjected to I/R were extracted. Biochemical and immunohistochemical examinations were performed on the extracted kidneys. Biochemical and immunohistochemical results from the ETO-50 and ETO-100 groups were compared with those from the RIR and SG groups.
2.4.1.5. Catalase (CAT) analysis. Catalase activity was determined by measuring the rate of decay of H2O2 absorbance at 240 nm. CAT activity was expressed as k/g protein [20]. 2.4.1.6. COX activity analysis. A COX activity kit measures COX POX activity. The reaction principle is based on the colorimetric measurement of oxidized N,N,N,N′-tetramethyl-p-phenylenediamine (TMPD) formation at 590 nm. 2.4.1.7. Experimental protocol. Into the COX standard well, specimen and specimen blank wells were placed 150 μL analysis buffer and 10 μL hem solution. Subsequently, 10 μL from the standard, specimen and inactive
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specimens was added to the wells; 140 μL analysis buffer, 10 μL hem solution, 10 μL specimen and 10 μL SC-560 solution were added to the inhibitor wells. After rinsing for a few seconds in a plate rinser, these were left to incubate for 5 min at 25 °C. Following incubation, first 20 μL colorimetric substrate and then 20 μL arachidonic acid were added to all wells. The plate was mixed in a mixer for a few seconds; after incubation for 5 min at Plate 25 C′ absorbances were read at a wavelength of 590 nm. Total COX activity and COX-1, COX-2 activities were calculated using the formula given below. Once total COX activity had been calculated for every specimen, the COX activities of specimens treated with SC-560 were calculated. These results represented specimens' COX-2 activities. COX-1 activities were calculated by subtracting COX-2 activities from total COX activities. The amount of enzyme oxidizing 1 nmol of TMPD in 1 min at 25 °C was taken as 1 enzyme unit and was given as enzyme activities (unit/wet g tissue) in specimens.
Total COX Activity ¼
ΔA590=5 min 0:21 mLðTVÞ 2 0:00826 Ë cM−1 0:01 mLðSVÞ
TH: total volumeNH: specimen volume*2 mol TMPD is used for the reduction of PGG2 to PGH2. The results were therefore divided into two. 2.5. Immunohistochemical procedures For immunohistochemical procedures, 4-μm-thick sections from rat kidney materials kept in 10% formalin were placed on a positivelycharged slide. Examinations were performed under an Olympus BX51 microscope, and interstitium, glomerular and tubular structures were evaluated separately using the grading system given below on the basis of Bcl-2 staining patterns. Extension and intensity were considered separately in this evaluation. Extension shows the area in which stain is present, while intensity shows the degree of staining. At grading for extension, grade I refers to less than 10% of cells being stained, grade II indicates 10–50% staining and grade III indicates that more than 50% are stained. At grading for intensity, grade I indicates mild staining, grade II moderate staining and grade III intense staining. 2.6. Statistical analysis Results from the experiments were expressed as “mean ± standard error” (x ± SEM). Significance of differences between groups was determined using one-way ANOVA, followed by Fisher's post-hoc LSD
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(least significant differences). All statistical procedures were performed on “IBM SPSS Statistics Version 20” statistical software, and significance was set at p b 0.05. 3. Results 3.1. Biochemical results 3.1.1. MDA, MPO, tGSH, SOD, CAT, COX-1 and COX-2 analysis results As seen in Fig. 1, the MDA amount is 8.3 ± 0.5 in kidney tissue of rats which received 50 mg/kg of etoricoxib; it was 3.1 ± 0.4 μmol/g protein in the dose of 100 mg/kg. Etoricoxib developed I/R in both doses, and significantly reduced the MDA elevation in kidney tissue compared to the RIR control group (P b 0.0001). The MPO activity in kidney tissue of rats which received 100 mg/kg etoricoxib (5.5 ± 0.7) was found to be lower than that of rats which received 50 mg/kg etoricoxib (12.3 ± 1.3) However, the statistical significance level of both doses was the same (P b 0.0001) (Fig. 1). While the tGSH activity was more significantly higher in the kidney tissue of rats which received 100 mg/kg of etoricoxib (12.6 ± 1.2) (P b 0.0001), the significance level was P b 0.05 in the dose of 50 mg/kg (6.3 ± 0.7) (Fig. 1). The SOD (21.8 ± 2) and CAT (18.8 ± 2.7) activities in 100 mg/kg of etoricoxib were calculated to be higher and more significant (P b 0.0001) (Fig. 1). As seen in Fig. 2, the COX-1 activity was found to be almost the same in the kidney tissues of the RIR, ETO-50, ETO-100 and the SG groups. The difference in the COX-1 activity between all these groups was not statistically significant. However, etoricoxib suppressed the COX-2 activity significantly in doses of 50 mg/kg (4.5 ± 0.7) and 100 mg/kg (2.3 ± 0.5) compared to the RIR group (P b 0.0001) (Fig. 2). 3.2. Immunohistochemical results 3.2.1. Bcl-2 results As seen in Fig. 3a, the staining area and the staining density in the tubular cells of the RIR group were determined as grade I. The staining area and the staining density in glomerular cells of the RIR group were also determined as grade I (Fig. 3b). This condition indicates that the staining area of the tubular and glomerular cells was below 10% and the staining density was low (Bcl-2 x 200). However, the staining area of the tubular cells in the ETO-50 group was grade III and the density was grade I (Fig. 3c). This indicates that more than 50% of the tubular cell area of the ETO-50 group was stained mildly (Bcl-2 x 200). The
Fig. 1. The effect of etoricoxib on MDA, MPO, tGSH, SOD and CAT levels in kidney tissue after induction of I/R. The ETO-50, ETO-100 and SG groups were compared with the RIR group and p b 0.05 was regarded as significant. Results are expressed as mean ± standard error (N = 6).
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as grade III and the density was determined as grade II (Fig. 3f). These findings reveal that more than 50% of the tubular and glomerular cell area of the ETO-100 group were stained moderately (Bcl-2 x 200). While the staining area of the tubular cells of the SG group was above 50%, the staining intensity was found to be intense (Fig. 3g). The extensiveness and the density of Bcl-2 in glomerular cells of the SG group were the same as those of the tubular cells (Fig. 3h). This microscopic appearance indicates that the staining area and the density of tubular and glomerular cells of the SG group were grade III (Bcl-2 x 200).
Fig. 2. COX-1 and COX-2 activities in RIR, ETO-50, ETO-100 and SG group rat kidney tissue. Groups were compared with RIR and p b 0.05 was regarded as significant. Results were expressed as mean ± standard error. (N = 6).
staining area of the glomerular cells in the ETO-50 group was observed as grade III and the density was grade II (Fig. 3d). This indicates that more than 50% of the glomerular cells were stained moderately (Bcl-2 x 200). While the staining area in the tubular cells of the ETO-100 group was grade III and the density was grade II (Fig. 3e), the staining area in the glomerular cells was determined
3.2.2. Caspase-3 results As observed in Fig. 4a, the staining extensiveness and density were determined as grade III in the tubular cells; however, while the staining extensiveness was grade III in the glomerular cells, the density was grade II (Fig. 4b, caspase 3x200) in the RIR group. The staining extensiveness and the density of renal tubular cells of the ETO-50 group (grade II) decreased compared to that of the RIR group (Fig. 4c, caspase 3x200). While the extensiveness was grade II in the glomerular cells of 50 mg/kg etoroxib- receiving group, the density was grade I (Fig. 4d, caspase 3x200). Etoricoxib 100 mg/kg decreased the staining density in the tubular cells (grade I) compared to the 50 mg/kg-receiving group; however, the extensiveness was the same in both groups (grade II); the extensiveness and the density were the same (grade I) in the glomerular cells of the 100 mg/kg etoricoxib-receiving group (Fig. 4e, caspase 3x200). The extensiveness and the density in the tubular and glomerular cells of the SG group were found to be the same as those of the ETO-100 group (Fig. 4f, caspase 3x200).
Fig. 3. (3a) Extension and intensity of Bcl-2 in RIR group renal tubular cells. (3b) Extension and intensity of Bcl-2 in RIR group renal glomerular cells. (3c) Extension and intensity of Bcl-2 in ETO-50 group renal tubular cells. (3d) Extension and degree of staining of Bcl-2 in ETO-50 group renal glomerular cells. (3e) Extension and degree of staining of Bcl-2 in ETO-100 renal tubular cells. (3f) Extension and intensity of Bcl-2 in ETO-100 renal glomerular cells. (3g) Extension and intensity of Bcl-2 in SG renal tubular cells. (3h) Extension and degree of staining of Bcl-2 in SG renal glomerular cells.
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Fig. 4. (4a) Extension and intensity of caspase-3 in RIR group renal tubular cells. (4b) Extension and intensity of caspase-3 in RIR group renal glomerular cells. (4c) Extension and intensity of caspase-3 in ETO-50 group renal tubular cells. (4d) Extension and intensity of caspase-3 in ETO-50 group renal glomerular cells. (4e) Extension and degree of staining of Bcl-2 in ETO-100 renal tubular (arrow) and glomerular cells (star). (4f) Extension and intensity of Bcl-2 in SG renal tubular (arrow) and glomerular cells (star).
4. Discussion This study investigated the effect of etoricoxib on damage induced with I/R in the rat kidney in biochemical and immunohistochemical terms. Our results show that the I/R process leads to marked oxidative injury in kidney tissue. Studies of the damage mechanism in I/R have shown that large quantities of free oxygen radicals are produced with reperfusion of ischemic tissue [21]. The most harmful effect caused in cells by free oxygen radicals is lipid peroxidation [22]. This reaction starts with lipid radical formation and concludes with the formation of the more toxic MDA [23]. In our study, the level of MDA in kidney tissue subjected to I/R was significantly higher in the ETO-50 group compared to the ETO-100 and SG rat groups. The decreased MDA amount in the injured kidney tissue which received etoricoxib suggests that 50 and 100 mg/kg doses of etoricoxib are effective against IR-related oxidative stress. A large number of studies have shown that MDA concentrations rise in tissue damaged by I/R [24,25]. Altuner D et al. reported that the I/R procedure causes oxidative damage in kidney tissue [26]. This shows that our experimental results are compatible with those from the literature. MPO activity was also higher in kidney tissue in which I/R was induced than in the SG and etoricoxib groups. MPO is known as a parameter of polymorph nuclear leukocyte (PNL) infiltration and activation [27]. Experimental studies on animals have shown an association between degree of PNL activation and degree of reperfusion injury [28]. 50 and 100 mg/kg doses of etoricoxib have statistically significantly decreased the MPO activity which had increased with IR in kidney tissue. However, there was a higher decrease in the MPO activity at the dose of 100 mg/kg compared to 50 mg/kg. This result suggests that the protective effect of etoricoxib against
IR injury could be associated with the inhibition of PNL activation. One of the most important endogenous antioxidants in defense against oxidative damage is tGSH. Elevated GSH is an indicator of cell function and vitality. A fall in GSH levels is regarded as a damage marker [29]. Studies have shown that glutathione levels decrease in the kidney during the I/R period and that this decrease arise from intense oxidative stress throughout I/R [30]. The results of this study indicate that oxidative IR injury caused by the decreased tGSH amount is preserved with etoricoxib and that the antioxidant effect of etoricoxib and its protective effects on tGSH are more prominent in high dose of etoricoxib. Tok A et al. revealed that the I/R process leads to a decrease in tGSH levels in the rat kidney and causes severe oxidative damage, and that this damage can be reduced with antioxidant activity [31]. Furthermore, etoricoxib protected the SOD activity to a higher extent with 100 mg/kg compared to 50 mg/kg in kidney tissue with IR. As known, SOD is an endogenous antioxidant and its activity decreases in damaged tissues [32]. Yang S et al. also reported that SOD activity decreased in kidney tissue with IR [24]. CAT is another antioxidant, which decreased in IR-induced renal tissue. CAT levels were found to be statistically significantly higher in groups which received 50 mg/kg and 100 mg/kg etoricoxib compared to the RIR group. Etoricoxib was seen to protect the CAT level statistically significantly better at the dose of 100 mg/kg in the tissue subjected to oxidative stress. It was reported that IR decreased the CAT level in kidney tissue subjected to IR-induced oxidative stress, and antioxidant treatment protected the kidney tissue from IR injury [33]. While the oxidant/antioxidant balance changed in favor of oxidants in the RIR group, this balance was in favor of antioxidants in the ETO-50 and ETO-100 groups. The oxidant/antioxidant balance is maintained with superiority of antioxidants in healthy tissues.
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Various aggressive factors which may lead to tissue injury impair the oxidant/antioxidant balance in favor of oxidants [34]. COX-2 activity in the RIR group in our study was also significantly lower than that in the ETO-50, ETO-100 and SG group kidney tissues. COX-1 is involved in the synthesis of cytoprotective prostaglandins [24]. COX-2 is activated with pro-inflammatory agents [35]. COX-2 production is lower in healthy tissues, but COX-2 activity exhibits a marked increase in inflamed tissues [36]. In our study, the COX-2 activity was determined to be lower than COX-1 activity in the kidney tissues of the healthy group, and the COX-2 activity was found to be higher in the RIR group. While etoricoxib in doses of 50 mg/kg and 100 mg/kg significantly prevented the COX-2 activity which increases with IR, it did not change COX-1 activity. Due to the fact that etoricoxib does not affect the COX-1 activity, it does not cause side effects such as renal failure, peptic ulcer and bleeding. As known, non-steroidal antiinflammatory drugs (NSAIDs) are effective in the treatment of acute postoperative pain. However, the side effects of NSAIDs, namely gastric irritation, peptic ulcer, renal failure, platelet inhibition and hemorrhage limit their use [9]. In previous studies too, amiphostin was reported to provide a renoprotective effect through inhibiting the COX-2 activity which increases in renal IR injury [37]. Senbel AM et al. reported that the COX-2 selective antagonist celecoxib protected the kidney tissue from COX-2 activation-related oxidative injury [38]. This information from the literature confirms the accuracy of our results. Again, in this study, Bcl-2 gene expression, an immunohistochemical parameter, was also weak in kidney tubular and glomerular tissue in the RIR group, in which MDA, MPO and COX-2 levels were high and tGSH levels were low. Etoricoxib also increased Bcl-2 gene expression in a dose-dependent manner. The Bcl-2 gene is an oncoprotein that plays an anti-apoptotic role. Bcl-2 inhibits apoptosis, and significantly reduces cell death initiated by various stimuli [39,40]. Ito T et al. also reported that Bcl-2 reduced cell death caused by free radicals [41]. These data indicate that Bcl-2s play a role in the protective mechanism of action of etoricoxib. Fennell DA et al. revealed that this protective effect of Bcl-2 was caused by preventing the release into cytoplasm of cytochrome-C and apoptosis inducing factor (AIF) from mitochondria [42]. The decreased caspase-3 gene expression in the tubular and glomerular cells of the kidneys in the ETO-50 and ETO-100 groups and the increased caspase 3 gene expression in the RIR group compared with the SG group support that etoricoxib suppresses apoptosis by inducing the Bcl-2 gene expression. This is because cytochrome C, which is released to cytoplasm from the mitochondria under stress, initiates the cascade, which activates procaspase-3 with cofactor of dATP, and chromosomal DNA is degraded and cell phagocytosis is provided [43]. That is, the degree of induction of caspase-3 may increase with cytochrome-C′s passing into the cytoplasm from the mitochondria. In addition, substances which inhibit cytochrome-C translocation have been reported to suppress the caspase-3 activation and increase the rate of Bcl-2 [44]. However, there are also studies indicating that some of the factors which release the cytochrome-C enzyme from the mitochondria into cytoplasm are in fact caspase-3 [45].
5. Conclusion In conclusion, renal IR has been understood to be a complex pathological process, which begins with the tissue being deprived of oxygen, continues with oxidative stress, progresses with inflammation, and leads to apoptosis. Etoricoxib is observed to suppress renal IR injury by causing antioxidant, anti-inflammatory and antiapoptotic effects. The protective effect of etoricoxib was found to be more potent at the dose of 100 mg/kg compared to 50 mg/kg. This study may be a reference for investigating the protective effect of other COX-2 selective inhibitors in IR and different tissue injury models. This study also suggests that etoricoxib, which is an antiinflammatory, analgesic, antipyetic and antioxidant drug that does
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