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Antioxidant Vitamins Preserve Superoxide Dismutase Activities in Gentamicin-Induced Nephrotoxicity
Antioxidant Vitamins Preserve Superoxide Dismutase Activities in Gentamicin-Induced Nephrotoxicity M. Kadkhodaee, H. Khastar, H.A. Arab, R. Ghaznavi, M. Zahmatkesh, and M. Mahdavi-Mazdeh ABSTRACT Objective. The clinical use of gentamicin (G) is limited due to its known nephrotoxic actions. Generation of reactive oxygen species has been proposed as a causative factor of cell death in G-induced acute renal failure (ARF). Previous studies using superoxide dismutase (SOD) mimetics have indirectly suggested a role for the superoxide ion in G-induced ARF. In this study, we directly measured the enzyme activities using in situ isolated kidneys seeking to investigate the effects of antioxidant therapy on preservation of endogenous antioxidant levels in ARF. Methods. Thirty-five male Sprague-Dawley rats were randomly assigned to 5 groups: control, Tyrode-perfused; G, gentamicin (200 mg/L) added to the perfusate; G ⫹ vitamin E (Vit E; 100 mg/100 g BW, IM); G ⫹ vitamin C (Vit C) added to the drinking water for 3 days (200 mg/L) and to the perfusate (100 mg/L); G ⫹ Vit E ⫹ Vit C. SOD activities were determined in renal tissues based on NAPDH oxidation at 340 nm by spectrophotometry. Results. SOD activity was significantly reduced in the G group compared with the controls (P ⬍ .05). Administration of Vit E alone or in combination with Vit C significantly preserved enzyme activity levels compared with the G group (P ⬍ .05). Conclusion. Antioxidant vitamins have a role in preservation of renal endogenous antioxidant activities, namely SOD, in G-induced nephrotoxicity.
A
MINOGLYCOSIDE ANTIBIOTICS including gentamicin (G) produce nephrotoxicity in humans. These adverse renal effects may be induced by generation of reactive oxygen species (ROS). There are reports that antioxidant administration ameliorates G-induced nephropathy.1 A role for superoxide in G-mediated nephropathy was suggested when various superoxide dismutase (SOD) treatments were shown to be effective to ameliorate renal injury in G-induced nephrotoxicity.2 In the present study, the role of ROS in G-induced nephrotoxicity was assessed by administration of antioxidant vitamins and by evaluation of altered SOD activities, as the first line of antioxidant defense, using in situ isolated kidneys. MATERIALS AND METHODS Animal Preparation: In Situ Isolated Perfused Kidneys Male Sprague-Dawley rats weighing 200 to 300 g received animal care in compliance with the guidelines of our Animal and Human Ethical Committee. Animals were anesthetized by IP injection of ketamine hydrochloride (70 mg/kg). To perfuse the kidneys, the abdominal aorta above and below the renal artery was ligated. 0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.02.038 864
After the cannulation of the aorta, the kidneys were perfused using a peristaltic pump (IBS P803, Integra Bioscience, Switzerland). After infusion of heparin, perfusion was started with oxygenated Tyrode’s solution containing inulin (60 mg/dL) at a rate of 8 mL/min at 37°C.
Groups In this study, 35 rats were randomly arranged into 5 groups of 7 rats each: group 1, control kidneys were perfused with Tyrode’s soluFrom the Department of Physiology, Faculty of Medicine, Tehran Medical Sciences University, Tehran, Iran (M.K., H.K., R.G., M.Z., M.M.-M.), and the Department of Pharmacology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran (H.A.A.). This study was supported by a grant from Tehran Medical Sciences University. Address reprint requests to Dr Mehri Kadkhodaee, Department of Physiology, Faculty of Medicine, Tehran Medical Sciences University, 14174 Tehran, Iran. E-mail: kadkhodm@ tums.ac.ir © 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 39, 864 – 865 (2007)
ANTIOXIDANT VITAMINS PRESERVE SOD ACTIVITY
865
tion; group 2 (G), gentamicin (200 mg/L) added to the perfusate 15 minutes after initiation of perfusion; group 3 (G ⫹ Vit C), same as group 2 with vitamin C added to the drinking water for 3 days (200 mg/L) and to the perfusate (100 mg/L); group 4 (G ⫹ Vit E), same as group 2 with vitamin E (100 mg/100 g BW, IM) injected 12 hours before the start of the experiments; group 5 (G ⫹ Vit C ⫹ Vit E), same as group 2 but vitamins C and E were both administered (concentrations and conditions as in groups 3 and 4).
Experimental Protocol In all groups, perfusion was performed for 90 minutes; the first 15 minutes was considered to be the stabilizing period. At the end of the perfusion, the kidneys were removed from the body, weighed, and homogenized in phosphate buffer with the supernate separated by centrifugation. The reduction of absorbance was measured in the supernate by spectrophotometry at 340 nm in the presence of NADPH.
Statistical Analysis Data are expressed as mean values ⫾ SEM for the groups and analyzed by one-way analysis of variance. To show the difference among the groups, we used Tukey’s post-hoc test. P ⬍ .05 was considered significant.
RESULTS
The SOD activity was significantly reduced in the G group compared with the controls (0.68 ⫾ 0.16 vs 1.30 ⫾ 0.17 U/g; P ⬍ .05; Fig 1). Administration of vitamin E alone (1.36 ⫾ 0.14; P ⬍ .05) or in combination with vitamin C (1.64 ⫾ 0.12; P ⬍ .05) significantly preserved the enzyme activity levels compared with the G group. DISCUSSION
In this study, G caused significant nephrotoxicity as demonstrated by decreased SOD activities compared with controls. Prior administration of vitamin E inhibited the Ginduced decrease in enzyme activities. While vitamin C alone was not able to prevent reduction in SOD activities, the combination of vitamins C and E significantly preserved the enzyme activities. Among the antioxidants, SOD is the first and most important line of defense against ROS. At sufficient concentrations, antioxidant vitamins have been shown to inhibit pathological conditions. Vitamin E is the main fat-soluble endogenous antioxidant which reacts with oxygen radicals preventing free radical chain reactions to protect the membranes. In a heart model of G-induced toxicity, vitamin E
Fig 1. Comparison of SOD activities in kidneys perfused with Tyrode’s buffer without any treatment (control); Tyrode’s buffer containing 200 mg/L gentamicin (G); Tyrode’s buffer containing 100 mg/L vitamin C and pretreatment with 200 mg/L vitamin C in the drinking water for 3 days (Vit C); Tyrode’s buffer containing 100 mg/100 g BW vitamin E (Vit E) and coadministration of vitamins C and E (Vit C ⫹ E). The data are presented as mean values ⫾ SEM. *P ⬍ .05 compared with control; #P ⬍ .05 compared with G (n ⫽ 7).
protected guinea pig heart tissues against free-radicalinduced injury.3 However, storage of endogenous antioxidants, such as vitamin E, decreases gradually while reacting with free radicals. Due to the recycling property of vitamin E by vitamin C, administration of vitamin C helps to replenish the storage of vitamin E. Since high doses of vitamin C are reported to act as an oxidant agent, we coadministered moderate doses of vitamins E and C in this study to achieve maximum antioxidant effects. The present study revealed that the G-induced renal toxicity was significantly reduced by cosupplementation of vitamins C and E. We concluded that antioxidant vitamins have a role in preservation of renal endogenous antioxidant activities, namely SOD, in G-induced nephrotoxicity. REFERENCES 1. Pedraza-Chaverri J, Gonzalez-Orozco AE, Maldonado PD, et al: Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy in rats. Eur J Pharmacol 473:71, 2003 2. Cuzzocrea S, Mazzon E, Dugo L, et al: A role for superoxide in gentamicin-mediated nephropathy in rats. Eur J Pharmacol 450:67, 2002 3. Ozturk HS, Kavutcu M, Kacmaz M, et al: The effects of gentamicin on the activities of glutathione peroxidase and superoxide dismutase enzymes and malondialdehyde levels in heart tissues of guinea pigs. Curr Med Res Opin 14:47, 1997
A
MINOGLYCOSIDE ANTIBIOTICS including gentamicin (G) produce nephrotoxicity in humans. These adverse renal effects may be induced by generation of reactive oxygen species (ROS). There are reports that antioxidant administration ameliorates G-induced nephropathy.1 A role for superoxide in G-mediated nephropathy was suggested when various superoxide dismutase (SOD) treatments were shown to be effective to ameliorate renal injury in G-induced nephrotoxicity.2 In the present study, the role of ROS in G-induced nephrotoxicity was assessed by administration of antioxidant vitamins and by evaluation of altered SOD activities, as the first line of antioxidant defense, using in situ isolated kidneys. MATERIALS AND METHODS Animal Preparation: In Situ Isolated Perfused Kidneys Male Sprague-Dawley rats weighing 200 to 300 g received animal care in compliance with the guidelines of our Animal and Human Ethical Committee. Animals were anesthetized by IP injection of ketamine hydrochloride (70 mg/kg). To perfuse the kidneys, the abdominal aorta above and below the renal artery was ligated. 0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.02.038 864
After the cannulation of the aorta, the kidneys were perfused using a peristaltic pump (IBS P803, Integra Bioscience, Switzerland). After infusion of heparin, perfusion was started with oxygenated Tyrode’s solution containing inulin (60 mg/dL) at a rate of 8 mL/min at 37°C.
Groups In this study, 35 rats were randomly arranged into 5 groups of 7 rats each: group 1, control kidneys were perfused with Tyrode’s soluFrom the Department of Physiology, Faculty of Medicine, Tehran Medical Sciences University, Tehran, Iran (M.K., H.K., R.G., M.Z., M.M.-M.), and the Department of Pharmacology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran (H.A.A.). This study was supported by a grant from Tehran Medical Sciences University. Address reprint requests to Dr Mehri Kadkhodaee, Department of Physiology, Faculty of Medicine, Tehran Medical Sciences University, 14174 Tehran, Iran. E-mail: kadkhodm@ tums.ac.ir © 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 39, 864 – 865 (2007)
ANTIOXIDANT VITAMINS PRESERVE SOD ACTIVITY
865
tion; group 2 (G), gentamicin (200 mg/L) added to the perfusate 15 minutes after initiation of perfusion; group 3 (G ⫹ Vit C), same as group 2 with vitamin C added to the drinking water for 3 days (200 mg/L) and to the perfusate (100 mg/L); group 4 (G ⫹ Vit E), same as group 2 with vitamin E (100 mg/100 g BW, IM) injected 12 hours before the start of the experiments; group 5 (G ⫹ Vit C ⫹ Vit E), same as group 2 but vitamins C and E were both administered (concentrations and conditions as in groups 3 and 4).
Experimental Protocol In all groups, perfusion was performed for 90 minutes; the first 15 minutes was considered to be the stabilizing period. At the end of the perfusion, the kidneys were removed from the body, weighed, and homogenized in phosphate buffer with the supernate separated by centrifugation. The reduction of absorbance was measured in the supernate by spectrophotometry at 340 nm in the presence of NADPH.
Statistical Analysis Data are expressed as mean values ⫾ SEM for the groups and analyzed by one-way analysis of variance. To show the difference among the groups, we used Tukey’s post-hoc test. P ⬍ .05 was considered significant.
RESULTS
The SOD activity was significantly reduced in the G group compared with the controls (0.68 ⫾ 0.16 vs 1.30 ⫾ 0.17 U/g; P ⬍ .05; Fig 1). Administration of vitamin E alone (1.36 ⫾ 0.14; P ⬍ .05) or in combination with vitamin C (1.64 ⫾ 0.12; P ⬍ .05) significantly preserved the enzyme activity levels compared with the G group. DISCUSSION
In this study, G caused significant nephrotoxicity as demonstrated by decreased SOD activities compared with controls. Prior administration of vitamin E inhibited the Ginduced decrease in enzyme activities. While vitamin C alone was not able to prevent reduction in SOD activities, the combination of vitamins C and E significantly preserved the enzyme activities. Among the antioxidants, SOD is the first and most important line of defense against ROS. At sufficient concentrations, antioxidant vitamins have been shown to inhibit pathological conditions. Vitamin E is the main fat-soluble endogenous antioxidant which reacts with oxygen radicals preventing free radical chain reactions to protect the membranes. In a heart model of G-induced toxicity, vitamin E
Fig 1. Comparison of SOD activities in kidneys perfused with Tyrode’s buffer without any treatment (control); Tyrode’s buffer containing 200 mg/L gentamicin (G); Tyrode’s buffer containing 100 mg/L vitamin C and pretreatment with 200 mg/L vitamin C in the drinking water for 3 days (Vit C); Tyrode’s buffer containing 100 mg/100 g BW vitamin E (Vit E) and coadministration of vitamins C and E (Vit C ⫹ E). The data are presented as mean values ⫾ SEM. *P ⬍ .05 compared with control; #P ⬍ .05 compared with G (n ⫽ 7).
protected guinea pig heart tissues against free-radicalinduced injury.3 However, storage of endogenous antioxidants, such as vitamin E, decreases gradually while reacting with free radicals. Due to the recycling property of vitamin E by vitamin C, administration of vitamin C helps to replenish the storage of vitamin E. Since high doses of vitamin C are reported to act as an oxidant agent, we coadministered moderate doses of vitamins E and C in this study to achieve maximum antioxidant effects. The present study revealed that the G-induced renal toxicity was significantly reduced by cosupplementation of vitamins C and E. We concluded that antioxidant vitamins have a role in preservation of renal endogenous antioxidant activities, namely SOD, in G-induced nephrotoxicity. REFERENCES 1. Pedraza-Chaverri J, Gonzalez-Orozco AE, Maldonado PD, et al: Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy in rats. Eur J Pharmacol 473:71, 2003 2. Cuzzocrea S, Mazzon E, Dugo L, et al: A role for superoxide in gentamicin-mediated nephropathy in rats. Eur J Pharmacol 450:67, 2002 3. Ozturk HS, Kavutcu M, Kacmaz M, et al: The effects of gentamicin on the activities of glutathione peroxidase and superoxide dismutase enzymes and malondialdehyde levels in heart tissues of guinea pigs. Curr Med Res Opin 14:47, 1997