Journal of Pediatric Surgery xxx (2014) xxx–xxx
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Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Methylene blue attenuates renal ischemia–reperfusion injury in rats Fatma Sarac a,⁎, Huseyin Kilincaslan b, Elif Kilic c, Macit Koldas d, Elcin Hakan Terzi e, Ibrahim Aydogdu b a
Department of Pediatric Surgery, Haseki Research and Education Hospital, Istanbul, Turkey Department of Pediatric Surgery, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey c Department of Biochemistry, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey d Department of Biochemistry, Haseki Research and Education Hospital, Istanbul, Turkey e Department of Histology and Embryology, Faculty of Medicine, Abant Izzet Baysal University, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 17 June 2014 Accepted 30 June 2014 Available online xxxx Key words: Methylene blue Renal ischemia/reperfusion Antioxidant Ischemia modified albumin
a b s t r a c t Background and purpose: In our study, we investigated the effects of methylene blue (MB) on histopathological changes in renal ischemia/reperfusion (I/R) injury rat model. Material and methods: Twenty-one Sprague–Dawley male rats were divided equally into three groups. Group 1 (control) was administered intraperitoneal saline solution. In Groups 2 (untreated group) and 3 (MB treatment), the renal arteries were clamped, and ischemia (for 1 hour) and then reperfusion (for 4 hours) were applied. Thirty minutes before ischemia, the untreated group received physiological saline, whereas the treatment group was administered 30 mg/kg MB through an intraperitoneal route. Blood samples were drawn, and renal specimens were harvested 5.5 hours after physiologic saline injection in the control and immediately after the reperfusion period in the other groups. The levels of tissue superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total oxidant status (TOS), total antioxidant status (TAS), plasma urea, creatinine and ischemia modified albumin (IMA) were measured. Moreover, the histopathological damage score of the renal tissue was determined. Results: MB significantly alleviated the severity of histopathological damage by increasing the levels of tissue SOD and TAS and decreasing TOS concentrations in the renal I/R model (p b 0.05). Conclusion: Administration of MB in renal I/R damage may play a protective role. © 2014 Elsevier Inc. All rights reserved.
Renal ischemia is characterized by transient decrease or cessation of renal blood flow. It can be observed in many conditions, including renal transplantation, partial nephrectomy, renal artery angioplasty, cardiopulmonary bypass surgery, trauma, sepsis, burns, hydronephrosis, and elective urological operations. As a result, acute renal failure can develop, resulting in tubular necrosis, decrease in glomerular filtration and increase in renal resistance [1,2]. Prolonged ischemia destroys cellular integrity or even leads to cell death as a result of the accumulation of toxic metabolites. Ischemic tissue should be reperfused to achieve the regeneration of tissue and elimination of toxic metabolites. The recovery of tissue perfusion by means of drugs or mechanical interventions is called reperfusion. Destructive changes during reperfusion are more severe than in paradoxical ischemic injury. In ischemia/reperfusion (I/R) injury, free oxygen radicals, especially those released by polymorphonuclear leukocytes, that accumulate within tissues exert important effects [3,4]. The free radicals interact with almost all biomolecules
⁎ Corresponding author at: Haseki Eğitim ve Araştırma Hastanesi, Çocuk Cerrahisi Kliniği, Haseki Sultan Mah. 34096 Fatih/İstanbul. Tel.: +90 529 44 00; fax: +90 212 589 62 29. E-mail addresses: [email protected] (F. Sarac), [email protected] (H. Kilincaslan), [email protected] (E. Kilic), [email protected] (M. Koldas), [email protected] (E.H. Terzi), [email protected] (I. Aydogdu).
integrated into the structure of living organisms, and they can induce reversible or irreversible effects on these biomolecules [5]. Tissues possess enzymatic and non-enzymatic antioxidant mechanisms against oxidative damage. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are examples of antioxidant enzyme systems [6]. Cellular stress factors such as hypoxia, acidosis, free radical damage, and impairment of membrane integrity induce structural changes in albumin molecules, attenuating the binding capacity of albumin for transition metals such as copper, nickel, and cobalt. The resulting defective albumin is referred to as “ischemia modified albumin” (IMA). IMA is a known sensitive marker in pulmonary embolism and myocardial, muscular, mesenteric and cerebral ischemia [7–9]. As an agent with lower toxicity, methylene blue (MB) has been used in clinical practice [10]. It increases the level of SOD and inhibits the formation of oxygen radicals, with resulting antioxidant effects [11,12]. Experimental studies have demonstrated that it also corrects hemodynamic instability developed in hepatic I/R injury [13], protects kidneys from harmful effects of immune suppressive agents via its antioxidant effect [14], and decreases intraabdominal adhesion [15]. In our study, we investigated the effects of MB on histopathological changes in renal I/R injury; tissue oxidative stress parameters such as SOD, CAT, GPx, total oxidant status (TOS); total antioxidant status (TAS) and levels of serum IMA, which is a proper marker of ischemia.
http://dx.doi.org/10.1016/j.jpedsurg.2014.06.018 0022-3468/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
1. Materials and methods The experimental protocol was approved by the Ethics Committee of the Faculty of Medicine, Bezmialem Vakif University. A total of 21 male Sprague–Dawley rats weighing 265–320 g were used. The rats were divided into three equal groups: Group 1 (control) was composed of normal healthy rats that were treated with intraperitoneal (i.p.) physiologic saline solution. Group 2 (untreated): This group consisted of rats that were administered i.p. physiological saline 30 minutes before ischemia/ reperfusion Group 3 (MB treatment): This group consisted of rats that were administered i.p. 30 mg/kg MB (Merck, Darmstadt, Germany) in 1 ml of sterile water 30 minutes before ischemia/reperfusion. Excluding rats in the control group, the rats in other groups were laid on the examination table under intramuscular (i.m.) 15 mg/kg xylazine (Rompun, Bayer, Turkey) and 50 mg/kg ketamine (Ketalar, Eczacıbaşı, Türkiye) anesthesia. The anterior abdominal wall was explored through a midline incision. Vessels were separated with blunt dissection, and the renal arteries were clamped with atraumatic vascular clamps. After a period of 60 minutes, the clamps were removed, and free blood flow was maintained. The incision was closed and disinfected with 10 % povidone iodine. In the control group, at the end of 5 hours following administration of physiological saline, blood samples were drawn from the rats; however, in groups 2, and 3, at the end of 4 hours after reperfusion, blood samples were drawn from rats, and their left kidneys were removed. After peeling of the renal capsule with a scalpel, the kidney was divided into two parts with a longitudinal section. One portion was placed in 10% formaldehyde solution for histopathological analysis, and the other portions together with plasma samples were stored at −70 °C until biochemical analysis was performed.
iodophenyl)-3-(4-nitrophenol)-5 phenyltetrazolium chloride (INT) to form a red formazan dye [17]. One unit of SOD was defined as the amount of enzyme necessary to produce a 50% inhibition in the INT reduction rate. SOD activity is expressed as units per gram tissue protein. 1.2.3. CAT activity The supernatants of the kidney were assayed by the method of Aebi et al. [18], which monitors the disappearance of H2O2 in the presence of the cell homogenate at 240 nm. The enzyme reaction was started by adding 0.1 ml of the sample (0.4–0.5 mg protein) to 2.9 ml of 50 mM phosphate buffer at pH 7.0 containing 12 mM H2O2. The absorbance was recorded immediately after addition of the sample at 240 nm at 15-second intervals for 2 minutes. A blank solution without sample was prepared. The absorbance (A) was read at 240 nm, and the A/min was calculated. The calculation was performed using the extinction coefficient of H2O2 i.e., 43.6 M −1 cm −1. The activity is expressed as units per milligram tissue protein. 1.2.4. GPx activity Glutathione peroxidase activity was estimated by measuring the changes in the absorbance at 340 nm owing to NADPH consumption in the presence of H2O2 [19]. The glutathione peroxidase activity is expressed as mmol per gram tissue protein (mmol/g protein). 1.2.5. Measurement of TOS and TAS The TOS and TAS of the tissues were measured using automated colorimetric measurement methods [20]. The TOS assay was calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalents per liter (μ mol H2O2 Eq/L). The TAS assay method is based on the bleaching of the characteristic color of a more stable 2,20-azinobis [3 ethylbenzothiazoline-6-sulfonic acid] (ABST) radical cation by antioxidants. The assay has excellent precision values that are lower than 3%. The results are expressed as the mmol Trolox/mg protein.
1.1. Histopathologic evaluation The renal tissues were individually immersed in Bouin's fixative, dehydrated in alcohol and embedded in paraffin. Sections of 5 μm were obtained, deparaffinized and stained with hematoxylin and eosin (H&E). The renal tissue was examined and evaluated in random order under blinded conditions with standard light microscopy. Tubulointerstitial injury was defined as tubular atrophy, dilatation, loss of brush border, cellular infiltration, and widening of the interstitium. The degree of tubulointerstitial damage in the cortex was determined using a semiquantitative graded scale [16], where 0 represents no abnormality, 1: minimal damage (involvement of 25% of the cortex), 2: mild damage (involvement of 25–50% of the cortex), 3: moderate damage involvement of 50–75% of the cortex), and 4: severe damage (involvement of 75% of the cortex). These analyses were performed with 2 tissue sections obtained from each animal examined less than × 400 magnification for at least 10 different regions for each section [1].
1.2.6. Measurement of urea, creatinine and IMA Using standard methods, the urea and creatinine values of venous blood samples were analyzed. In analysis of IMA, the decreased binding capacity of albumin to cobalt was evaluated using a rapid colorimetric assay developed by Bar-Or et al. [21,22]. The results are expressed in absorbance units (absu). 1.3. Statistical analyses One-sample Kolmogorov Smirnov tests were used to determine that the quantitative data for every group were normally distributed. Therefore, intergroup comparisons were performed using one-way ANOVA and the post-hoc Tukey multiple comparison method. Histopathological scores were compared using Kruskal–Wallis and post-hoc Dunn tests. Statistical test results were considered significant at a p b 0.05. 2. Results
1.2. Biochemical tissue analyses 1.2.1. Preparation of tissue samples The kidneys were cleaned, and each segment was homogenized in 10 volumes of 50 mM Tris–HCl at pH 7.4 using a rotor-stator homogenizer. The homogenate was centrifuged at 2000 ×g at 4 °C for 10 min, and a low-speed supernatant fraction was used for ex vivo assays. 1.2.2. SOD activity The measurement of the tissue homogenate SOD enzyme activity was based on the generation of superoxide radicals by the action of xanthine and xanthine oxidase, which react with 2-(4-
A total of 21 rats were included in the study. During the experiment, none of the rats died. Histopathologic damage scores were compared between groups, and the severity of destructive changes in the untreated group were found to be significantly higher than that of the other groups (p b 0.05). In the MB treatment group, these histopathologic findings were statistically suppressed (p b 0.05). Histopathological sections are shown in Fig. 1, and comparisons of histopathological damage scores are shown in Fig. 2. The SOD activity of the groups was compared. In the MB treatment group SOD activity was significantly increased when
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
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Fig. 1. In the untreated group, widespread bleeding and congestion between tubules in the cortex and medulla and hydropic degeneration in the tubular epithelium were observed. One striking finding was that some tubules were filled with debris. Moreover, marked tubular degeneration was noted. However, in the MB treatment group, bleeding and congestion in the cortex and medulla and accumulation of debris in tubules were remarkably decreased. Prominently decreased tubular dilation was observed relative to the untreated group (bar 100 μm).
compared to control and untreated groups (p b 0.05). However, no significant difference was found between the control and untreated groups (p = 0.77) (Fig. 3). In addition, there was no significant difference according to pairwise comparisons of tissue CAT and GPx activity. For TOS values, a significant difference was found in comparisons between the untreated group and both the control and treatment groups (p b 0.05). MB decreased TOS levels significantly. A significant difference was not detected between the MB treatment and control groups (p = 0.87) (Fig. 4). Groups were compared in terms of TAS values. In comparisons between the MB treatment group and both the control and untreated group, there were significant differences (p b 0.05). MB increased TAS levels significantly. No significant difference was found between the untreated and control groups (p = 0.98) (Fig. 5).
Significant increases in serum urea and creatinine values were detected in both the untreated and MB treatment groups compared with the control group (p b 0.05). No significant difference was detected between the MB treatment group and untreated group regarding serum urea and creatinine values (p = 0.57). In pairwise comparisons of groups of serum IMA levels, no significant difference was detected. The parameters of all groups estimated in the plasma and renal tissue are shown in Table 1.
Fig. 2. Comparison of histopathologic damage scores between groups. *; p b 0.05.
Fig. 3. Comparison of SOD activities between groups. *; p b 0.05.
3. Discussion Decreases in renal blood flow or its cessation result in the subsequent development of reperfusion together with various degrees of tissue injury. Renal I/R causes tubular damage, atrophy,
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx Table 1 Histopathological findings and biochemical values of all of the groups (±standart deviation). Control
Untreated
MB treatment
Hıstopathologıc damage score 0.29 ± 0.49 3.29 ± 0.76 1.29 ± 0.49 Ischemıa modıfıed albumın 0.91 ± 0.07 0.79 ± 0.11 0.79 ± 0.21 Superoxıde dısmutase 4.1 ± 0.24 4.42 ± 0.05 6.12 ± 1.48 Glutathıone peroxıdase 284.08 ± 22.87 246.09 ± 35.21 246.62 ± 64.14 Catalase 0.77 ± 0.19 0.94 ± 0.31 1.18 ± 0.85 Total oxıdant status 6.91 ± 1.17 9.28 ± 1.00 6.50 ± 2.15 Total antıoxıdant status 0.59 ± 0.11 0.58 ± 0.14 0.83 ± 0.12 Urea 40,47 ± 2.49 56.9 ± 5.82 54.0 ± 6.68 Creatınıne 0.3 ± 0.02 0.38 ± 0.02 0.4 ± 0.05
Fig. 4. Comparison of TOS values between groups. *; p b 0.05.
and dilation in tubules, which are the most vulnerable part of the renal unit. Free oxygen radicals play important roles in this process [1,4,23]. To counteract the potential harm from free oxygen radicals, antioxidant mechanisms become functional and attempt to decrease the effects of destructive changes. Antioxidant enzymes, such as SOD, CAT, GPx, and glutathione reductase, are influential in the antioxidant mechanism whereby harmful free oxygen radicals are transformed into harmless side products [24,25]. Many substances have been used to prevent destructive changes in experimental renal I/R models. Urtica dioica L., melatonin, glutamine, aliskiren, dexamethasone, cannabidiol, sildenafil, sulfosalazine, and beta carotene are the most commonly used for such purpose [1,2,26–31]. MB is a substance with low toxicity and molecular weight that can easily penetrate into tissues and be used in humans. It has been used to demonstrate extraluminal leakage in the gastrointestinal system surgery. Its use in methemoglobinemia, malaria, priapism, some neurological diseases, and cyanide and carbon monoxide poisoning has been reported [3,25,32–35]. It competes with molecular oxygen used in electron transfer and increases the levels of antioxidant enzymes such as SOD with resulting inhibition of oxygen radical formation [10,14,23,36]. The use of MB in renal I/R injury has not been previously reported. In our study, because of its antioxidant effect, MB significantly suppressed the histopathological findings. MB therapy markedly
decreased the severity of intracortical and intramedullary bleeding, congestion, intratubular accumulation of debris, and tubular dilation. MB significantly increased the tissue SOD ratios. The antioxidant effect of MB is known to be facilitated by increasing the levels of SOD and similar enzymes. In a study performed by Aksu et al., the authors investigated the impact of MB on oxidative stress and hepatic injury in rats with ligated choleducti and demonstrated its alleviating effects via increasing SOD activity [12]. Our findings are in accordance with those in the literature. The impact of MB on CAT activity differs between experimental studies. One study demonstrated a lack of any significant impact of MB on CAT values, whereas another study demonstrated an enhancing effect on CAT activity [25]. However, two separate studies have indicated that MB has suppressive effects on CAT activity [37,38]. In our study, MB did not exert a significant impact on CAT activity. These differences in outcomes might suggest that MB does not produce its antioxidant effect via the CAT pathway. Further studies of this issue are needed. In the literature, a very small number of studies have demonstrated the impact of MB on tissue GPx activity. Demirbilek et al. performed a study in septic rats and indicated that MB demonstrated an antioxidant effect in the lung tissue by increasing GPx activity [25]. However, in our study, MB did not exert a significant effect on tissue GPx activity. We believe that further studies are needed to elucidate this difference. MB increased TAS levels significantly, whereas TOS levels were depressed. This finding reinforces the antioxidant effect of MB. To the best of our knowledge, only a study by Kilincaslan et al. had a similar design and demonstrated that MB increased TAS but decreased TOS values in an experimental corrosive esophagitis model induced in rats [38]. As is already known, increases in serum urea and creatinine levels are important indicators of renal dysfunction, which is characterized by a drop in glomerular filtration rate. In our study of the I/R group, serum urea and creatinine levels increased significantly as compared with the control group. With MB treatment, these levels were not significantly suppressed. This phenomenon can be explained by the relatively short duration of the experiments. IMA is an excellent indicator of ischemic conditions. In ischemic conditions, serum IMA levels increase within minutes [7,8]. As a limitation of this study, IMA levels were not examined after the ischemia. On the other hand, no significant intergroup difference could be found after reperfusion. Our statistically insignificant results can be explained by the drop in serum IMA values down to normal levels following reperfusion. In the literature, we have not encountered any study investigating serum IMA levels in a renal I/R model. Therefore, studies performed on serum IMA samples obtained before reperfusion will clarify the issue. In conclusion, with its inherent antioxidant activity, MB can be used as a potential therapeutic agent in renal I/R models.
References Fig. 5. Comparison of TAS values between groups. *; p b 0.05.
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Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Methylene blue attenuates renal ischemia–reperfusion injury in rats Fatma Sarac a,⁎, Huseyin Kilincaslan b, Elif Kilic c, Macit Koldas d, Elcin Hakan Terzi e, Ibrahim Aydogdu b a
Department of Pediatric Surgery, Haseki Research and Education Hospital, Istanbul, Turkey Department of Pediatric Surgery, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey c Department of Biochemistry, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey d Department of Biochemistry, Haseki Research and Education Hospital, Istanbul, Turkey e Department of Histology and Embryology, Faculty of Medicine, Abant Izzet Baysal University, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 17 June 2014 Accepted 30 June 2014 Available online xxxx Key words: Methylene blue Renal ischemia/reperfusion Antioxidant Ischemia modified albumin
a b s t r a c t Background and purpose: In our study, we investigated the effects of methylene blue (MB) on histopathological changes in renal ischemia/reperfusion (I/R) injury rat model. Material and methods: Twenty-one Sprague–Dawley male rats were divided equally into three groups. Group 1 (control) was administered intraperitoneal saline solution. In Groups 2 (untreated group) and 3 (MB treatment), the renal arteries were clamped, and ischemia (for 1 hour) and then reperfusion (for 4 hours) were applied. Thirty minutes before ischemia, the untreated group received physiological saline, whereas the treatment group was administered 30 mg/kg MB through an intraperitoneal route. Blood samples were drawn, and renal specimens were harvested 5.5 hours after physiologic saline injection in the control and immediately after the reperfusion period in the other groups. The levels of tissue superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total oxidant status (TOS), total antioxidant status (TAS), plasma urea, creatinine and ischemia modified albumin (IMA) were measured. Moreover, the histopathological damage score of the renal tissue was determined. Results: MB significantly alleviated the severity of histopathological damage by increasing the levels of tissue SOD and TAS and decreasing TOS concentrations in the renal I/R model (p b 0.05). Conclusion: Administration of MB in renal I/R damage may play a protective role. © 2014 Elsevier Inc. All rights reserved.
Renal ischemia is characterized by transient decrease or cessation of renal blood flow. It can be observed in many conditions, including renal transplantation, partial nephrectomy, renal artery angioplasty, cardiopulmonary bypass surgery, trauma, sepsis, burns, hydronephrosis, and elective urological operations. As a result, acute renal failure can develop, resulting in tubular necrosis, decrease in glomerular filtration and increase in renal resistance [1,2]. Prolonged ischemia destroys cellular integrity or even leads to cell death as a result of the accumulation of toxic metabolites. Ischemic tissue should be reperfused to achieve the regeneration of tissue and elimination of toxic metabolites. The recovery of tissue perfusion by means of drugs or mechanical interventions is called reperfusion. Destructive changes during reperfusion are more severe than in paradoxical ischemic injury. In ischemia/reperfusion (I/R) injury, free oxygen radicals, especially those released by polymorphonuclear leukocytes, that accumulate within tissues exert important effects [3,4]. The free radicals interact with almost all biomolecules
⁎ Corresponding author at: Haseki Eğitim ve Araştırma Hastanesi, Çocuk Cerrahisi Kliniği, Haseki Sultan Mah. 34096 Fatih/İstanbul. Tel.: +90 529 44 00; fax: +90 212 589 62 29. E-mail addresses: [email protected] (F. Sarac), [email protected] (H. Kilincaslan), [email protected] (E. Kilic), [email protected] (M. Koldas), [email protected] (E.H. Terzi), [email protected] (I. Aydogdu).
integrated into the structure of living organisms, and they can induce reversible or irreversible effects on these biomolecules [5]. Tissues possess enzymatic and non-enzymatic antioxidant mechanisms against oxidative damage. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are examples of antioxidant enzyme systems [6]. Cellular stress factors such as hypoxia, acidosis, free radical damage, and impairment of membrane integrity induce structural changes in albumin molecules, attenuating the binding capacity of albumin for transition metals such as copper, nickel, and cobalt. The resulting defective albumin is referred to as “ischemia modified albumin” (IMA). IMA is a known sensitive marker in pulmonary embolism and myocardial, muscular, mesenteric and cerebral ischemia [7–9]. As an agent with lower toxicity, methylene blue (MB) has been used in clinical practice [10]. It increases the level of SOD and inhibits the formation of oxygen radicals, with resulting antioxidant effects [11,12]. Experimental studies have demonstrated that it also corrects hemodynamic instability developed in hepatic I/R injury [13], protects kidneys from harmful effects of immune suppressive agents via its antioxidant effect [14], and decreases intraabdominal adhesion [15]. In our study, we investigated the effects of MB on histopathological changes in renal I/R injury; tissue oxidative stress parameters such as SOD, CAT, GPx, total oxidant status (TOS); total antioxidant status (TAS) and levels of serum IMA, which is a proper marker of ischemia.
http://dx.doi.org/10.1016/j.jpedsurg.2014.06.018 0022-3468/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
1. Materials and methods The experimental protocol was approved by the Ethics Committee of the Faculty of Medicine, Bezmialem Vakif University. A total of 21 male Sprague–Dawley rats weighing 265–320 g were used. The rats were divided into three equal groups: Group 1 (control) was composed of normal healthy rats that were treated with intraperitoneal (i.p.) physiologic saline solution. Group 2 (untreated): This group consisted of rats that were administered i.p. physiological saline 30 minutes before ischemia/ reperfusion Group 3 (MB treatment): This group consisted of rats that were administered i.p. 30 mg/kg MB (Merck, Darmstadt, Germany) in 1 ml of sterile water 30 minutes before ischemia/reperfusion. Excluding rats in the control group, the rats in other groups were laid on the examination table under intramuscular (i.m.) 15 mg/kg xylazine (Rompun, Bayer, Turkey) and 50 mg/kg ketamine (Ketalar, Eczacıbaşı, Türkiye) anesthesia. The anterior abdominal wall was explored through a midline incision. Vessels were separated with blunt dissection, and the renal arteries were clamped with atraumatic vascular clamps. After a period of 60 minutes, the clamps were removed, and free blood flow was maintained. The incision was closed and disinfected with 10 % povidone iodine. In the control group, at the end of 5 hours following administration of physiological saline, blood samples were drawn from the rats; however, in groups 2, and 3, at the end of 4 hours after reperfusion, blood samples were drawn from rats, and their left kidneys were removed. After peeling of the renal capsule with a scalpel, the kidney was divided into two parts with a longitudinal section. One portion was placed in 10% formaldehyde solution for histopathological analysis, and the other portions together with plasma samples were stored at −70 °C until biochemical analysis was performed.
iodophenyl)-3-(4-nitrophenol)-5 phenyltetrazolium chloride (INT) to form a red formazan dye [17]. One unit of SOD was defined as the amount of enzyme necessary to produce a 50% inhibition in the INT reduction rate. SOD activity is expressed as units per gram tissue protein. 1.2.3. CAT activity The supernatants of the kidney were assayed by the method of Aebi et al. [18], which monitors the disappearance of H2O2 in the presence of the cell homogenate at 240 nm. The enzyme reaction was started by adding 0.1 ml of the sample (0.4–0.5 mg protein) to 2.9 ml of 50 mM phosphate buffer at pH 7.0 containing 12 mM H2O2. The absorbance was recorded immediately after addition of the sample at 240 nm at 15-second intervals for 2 minutes. A blank solution without sample was prepared. The absorbance (A) was read at 240 nm, and the A/min was calculated. The calculation was performed using the extinction coefficient of H2O2 i.e., 43.6 M −1 cm −1. The activity is expressed as units per milligram tissue protein. 1.2.4. GPx activity Glutathione peroxidase activity was estimated by measuring the changes in the absorbance at 340 nm owing to NADPH consumption in the presence of H2O2 [19]. The glutathione peroxidase activity is expressed as mmol per gram tissue protein (mmol/g protein). 1.2.5. Measurement of TOS and TAS The TOS and TAS of the tissues were measured using automated colorimetric measurement methods [20]. The TOS assay was calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalents per liter (μ mol H2O2 Eq/L). The TAS assay method is based on the bleaching of the characteristic color of a more stable 2,20-azinobis [3 ethylbenzothiazoline-6-sulfonic acid] (ABST) radical cation by antioxidants. The assay has excellent precision values that are lower than 3%. The results are expressed as the mmol Trolox/mg protein.
1.1. Histopathologic evaluation The renal tissues were individually immersed in Bouin's fixative, dehydrated in alcohol and embedded in paraffin. Sections of 5 μm were obtained, deparaffinized and stained with hematoxylin and eosin (H&E). The renal tissue was examined and evaluated in random order under blinded conditions with standard light microscopy. Tubulointerstitial injury was defined as tubular atrophy, dilatation, loss of brush border, cellular infiltration, and widening of the interstitium. The degree of tubulointerstitial damage in the cortex was determined using a semiquantitative graded scale [16], where 0 represents no abnormality, 1: minimal damage (involvement of 25% of the cortex), 2: mild damage (involvement of 25–50% of the cortex), 3: moderate damage involvement of 50–75% of the cortex), and 4: severe damage (involvement of 75% of the cortex). These analyses were performed with 2 tissue sections obtained from each animal examined less than × 400 magnification for at least 10 different regions for each section [1].
1.2.6. Measurement of urea, creatinine and IMA Using standard methods, the urea and creatinine values of venous blood samples were analyzed. In analysis of IMA, the decreased binding capacity of albumin to cobalt was evaluated using a rapid colorimetric assay developed by Bar-Or et al. [21,22]. The results are expressed in absorbance units (absu). 1.3. Statistical analyses One-sample Kolmogorov Smirnov tests were used to determine that the quantitative data for every group were normally distributed. Therefore, intergroup comparisons were performed using one-way ANOVA and the post-hoc Tukey multiple comparison method. Histopathological scores were compared using Kruskal–Wallis and post-hoc Dunn tests. Statistical test results were considered significant at a p b 0.05. 2. Results
1.2. Biochemical tissue analyses 1.2.1. Preparation of tissue samples The kidneys were cleaned, and each segment was homogenized in 10 volumes of 50 mM Tris–HCl at pH 7.4 using a rotor-stator homogenizer. The homogenate was centrifuged at 2000 ×g at 4 °C for 10 min, and a low-speed supernatant fraction was used for ex vivo assays. 1.2.2. SOD activity The measurement of the tissue homogenate SOD enzyme activity was based on the generation of superoxide radicals by the action of xanthine and xanthine oxidase, which react with 2-(4-
A total of 21 rats were included in the study. During the experiment, none of the rats died. Histopathologic damage scores were compared between groups, and the severity of destructive changes in the untreated group were found to be significantly higher than that of the other groups (p b 0.05). In the MB treatment group, these histopathologic findings were statistically suppressed (p b 0.05). Histopathological sections are shown in Fig. 1, and comparisons of histopathological damage scores are shown in Fig. 2. The SOD activity of the groups was compared. In the MB treatment group SOD activity was significantly increased when
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx
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Fig. 1. In the untreated group, widespread bleeding and congestion between tubules in the cortex and medulla and hydropic degeneration in the tubular epithelium were observed. One striking finding was that some tubules were filled with debris. Moreover, marked tubular degeneration was noted. However, in the MB treatment group, bleeding and congestion in the cortex and medulla and accumulation of debris in tubules were remarkably decreased. Prominently decreased tubular dilation was observed relative to the untreated group (bar 100 μm).
compared to control and untreated groups (p b 0.05). However, no significant difference was found between the control and untreated groups (p = 0.77) (Fig. 3). In addition, there was no significant difference according to pairwise comparisons of tissue CAT and GPx activity. For TOS values, a significant difference was found in comparisons between the untreated group and both the control and treatment groups (p b 0.05). MB decreased TOS levels significantly. A significant difference was not detected between the MB treatment and control groups (p = 0.87) (Fig. 4). Groups were compared in terms of TAS values. In comparisons between the MB treatment group and both the control and untreated group, there were significant differences (p b 0.05). MB increased TAS levels significantly. No significant difference was found between the untreated and control groups (p = 0.98) (Fig. 5).
Significant increases in serum urea and creatinine values were detected in both the untreated and MB treatment groups compared with the control group (p b 0.05). No significant difference was detected between the MB treatment group and untreated group regarding serum urea and creatinine values (p = 0.57). In pairwise comparisons of groups of serum IMA levels, no significant difference was detected. The parameters of all groups estimated in the plasma and renal tissue are shown in Table 1.
Fig. 2. Comparison of histopathologic damage scores between groups. *; p b 0.05.
Fig. 3. Comparison of SOD activities between groups. *; p b 0.05.
3. Discussion Decreases in renal blood flow or its cessation result in the subsequent development of reperfusion together with various degrees of tissue injury. Renal I/R causes tubular damage, atrophy,
Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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F. Sarac et al. / Journal of Pediatric Surgery xxx (2014) xxx–xxx Table 1 Histopathological findings and biochemical values of all of the groups (±standart deviation). Control
Untreated
MB treatment
Hıstopathologıc damage score 0.29 ± 0.49 3.29 ± 0.76 1.29 ± 0.49 Ischemıa modıfıed albumın 0.91 ± 0.07 0.79 ± 0.11 0.79 ± 0.21 Superoxıde dısmutase 4.1 ± 0.24 4.42 ± 0.05 6.12 ± 1.48 Glutathıone peroxıdase 284.08 ± 22.87 246.09 ± 35.21 246.62 ± 64.14 Catalase 0.77 ± 0.19 0.94 ± 0.31 1.18 ± 0.85 Total oxıdant status 6.91 ± 1.17 9.28 ± 1.00 6.50 ± 2.15 Total antıoxıdant status 0.59 ± 0.11 0.58 ± 0.14 0.83 ± 0.12 Urea 40,47 ± 2.49 56.9 ± 5.82 54.0 ± 6.68 Creatınıne 0.3 ± 0.02 0.38 ± 0.02 0.4 ± 0.05
Fig. 4. Comparison of TOS values between groups. *; p b 0.05.
and dilation in tubules, which are the most vulnerable part of the renal unit. Free oxygen radicals play important roles in this process [1,4,23]. To counteract the potential harm from free oxygen radicals, antioxidant mechanisms become functional and attempt to decrease the effects of destructive changes. Antioxidant enzymes, such as SOD, CAT, GPx, and glutathione reductase, are influential in the antioxidant mechanism whereby harmful free oxygen radicals are transformed into harmless side products [24,25]. Many substances have been used to prevent destructive changes in experimental renal I/R models. Urtica dioica L., melatonin, glutamine, aliskiren, dexamethasone, cannabidiol, sildenafil, sulfosalazine, and beta carotene are the most commonly used for such purpose [1,2,26–31]. MB is a substance with low toxicity and molecular weight that can easily penetrate into tissues and be used in humans. It has been used to demonstrate extraluminal leakage in the gastrointestinal system surgery. Its use in methemoglobinemia, malaria, priapism, some neurological diseases, and cyanide and carbon monoxide poisoning has been reported [3,25,32–35]. It competes with molecular oxygen used in electron transfer and increases the levels of antioxidant enzymes such as SOD with resulting inhibition of oxygen radical formation [10,14,23,36]. The use of MB in renal I/R injury has not been previously reported. In our study, because of its antioxidant effect, MB significantly suppressed the histopathological findings. MB therapy markedly
decreased the severity of intracortical and intramedullary bleeding, congestion, intratubular accumulation of debris, and tubular dilation. MB significantly increased the tissue SOD ratios. The antioxidant effect of MB is known to be facilitated by increasing the levels of SOD and similar enzymes. In a study performed by Aksu et al., the authors investigated the impact of MB on oxidative stress and hepatic injury in rats with ligated choleducti and demonstrated its alleviating effects via increasing SOD activity [12]. Our findings are in accordance with those in the literature. The impact of MB on CAT activity differs between experimental studies. One study demonstrated a lack of any significant impact of MB on CAT values, whereas another study demonstrated an enhancing effect on CAT activity [25]. However, two separate studies have indicated that MB has suppressive effects on CAT activity [37,38]. In our study, MB did not exert a significant impact on CAT activity. These differences in outcomes might suggest that MB does not produce its antioxidant effect via the CAT pathway. Further studies of this issue are needed. In the literature, a very small number of studies have demonstrated the impact of MB on tissue GPx activity. Demirbilek et al. performed a study in septic rats and indicated that MB demonstrated an antioxidant effect in the lung tissue by increasing GPx activity [25]. However, in our study, MB did not exert a significant effect on tissue GPx activity. We believe that further studies are needed to elucidate this difference. MB increased TAS levels significantly, whereas TOS levels were depressed. This finding reinforces the antioxidant effect of MB. To the best of our knowledge, only a study by Kilincaslan et al. had a similar design and demonstrated that MB increased TAS but decreased TOS values in an experimental corrosive esophagitis model induced in rats [38]. As is already known, increases in serum urea and creatinine levels are important indicators of renal dysfunction, which is characterized by a drop in glomerular filtration rate. In our study of the I/R group, serum urea and creatinine levels increased significantly as compared with the control group. With MB treatment, these levels were not significantly suppressed. This phenomenon can be explained by the relatively short duration of the experiments. IMA is an excellent indicator of ischemic conditions. In ischemic conditions, serum IMA levels increase within minutes [7,8]. As a limitation of this study, IMA levels were not examined after the ischemia. On the other hand, no significant intergroup difference could be found after reperfusion. Our statistically insignificant results can be explained by the drop in serum IMA values down to normal levels following reperfusion. In the literature, we have not encountered any study investigating serum IMA levels in a renal I/R model. Therefore, studies performed on serum IMA samples obtained before reperfusion will clarify the issue. In conclusion, with its inherent antioxidant activity, MB can be used as a potential therapeutic agent in renal I/R models.
References Fig. 5. Comparison of TAS values between groups. *; p b 0.05.
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Please cite this article as: Sarac F, et al, Methylene blue attenuates renal ischemia–reperfusion injury in rats, J Pediatr Surg (2014), http://dx. doi.org/10.1016/j.jpedsurg.2014.06.018
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