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Effects of Allopurinol and Apocynin on Renal Ischemia-Reperfusion Injury in Rats
Effects of Allopurinol and Apocynin on Renal Ischemia-Reperfusion Injury in Rats E.K. Choia, H. Junga, K.H. Kwaka, J. Yeoa, S.J. Yia, C.Y. Parka, T.H. Ryub, Y.H. Jeonc, K.M. Parkd, and D.G. Lima,* a Department of Anesthesiology and Pain Medicine, School of Medicine, Kyungpook National University, Daegu, Korea; bDepartment of Anesthesiology and Pain Medicine, School of Medicine, Catholic University of Daegu, Daegu, Korea; cDepartment of Anesthesiology and Pain Medicine, School of Dentistry, Kyungpook National University, Daegu, Korea; and dDepartment of Anatomy and BK21 Plus Program, Kyungpook National University School of Medicine, Daegu, Korea
ABSTRACT Background. This study evaluated the effects of allopurinol (ALP), a xanthine oxidase inhibitor, and apocynin (APC), a NADPH oxidase inhibitor, administered alone or together, on kidney damage caused by renal ischemia-reperfusion (IR) in rats. Methods. Thirty rats were randomly assigned to 5 groups. Group 1 was a sham group. Group 2 was the renal IR control group (30-min ischemia followed by 24-h reperfusion). In groups 3 and 4, ALP or APC, respectively, was administered 1 h before the ischemia. In group 5, ALP and APC were co-administered. Blood urea nitrogen (BUN) and serum creatinine (Cr), renal tissue malondialdehyde (MDA) and superoxide dismutase (SOD), and histological changes were evaluated. Results. A significant increase in BUN and Cr level, and histological damage was seen in the IR control group, indicating renal injury. Elevated MDA and decreased SOD levels in the IR control group demonstrated that renal damage occurred through oxidative stress. Pretreatment with ALP or APC alone or together prevented IR-induced renal damage. However, there was no significant difference between treatment with a single drug and co-administration of ALP and APC. Conclusions. The use of ALP and/or APC before ischemia may be beneficial to ameliorate renal IR injury.
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ENAL ischemia-reperfusion (IR) injury, the main causative factor for acute kidney injury, occurs in a variety of clinical conditions, including renal transplant, partial nephrectomy, shock, and sepsis [1e3]. Cell and tissue damage during the ischemic period is paradoxically aggravated further when blood flow is restored, called reperfusion. Although many mechanisms have been implicated in renal IR injury, reactive oxygen species (ROS) are a critical mediator of reperfusion injury [4,5]. Excessive production of ROS promotes renal IR injury by affecting the function of cellular DNA, proteins, and lipids [6,7]. During IR injury, ROS are generated by numerous sources, including xanthine oxidase (XO), NADPH oxidase (NOX), and the mitochondrial respiration chain [8]. Neutralization of ROS by endogenous antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), or catalase reduces the toxic effects [9]. Furthermore, to ameliorate renal
injury induced by IR, several therapeutic strategies to inhibit XO and/or NOX have been investigated experimentally [10e12]. Many studies have demonstrated that allopurinol (ALP), an XO inhibitor, protects against renal IR injury [13]. During the ischemic period, adenosine triphosphate is degraded to hypoxanthine and xanthine. Xanthine dehydrogenase (XDH) is also converted to XO, which is This research was supported by Kyungpook National University Research Fund, 2012. *Address correspondence to Dong Gun Lim, MD, PhD, Department of Anesthesiology and Pain Medicine, School of Medicine, Kyungpook National University, 130 Dongdeok-ro, Jung-gu, Daegu, 700-721, Republic of Korea. E-mail: dglim@ knu.ac.kr
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.06.007
Transplantation Proceedings, 47, 1633e1638 (2015)
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related to the generation of ROS [6]. Furthermore, apocynin (APC, 4-hydroxy-3-methoxy-acetophenone) has been used as an antioxidant to prevent the formation of ROS in a number of studies. The action mechanism for APC relates to the inhibition of NOX, a critical enzyme that catalyzes the electron reduction of molecular oxygen to promote superoxide anion production [8,14]. Stimuli by various cytokines and vasoactive mediators result in enhancement of the NOX in the endothelium, which in turn generates excessive ROS [8]. ALP and APC inhibit ROS production through different mechanisms. Thus, it was proposed that combined administration of these inhibitors may be more effective than individual treatment. We therefore conducted this study to investigate the renoprotective effect of ALP and APC during renal IR injury individually and in combination. MATERIALS AND METHODS Animals Male Sprague-Dawley rats (275e325 g) were used in this study (Central Lab Animal Inc, Seoul, Korea). All animal procedures were approved by the Kyungpook National University Institutional Animal Care and Use Committee.
Experimental Protocol Rats were pretreated with either ALP (50 mg/kg, i.p.) or APC (20 mg/ kg, i.p.) 1 h before the renal IR procedure. Under ketamine (60 mg/kg, i.p.) and xylene (10 mg/kg, i.p.) anesthesia, a right flank incision was made and right nephrectomy was performed. A left flank incision was then made, and the renal artery and vein were clamped to induce ischemia with the use of a nontraumatic vascular clamp. After 30 min of ischemia, the clamp was removed to allow reperfusion, and the skin was sutured. At 24 h after reperfusion, blood samples were obtained from the heart and organs were harvested. The serum was separated through centrifugation at 3000 rpm for 15 min. The left kidney was divided into longitudinal sections. One section was processed for histopathological examination and the other section was placed in liquid nitrogen and stored at 80 C for the malondialdehyde (MDA) and SOD analysis. Rats (n ¼ 30) were randomly divided into 1 of 5 groups for the experiments. Group 1 was the sham group, in which the rats underwent a sham operation without the renal IR procedure and received normal saline (sham); group 2 was the renal IR control group, in which the rats underwent the renal IR procedure and received normal saline (IR control); group 3 was the ALP treatment group, in which the rats received an ALP i.p. injection 1 h before the renal IR procedure (ALP); group 4 was the APC treatment group, in which the rats received an APC i.p. injection 1 h before the renal IR procedure (APC); and group 5 was the ALP and APC treatment group, in which the rats received ALP and APC i.p. injection 1 h before the renal IR procedure (ALP þ APC).
Biochemical Analysis Blood urea nitrogen (BUN) and creatinine (Cr) levels were used to assess renal function. Serum levels of BUN and Cr were analyzed with the use of a colorimetric assay, following the manufacturer’s protocols (Asan Pharmacy, Seoul, Korea). The renal MDA levels were determined spectrophotometrically with the use of thiobarbituric acid reactive substances [15]. The absorbance of the reaction mixture was
Fig 1. Serum BUN (A) and Cr (B) levels in the experimental groups. Compared with the sham group, the IR control group showed significant increases in serum BUN and Cr levels. Administration of ALP or, APC alone, and ALP þ APC significantly reduced the IRinduced increase in BUN and Cr levels. There were no significant differences among the 3 drug treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Results are expressed as mean SEM (n ¼ 6 rats/group). measured at 535 nm. The MDA level was expressed as nmol/g tissue, according to a standard curve. The SOD activity assay was performed with the use of the pyrogallol autoxidation method [16]. Tris-HCl and pentetic acid buffer were used as a reaction medium, and the decrease in pyrogallol absorbance was monitored at 420 nm spectrophotometrically. SOD activity was evaluated as the amount of enzyme that reduced color change by 50% and calculated as U/mg protein.
Histopathological Analysis Kidney specimens were fixed immediately in 10% phosphate-buffered formalin, embedded in paraffin, cut in 4-mm sections, and stained with periodic acid Schiff (PAS). The sections were evaluated and scored for tubular cell form, lumen dilatation, and cast formation. Results were analyzed and graded by a scale from 0 to 3 (0, normal; 1, minimal; 2, moderate; 3, severe) [17]. The sections were evaluated by use of light microscopy at 200 magnification.
Statistical Analysis Data analysis was performed with the use of statistical software (SPSS, version 18.0 for Windows; SPSS, Chicago, Ill, United States). All data are expressed as mean SEM. The 1-way analysis of variance (ANOVA), followed by Bonferroni test for post hoc
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both MDA and SOD). Treatment with ALP or APC alone and in co-administration significantly lowered MDA levels compared with the IR control group (P < .05 versus ALP, APC, ALP þ APC, respectively). Furthermore, drug treatment significantly increased the SOD levels compared with the IR control group (P < .05 versus ALP, ALP þ APC, respectively). No significant differences were observed among the 3 drug treatment groups (Fig 2). Histopathological Evaluations
There were no noticeable changes in the kidney tissues from sham rats. Histological analysis of kidney sections from rats subjected to renal IR revealed significant tissue damage, including tubular cell atrophy, tubular dilatation, cast formation, and luminal congestion, as compared with the sham group (P < .05). Treatment with ALP or APC alone and in co-administration induced a significant recovery in the tubular cells compared with the IR control group (P < .05 versus ALP, APC, ALP þ APC, respectively). However, no significant differences were observed among the 3 drug treatment groups (Figs 3 and 4). DISCUSSION
Fig 2. Renal tissue MDA and SOD levels in the experimental groups. (A) Compared with the sham group, the IR control group showed significant increases in MDA levels. Administration of ALP or, APC alone, and ALP þ APC significantly reduced the IRinduced increase in MDA levels. (B) In the IR control group, the levels of SOD showed significant decreases compared with the sham group. SOD levels after administration of ALP or ALP þ APC group were significantly higher than the IR control group. There were no significant differences between the treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Results are expressed as mean SEM (n ¼ 6 rats/group).
Our study has revealed that treatment of rats with the XO inhibitor, ALP, and the NOX inhibitor, APC, attenuated renal dysfunction caused by renal IR injury. This is consistent with previous studies that indicated ALP or APC protects the kidneys from IR induced damage. APL and APC, either individually or in combination, act as an antioxidant. We demonstrated that the doses and administration time of ALP and APC chosen to reduce the production of ROS in this study were sufficient to abolish the kidney damage. However, contrary to our hypothesis, we found that combined therapy with ALP and APC did not have a more
comparisons, was used to compare values among all groups. Differences between the groups were considered significant at P < .05.
RESULTS Changes in Renal Function
In the IR control group, the level of serum BUN and Cr showed a significant increase compared with the sham group (P < .05 for both BUN and Cr). Pretreatment of the rats with ALP or APC alone and in co-administration resulted in significantly lower serum BUN and Cr levels than those in the rats with renal IR (P < .05 for both BUN and Cr versus ALP, APC, ALP þ APC, respectively). There were no significant differences in serum BUN and Cr levels among the 3 drug treatment groups (Fig 1). Changes in Renal Oxidative Stress
Renal IR significantly increased MDA levels and decreased SOD levels compared with the sham group (for P < .05 for
Fig 3. Histopathological damage scores in the renal tissues of the experimental groups. Renal IR caused significant tissue damage compared with the sham group. Treatment with ALP or, APC alone, and ALP þ APC significantly decreased tissue damage compared with the IR control group. No significant differences were observed between the treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Data are expressed as mean SEM (n ¼ 6 rats/group).
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Fig 4. Histopathological photographs of renal tissue in the experimental groups. (A) Sham group: no damage; (B) IR control group: marked tubular damage and luminal congestion; (C) ALP group; (D) APC group; (E) ALP þ APC group: mild tubular damage with normal histological appearance. Periodic acid Schiff (PAS) stain, magnification 200.
beneficial effect on IR induced damage. Rather, the protective effects were similar to those of single drug treatment. ROS can be produced during normal cellular metabolism; however, numerous experimental studies have indicated that ROS play a central role in organ damage by IR [4,6]. ROS such as the superoxide anion, hydroxyl radical, and hydrogen peroxide are generally accepted as hazardous to cell homeostasis, and excessive exposure can lead to cell death [18]. Pronounced formation of ROS may arise from either stimulation of XO or NOX or the mitochondrial electron transport chain [8]. Many drugs that attenuate oxidative stress and inflammatory responses have been used to ameliorate organ damage in experimental models of IR [12,19,20]. Among these drugs, ALP has been used as an antioxidant to inhibit kidney damage caused by renal IR injury [13]. The mechanism for the protective benefit of ALP may be due to an inhibition of the breakdown of purine metabolites such as hypoxanthine and xanthine, which in turn results in an inhibited formation of ROS [7]. During ischemia, xanthine is oxidized into uric acid by the XO, and, when re-oxygenation occurs, the XO transfers the electron to form ROS [7]. Besides inhibiting the formation of ROS through XO inhibition, ALP may also scavenge free radicals [21], preserve mitochondrial membrane integrity [22], and reduce the inflammatory response [23]. In this study, pretreatment of the rats with ALP resulted in a significant decrease in their serum BUN, Cr, and MDAdwhich is an indicator of oxidative stress due to lipid peroxidationdand preservation of the antioxidant SOD in the renal tissues. Moreover, the histological damage induced by IR was ameliorated in the ALP treatment group. On the basis of these results, and consistent with other reports [24,25], ALP was demonstrated to be a renoprotective substance that prevents IR injury. Although the exact mechanism of the ALP protective effect is unknown, we suggest that the inhibition of XO partly contributes to renoprotection and that other mechanisms such as free radical scavenging, the
reduction of inflammation, and the maintenance of the mitochondrial membrane integrity may also be related to the protection it offers against oxidative damage caused by renal IR injury. Although the role of XO in ROS production is well known, other pathological pathways contribute to the formation of ROS. NOX is another source of ROS production, which in turn generates an inflammatory state and diverse pathological conditions, including degenerative and neoplastic diseases [26]. Previous studies have demonstrated that inhibitors of NOX protect against organ injury in a number of clinical settings relating to IR [8,27]. Among the numerous NOX inhibitors, APC is a naturally derived, methoxy-substituted catechol that inhibits NOX by prohibiting the assembly of its subunits and, due to its very low toxicity, can be used at a high dose and for a long period of time. APC has been used in many experimental IR models and is considered a promising potential therapeutic for cardiovascular and neurological diseases due to its antioxidant and anti-inflammatory effects [28,29]. However, in addition to its generally recognized role as a NOX inhibitor, APC has been also observed to decrease the ROS through a NOX-independent pathway. Heumuller et al [30] showed that APC acted as a free radical scavenger in ROS generation. Furthermore, APC has been shown to affect the arachidonic acid metabolism [31], increase blood flow [32], and inhibit cyclooxygenase activity [33]. In this study, APC treatment in rats caused a significant decrease in serum BUN and Cr levels. Furthermore, APC treatment reduced MDA levels and improved histological damage induced by IR. Combined, our data indicate that APC protects against renal IR injury by reducing the formation of NOX-derived superoxide anion. Furthermore, as mentioned above, NOX-independent mechanisms may also contribute to the reduction in ROS formation and inflammatory response during renal injury after the IR procedure. We initially hypothesized that combined treatment with the use of ALP and APC may offer greater protective
ALLOPURINOL/APOCYNIN AND RENAL IR INJURY
effects by interfering with the different pathways that mediate ROS formation. However, our experimental results showed that combined administration of ALP and APC did not present additional synergistic antioxidant effects. Rather, the observed protective effect with combination therapy was similar to that of individual administration. Although ALP and APC are partly known to inhibit XO and NOX, respectively, the mechanisms by which XO and NOX interact remain unclear. McNally et al [34] proposed that the mechanism of the NOX-XO cascade activation may occur in series rather than in parallel. In other words, XO inhibition prevents the formation of superoxide anions and has no effect on the NOX activity, whereas, on the contrary, NOX inhibition, in addition to its role on NOX itself, inhibits the activation of XO, which in turn prevents the generation of superoxide anions. Furthermore, Stolk et al [35] also demonstrated that APC was activated by H2O2 and myeloperoxidase and was stimulated by large amounts of ROS; the activation of APC might be limited by the presence of other antioxidants. Therefore, it is assumed that the reduction of ROS by ALP may restrict the inhibitory effect of APC. On the basis of the various proposals of the above-mentioned studies, we can state that the combined treatment with ALP and APC has a similar beneficial effect as each inhibitor alone. However, further studies are needed to elucidate the role of the XO and NOX interaction that are involved in the formation of superoxide anions. In conclusion, oxidative stress induces a significant increase in MDA and decrease in SOD levels, accompanied by impaired kidney function, during renal IR injury. ALP and/or APC individually and in combination therapy protects against renal dysfunction caused by IR. Therefore, we propose that treatments that inhibit XO and/or NOX may be useful to limit renal injury induced by IR.
REFERENCES [1] Matin SF, Novick AC. Renal dysfunction associated with staged bilateral partial nephrectomy: the importance of operative positioning. J Urol 2001;165:880e1. [2] Anaya-Prado R, Toledo-Pereyra LH. The molecular events underlying ischemia/reperfusion injury. Transplant Proc 2002;34: 2518e9. [3] Landry GJ, Lau IH, Liem TK, et al. Adjunctive renal artery revascularization during juxtarenal and suprarenal abdominal aortic aneurysm repairs. Am J Surg 2010;199:641e5. [4] de Groot H, Rauen U. Ischemia-reperfusion injury: processes in pathogenetic networks: a review. Transplant Proc 2007;39:481e4. [5] Granger DN, Korthuis RJ. Physiologic mechanisms of postischemic tissue injury. Annu Rev Physiol 1995;57:311e32. [6] McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159e63. [7] Paller MS, Hoidal JR, Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 1984;74:1156e64. [8] Abdelrahman M, Mazzon E, Bauer M, et al. Inhibitors of NADPH oxidase reduce the organ injury in hemorrhagic shock. Shock 2005;23:107e14.
1637 [9] Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/ reperfusion injury. Ann N Y Acad Sci 2005;1047:248e58. [10] Sahna E, Parlakpinar H, Ozturk F, et al. The protective effects of physiological and pharmacological concentrations of melatonin on renal ischemia-reperfusion injury in rats. Urol Res 2003;31:188e93. [11] Sahna E, Parlakpinar H, Cihan OF, et al. Effects of aminoguanidine against renal ischaemia-reperfusion injury in rats. Cell Biochem Funct 2006;24:137e41. [12] Hosseini F, Naseri MK, Badavi M, et al. Effect of beta carotene on lipid peroxidation and antioxidant status following renal ischemia/reperfusion injury in rat. Scand J Clin Lab Invest 2010;70:259e63. [13] Prieto-Moure B, Caraben-Redano A, Aliena-Valero A, et al. Allopurinol in renal ischemia. J Invest Surg 2014;27: 304e16. [14] Altintas R, Polat A, Vardi N, et al. The protective effects of apocynin on kidney damage caused by renal ischemia/reperfusion. J Endourol 2013;27:617e24. [15] Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86:271e8. [16] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469e74. [17] Kim J, Park JW, Park KM. Increased superoxide formation induced by irradiation preconditioning triggers kidney resistance to ischemia-reperfusion injury in mice. Am J Physiol Renal Physiol 2009;296:F1202e11. [18] Greene EL, Paller MS. Xanthine oxidase produces O2- in posthypoxic injury of renal epithelial cells. Am J Physiol 1992;263: F251e5. [19] Kizilgun M, Poyrazoglu Y, Oztas Y, et al. Beneficial effects of N-acetylcysteine and ebselen on renal ischemia/reperfusion injury. Ren Fail 2011;33:512e7. [20] Beytur A, Binbay M, Sarihan ME, et al. Dose-dependent protective effect of ivabradine against ischemia-reperfusioninduced renal injury in rats. Kidney Blood Press Res 2012;35: 114e9. [21] Moorhouse PC, Grootveld M, Halliwell B, et al. Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett 1987;213:23e8. [22] Lee WY, Lee SM. Synergistic protective effect of ischemic preconditioning and allopurinol on ischemia/reperfusion injury in rat liver. Biochem Biophys Res Commun 2006;349:1087e93. [23] Corda S, Laplace C, Vicaut E, Duranteau J. Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. Am J Respir Cell Mol Biol 2001;24:762e8. [24] Keel CE, Wang Z, Colli J, et al. Protective effects of reducing renal ischemia-reperfusion injury during renal hilar clamping: use of allopurinol as a nephroprotective agent. Urology 2013;81:210.e5ee10. [25] Vaughan DL, Wickramasinghe YA, Russell GI, et al. Is allopurinol beneficial in the prevention of renal ischaemiareperfusion injury in the rat?: Evaluation by near-infrared spectroscopy. Clin Sci (Lond) 1995;88:359e64. [26] Aldieri E, Riganti C, Polimeni M, et al. Classical inhibitors of NOX NAD(P)H oxidases are not specific. Curr Drug Metab 2008;9:686e96. [27] Kono H, Rusyn I, Uesugi T, et al. Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcoholinduced liver injury in the rat. Am J Physiol Gastrointest Liver Physiol 2001;280:G1005e12. [28] Chiang CH, Chuang CH, Liu SL. Apocynin attenuates ischemia-reperfusion lung injury in an isolated and perfused rat lung model. Transl Res 2011;158:17e29.
1638 [29] Liu PG, He SQ, Zhang YH, Wu J. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice. World J Gastroenterol 2008;14:2832e7. [30] Heumuller S, Wind S, Barbosa-Sicard E, et al. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 2008;51:211e7. [31] Engels F, Renirie BF, Hart BA, et al. Effects of apocynin, a drug isolated from the roots of picrorhiza kurroa, on arachidonic acid metabolism. FEBS Lett 1992;305:254e6. [32] Liu SX, Chiang CH, Yao QS, Chiou GC. Increase of ocular blood flow by some phytogenic compounds. J Ocul Pharmacol Ther 1996;12:95e101.
CHOI, JUNG, KWAK ET AL [33] Barbieri SS, Cavalca V, Eligini S, et al. Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms. Free Radic Biol Med 2004;37:156e65. [34] McNally JS, Davis ME, Giddens DP, et al. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol 2003;285:H2290e7. [35] Stolk J, Hiltermann TJ, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 1994;11:95e102.
ABSTRACT Background. This study evaluated the effects of allopurinol (ALP), a xanthine oxidase inhibitor, and apocynin (APC), a NADPH oxidase inhibitor, administered alone or together, on kidney damage caused by renal ischemia-reperfusion (IR) in rats. Methods. Thirty rats were randomly assigned to 5 groups. Group 1 was a sham group. Group 2 was the renal IR control group (30-min ischemia followed by 24-h reperfusion). In groups 3 and 4, ALP or APC, respectively, was administered 1 h before the ischemia. In group 5, ALP and APC were co-administered. Blood urea nitrogen (BUN) and serum creatinine (Cr), renal tissue malondialdehyde (MDA) and superoxide dismutase (SOD), and histological changes were evaluated. Results. A significant increase in BUN and Cr level, and histological damage was seen in the IR control group, indicating renal injury. Elevated MDA and decreased SOD levels in the IR control group demonstrated that renal damage occurred through oxidative stress. Pretreatment with ALP or APC alone or together prevented IR-induced renal damage. However, there was no significant difference between treatment with a single drug and co-administration of ALP and APC. Conclusions. The use of ALP and/or APC before ischemia may be beneficial to ameliorate renal IR injury.
R
ENAL ischemia-reperfusion (IR) injury, the main causative factor for acute kidney injury, occurs in a variety of clinical conditions, including renal transplant, partial nephrectomy, shock, and sepsis [1e3]. Cell and tissue damage during the ischemic period is paradoxically aggravated further when blood flow is restored, called reperfusion. Although many mechanisms have been implicated in renal IR injury, reactive oxygen species (ROS) are a critical mediator of reperfusion injury [4,5]. Excessive production of ROS promotes renal IR injury by affecting the function of cellular DNA, proteins, and lipids [6,7]. During IR injury, ROS are generated by numerous sources, including xanthine oxidase (XO), NADPH oxidase (NOX), and the mitochondrial respiration chain [8]. Neutralization of ROS by endogenous antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), or catalase reduces the toxic effects [9]. Furthermore, to ameliorate renal
injury induced by IR, several therapeutic strategies to inhibit XO and/or NOX have been investigated experimentally [10e12]. Many studies have demonstrated that allopurinol (ALP), an XO inhibitor, protects against renal IR injury [13]. During the ischemic period, adenosine triphosphate is degraded to hypoxanthine and xanthine. Xanthine dehydrogenase (XDH) is also converted to XO, which is This research was supported by Kyungpook National University Research Fund, 2012. *Address correspondence to Dong Gun Lim, MD, PhD, Department of Anesthesiology and Pain Medicine, School of Medicine, Kyungpook National University, 130 Dongdeok-ro, Jung-gu, Daegu, 700-721, Republic of Korea. E-mail: dglim@ knu.ac.kr
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.06.007
Transplantation Proceedings, 47, 1633e1638 (2015)
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related to the generation of ROS [6]. Furthermore, apocynin (APC, 4-hydroxy-3-methoxy-acetophenone) has been used as an antioxidant to prevent the formation of ROS in a number of studies. The action mechanism for APC relates to the inhibition of NOX, a critical enzyme that catalyzes the electron reduction of molecular oxygen to promote superoxide anion production [8,14]. Stimuli by various cytokines and vasoactive mediators result in enhancement of the NOX in the endothelium, which in turn generates excessive ROS [8]. ALP and APC inhibit ROS production through different mechanisms. Thus, it was proposed that combined administration of these inhibitors may be more effective than individual treatment. We therefore conducted this study to investigate the renoprotective effect of ALP and APC during renal IR injury individually and in combination. MATERIALS AND METHODS Animals Male Sprague-Dawley rats (275e325 g) were used in this study (Central Lab Animal Inc, Seoul, Korea). All animal procedures were approved by the Kyungpook National University Institutional Animal Care and Use Committee.
Experimental Protocol Rats were pretreated with either ALP (50 mg/kg, i.p.) or APC (20 mg/ kg, i.p.) 1 h before the renal IR procedure. Under ketamine (60 mg/kg, i.p.) and xylene (10 mg/kg, i.p.) anesthesia, a right flank incision was made and right nephrectomy was performed. A left flank incision was then made, and the renal artery and vein were clamped to induce ischemia with the use of a nontraumatic vascular clamp. After 30 min of ischemia, the clamp was removed to allow reperfusion, and the skin was sutured. At 24 h after reperfusion, blood samples were obtained from the heart and organs were harvested. The serum was separated through centrifugation at 3000 rpm for 15 min. The left kidney was divided into longitudinal sections. One section was processed for histopathological examination and the other section was placed in liquid nitrogen and stored at 80 C for the malondialdehyde (MDA) and SOD analysis. Rats (n ¼ 30) were randomly divided into 1 of 5 groups for the experiments. Group 1 was the sham group, in which the rats underwent a sham operation without the renal IR procedure and received normal saline (sham); group 2 was the renal IR control group, in which the rats underwent the renal IR procedure and received normal saline (IR control); group 3 was the ALP treatment group, in which the rats received an ALP i.p. injection 1 h before the renal IR procedure (ALP); group 4 was the APC treatment group, in which the rats received an APC i.p. injection 1 h before the renal IR procedure (APC); and group 5 was the ALP and APC treatment group, in which the rats received ALP and APC i.p. injection 1 h before the renal IR procedure (ALP þ APC).
Biochemical Analysis Blood urea nitrogen (BUN) and creatinine (Cr) levels were used to assess renal function. Serum levels of BUN and Cr were analyzed with the use of a colorimetric assay, following the manufacturer’s protocols (Asan Pharmacy, Seoul, Korea). The renal MDA levels were determined spectrophotometrically with the use of thiobarbituric acid reactive substances [15]. The absorbance of the reaction mixture was
Fig 1. Serum BUN (A) and Cr (B) levels in the experimental groups. Compared with the sham group, the IR control group showed significant increases in serum BUN and Cr levels. Administration of ALP or, APC alone, and ALP þ APC significantly reduced the IRinduced increase in BUN and Cr levels. There were no significant differences among the 3 drug treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Results are expressed as mean SEM (n ¼ 6 rats/group). measured at 535 nm. The MDA level was expressed as nmol/g tissue, according to a standard curve. The SOD activity assay was performed with the use of the pyrogallol autoxidation method [16]. Tris-HCl and pentetic acid buffer were used as a reaction medium, and the decrease in pyrogallol absorbance was monitored at 420 nm spectrophotometrically. SOD activity was evaluated as the amount of enzyme that reduced color change by 50% and calculated as U/mg protein.
Histopathological Analysis Kidney specimens were fixed immediately in 10% phosphate-buffered formalin, embedded in paraffin, cut in 4-mm sections, and stained with periodic acid Schiff (PAS). The sections were evaluated and scored for tubular cell form, lumen dilatation, and cast formation. Results were analyzed and graded by a scale from 0 to 3 (0, normal; 1, minimal; 2, moderate; 3, severe) [17]. The sections were evaluated by use of light microscopy at 200 magnification.
Statistical Analysis Data analysis was performed with the use of statistical software (SPSS, version 18.0 for Windows; SPSS, Chicago, Ill, United States). All data are expressed as mean SEM. The 1-way analysis of variance (ANOVA), followed by Bonferroni test for post hoc
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both MDA and SOD). Treatment with ALP or APC alone and in co-administration significantly lowered MDA levels compared with the IR control group (P < .05 versus ALP, APC, ALP þ APC, respectively). Furthermore, drug treatment significantly increased the SOD levels compared with the IR control group (P < .05 versus ALP, ALP þ APC, respectively). No significant differences were observed among the 3 drug treatment groups (Fig 2). Histopathological Evaluations
There were no noticeable changes in the kidney tissues from sham rats. Histological analysis of kidney sections from rats subjected to renal IR revealed significant tissue damage, including tubular cell atrophy, tubular dilatation, cast formation, and luminal congestion, as compared with the sham group (P < .05). Treatment with ALP or APC alone and in co-administration induced a significant recovery in the tubular cells compared with the IR control group (P < .05 versus ALP, APC, ALP þ APC, respectively). However, no significant differences were observed among the 3 drug treatment groups (Figs 3 and 4). DISCUSSION
Fig 2. Renal tissue MDA and SOD levels in the experimental groups. (A) Compared with the sham group, the IR control group showed significant increases in MDA levels. Administration of ALP or, APC alone, and ALP þ APC significantly reduced the IRinduced increase in MDA levels. (B) In the IR control group, the levels of SOD showed significant decreases compared with the sham group. SOD levels after administration of ALP or ALP þ APC group were significantly higher than the IR control group. There were no significant differences between the treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Results are expressed as mean SEM (n ¼ 6 rats/group).
Our study has revealed that treatment of rats with the XO inhibitor, ALP, and the NOX inhibitor, APC, attenuated renal dysfunction caused by renal IR injury. This is consistent with previous studies that indicated ALP or APC protects the kidneys from IR induced damage. APL and APC, either individually or in combination, act as an antioxidant. We demonstrated that the doses and administration time of ALP and APC chosen to reduce the production of ROS in this study were sufficient to abolish the kidney damage. However, contrary to our hypothesis, we found that combined therapy with ALP and APC did not have a more
comparisons, was used to compare values among all groups. Differences between the groups were considered significant at P < .05.
RESULTS Changes in Renal Function
In the IR control group, the level of serum BUN and Cr showed a significant increase compared with the sham group (P < .05 for both BUN and Cr). Pretreatment of the rats with ALP or APC alone and in co-administration resulted in significantly lower serum BUN and Cr levels than those in the rats with renal IR (P < .05 for both BUN and Cr versus ALP, APC, ALP þ APC, respectively). There were no significant differences in serum BUN and Cr levels among the 3 drug treatment groups (Fig 1). Changes in Renal Oxidative Stress
Renal IR significantly increased MDA levels and decreased SOD levels compared with the sham group (for P < .05 for
Fig 3. Histopathological damage scores in the renal tissues of the experimental groups. Renal IR caused significant tissue damage compared with the sham group. Treatment with ALP or, APC alone, and ALP þ APC significantly decreased tissue damage compared with the IR control group. No significant differences were observed between the treatment groups. *P < .05 versus the sham group; yP < .05 versus the IR control group. Data are expressed as mean SEM (n ¼ 6 rats/group).
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CHOI, JUNG, KWAK ET AL
Fig 4. Histopathological photographs of renal tissue in the experimental groups. (A) Sham group: no damage; (B) IR control group: marked tubular damage and luminal congestion; (C) ALP group; (D) APC group; (E) ALP þ APC group: mild tubular damage with normal histological appearance. Periodic acid Schiff (PAS) stain, magnification 200.
beneficial effect on IR induced damage. Rather, the protective effects were similar to those of single drug treatment. ROS can be produced during normal cellular metabolism; however, numerous experimental studies have indicated that ROS play a central role in organ damage by IR [4,6]. ROS such as the superoxide anion, hydroxyl radical, and hydrogen peroxide are generally accepted as hazardous to cell homeostasis, and excessive exposure can lead to cell death [18]. Pronounced formation of ROS may arise from either stimulation of XO or NOX or the mitochondrial electron transport chain [8]. Many drugs that attenuate oxidative stress and inflammatory responses have been used to ameliorate organ damage in experimental models of IR [12,19,20]. Among these drugs, ALP has been used as an antioxidant to inhibit kidney damage caused by renal IR injury [13]. The mechanism for the protective benefit of ALP may be due to an inhibition of the breakdown of purine metabolites such as hypoxanthine and xanthine, which in turn results in an inhibited formation of ROS [7]. During ischemia, xanthine is oxidized into uric acid by the XO, and, when re-oxygenation occurs, the XO transfers the electron to form ROS [7]. Besides inhibiting the formation of ROS through XO inhibition, ALP may also scavenge free radicals [21], preserve mitochondrial membrane integrity [22], and reduce the inflammatory response [23]. In this study, pretreatment of the rats with ALP resulted in a significant decrease in their serum BUN, Cr, and MDAdwhich is an indicator of oxidative stress due to lipid peroxidationdand preservation of the antioxidant SOD in the renal tissues. Moreover, the histological damage induced by IR was ameliorated in the ALP treatment group. On the basis of these results, and consistent with other reports [24,25], ALP was demonstrated to be a renoprotective substance that prevents IR injury. Although the exact mechanism of the ALP protective effect is unknown, we suggest that the inhibition of XO partly contributes to renoprotection and that other mechanisms such as free radical scavenging, the
reduction of inflammation, and the maintenance of the mitochondrial membrane integrity may also be related to the protection it offers against oxidative damage caused by renal IR injury. Although the role of XO in ROS production is well known, other pathological pathways contribute to the formation of ROS. NOX is another source of ROS production, which in turn generates an inflammatory state and diverse pathological conditions, including degenerative and neoplastic diseases [26]. Previous studies have demonstrated that inhibitors of NOX protect against organ injury in a number of clinical settings relating to IR [8,27]. Among the numerous NOX inhibitors, APC is a naturally derived, methoxy-substituted catechol that inhibits NOX by prohibiting the assembly of its subunits and, due to its very low toxicity, can be used at a high dose and for a long period of time. APC has been used in many experimental IR models and is considered a promising potential therapeutic for cardiovascular and neurological diseases due to its antioxidant and anti-inflammatory effects [28,29]. However, in addition to its generally recognized role as a NOX inhibitor, APC has been also observed to decrease the ROS through a NOX-independent pathway. Heumuller et al [30] showed that APC acted as a free radical scavenger in ROS generation. Furthermore, APC has been shown to affect the arachidonic acid metabolism [31], increase blood flow [32], and inhibit cyclooxygenase activity [33]. In this study, APC treatment in rats caused a significant decrease in serum BUN and Cr levels. Furthermore, APC treatment reduced MDA levels and improved histological damage induced by IR. Combined, our data indicate that APC protects against renal IR injury by reducing the formation of NOX-derived superoxide anion. Furthermore, as mentioned above, NOX-independent mechanisms may also contribute to the reduction in ROS formation and inflammatory response during renal injury after the IR procedure. We initially hypothesized that combined treatment with the use of ALP and APC may offer greater protective
ALLOPURINOL/APOCYNIN AND RENAL IR INJURY
effects by interfering with the different pathways that mediate ROS formation. However, our experimental results showed that combined administration of ALP and APC did not present additional synergistic antioxidant effects. Rather, the observed protective effect with combination therapy was similar to that of individual administration. Although ALP and APC are partly known to inhibit XO and NOX, respectively, the mechanisms by which XO and NOX interact remain unclear. McNally et al [34] proposed that the mechanism of the NOX-XO cascade activation may occur in series rather than in parallel. In other words, XO inhibition prevents the formation of superoxide anions and has no effect on the NOX activity, whereas, on the contrary, NOX inhibition, in addition to its role on NOX itself, inhibits the activation of XO, which in turn prevents the generation of superoxide anions. Furthermore, Stolk et al [35] also demonstrated that APC was activated by H2O2 and myeloperoxidase and was stimulated by large amounts of ROS; the activation of APC might be limited by the presence of other antioxidants. Therefore, it is assumed that the reduction of ROS by ALP may restrict the inhibitory effect of APC. On the basis of the various proposals of the above-mentioned studies, we can state that the combined treatment with ALP and APC has a similar beneficial effect as each inhibitor alone. However, further studies are needed to elucidate the role of the XO and NOX interaction that are involved in the formation of superoxide anions. In conclusion, oxidative stress induces a significant increase in MDA and decrease in SOD levels, accompanied by impaired kidney function, during renal IR injury. ALP and/or APC individually and in combination therapy protects against renal dysfunction caused by IR. Therefore, we propose that treatments that inhibit XO and/or NOX may be useful to limit renal injury induced by IR.
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