Melatonin administration prevents the nephrotoxicity induced by gentamicin

Authors


Dr E. Ozbek, Turgut Özal Tip Merkezi, Uroloji Anabilim Dali, 44069 Malatya, Turkey. E-mail: eozbek@superonline.com

Abstract

Objective To investigate the effect of melatonin on the antioxidant enzyme activity and renal tubular necrosis induced by gentamicin.

Materials and methods Twenty-four adult male Sprague-Dawley rats were divided into three equal groups. In group 1, the rats were injected with vehicle (controls), in group 2 they were injected with gentamicin for 5 days and in group 3 injected with gentamicin plus melatonin for 5 days. At 24 h after the last injection, rats were killed and the renal cortex separated from the medulla. Most of the cortex was homogenized but a small sample was fixed in formaldehyde solution for histological examination by light microscopy. Blood samples were also taken to assess the serum levels of urea, creatinine, Na+, K+ and γ-glutamyl transpeptidase (γ-GT); before death, urine samples were analysed for protein content. Crude extracts of the cortex were used to determine lipoperoxides, reduced glutathione (GSH-Px), catalase and superoxide dismutase (SOD). The results were compared using the Mann–Whitney U-test.

Results Compared with the controls rats, gentamicin caused hyperproteinuria, an increase in the level of γ-GT in serum, a marked increase in lipoperoxides and a significant decrease of GSH-Px, catalase and SOD activity in the kidney. In the rats in group 3 there was a marked restoration in lipid peroxidation, GSH-Px, catalase, SOD activity and proteinuria, and in γ-GT in serum. In rats in group 2 there was widespread tubular necrosis (grade 2–4) but in rats in group 3 there was a marked reduction in the extent of tubular damage. There was no significant difference in serum levels of Na+, K+, blood urea nitrogen and creatinine.

Conclusion These results indicate that melatonin prevents the tubular necrosis induced by gentamicin in rats, presumably because it is a potent antioxidant and restores antioxidant enzyme activity in the rat kidney.

Introduction

Aminoglycoside antibiotics including gentamicin are widely used in the treatment of Gram-negative infections. A major complication of these drugs is nephrotoxicity, and this accounts for ≈ 15% of all cases of acute renal failure [1]. Several studies have shown that reactive oxygen metabolites might be important in gentamicin nephrotoxicity [2]. In addition, gentamicin may enhance the generation of reactive oxygen metabolites by renal cortical mitochondria [3]. Recently, gentamicin also has been shown to enhance the generation of superoxide anions and hydroxyl radicals in renal cortical mitochondria [3]. Some authors have proposed that gentamicin-induced mobilization of iron from cortical mitochondria is responsible for gentamicin nephrotoxicity [4]. Recent in vivo studies have shown the protective effect of hydroxyl radical scavengers and/or iron chelators (presumably by preventing the generation of hydroxyl radical by the iron-catalysed Haber-Weiss reaction) in several models of tissue injury [5].

Gentamicin causes morphological and functional changes in renal cortical mitochondria, which occur before renal tubular cell injury or necrosis induced by gentamicin [6]. The specificity of gentamicin for renal toxicity is apparently related to its preferential accumulation in the renal proximal convoluted tubules (50–100 times more than in serum) [1]. Iron chelators and free-radical scavengers are reportedly protective against gentamicin nephrotoxicity [2].

Melatonin is a potent scavenger of free radicals and it may also stimulate other antioxidant activity [7]. The purpose of the present study was to examine antioxidant enzyme activities and tubular necrosis in rat kidneys treated with gentamicin and compare the results with those from rats treated with melatonin.

Materials and methods

Adult male Sprague–Dawley rats (230–250 g) were acquired from university vivarium sources and maintained in a 14-h light/10 h dark cycle with free access to food and water. Twenty-four rats were divided into three equal groups; in group 1, rats were injected with vehicle (5% ethanol); in group 2 they were injected with gentamicin (Deva Corp, Ankara, Turkey); and in group 3 with gentamicin plus melatonin (Sigma Chemical Corp, St Louis, MO). Gentamicin was injected subcutaneously on five consecutive days (100 mg/kg/day) and in group 3, melatonin was injected simultaneously and intraperitoneally (500 µg/k/day). The melatonin was dissolved in ethanol and further diluted in saline, to a final ethanol concentration of 5%[8]. At 24 h after the last injection the rats were killed under anaesthesia (intraperitoneal sodium pentobarbitol, 50 mg/kg body weight). The kidneys were quickly removed and decapsulated, the renal cortex carefully separated from the medulla, homogenized as described previously [2], and a sample of the kidney tissues placed in formaldehyde solution. These were then prepared for routine histopathological examination by light microscopy (Nikon, Tokyo, Japan). The kidney sections were analysed semi-quantitatively using the technique of Houghton et al.[9]. The changes seen were limited to the tubulo-interstitial areas and were graded as: 0, normal; 1, areas of focal granulovacuolar epithelial cell degeneration and granular debris in the tubular lumen, with or without evidence of tubular epithelial cell desquamation in small foci (< 1% of the tubule population involved by desquamation); 2, tubular epithelial necrosis and desquamation easily seen but involving less than half of the cortical tubules; 3, more than half of proximal tubules showing desquamation and necrosis but involved tubules easily found; 4, complete or almost complete proximal tubular necrosis.

Trunk blood was sampled to determine the serum levels of urea nitrogen (BUN), creatinine, Na+, K+ and γ-glutamyl transpeptidase (γ-GT). Before death a urine sample was assayed for protein. All biochemical variables were determined using an Olympus Autoanalyser (Olympus Instruments, Tokyo, Japan). Crude extracts were used to determine lipoperoxides, reduced glutathione (GSH-Px), catalase and superoxide dismutase (SOD). Catalase activity was determined according to the method of Aebi [10], by monitoring the initial rate of disappearance of hydrogen peroxide (initial concentration 10 mmol/L) at 240 nm in a spectrophotometer. Results were reported as the constant rate per second per gram of protein. Malondialdehyde (MDA), referred to as thiobarbituric acid-reactive substance), was measured with thiobarbituric acid at 532 nm in a spectrofluorometer, as described previously [11]. SOD activity was measured according to Sun et al.[12] by determining the reduction of nitroblue tetrazolium (NBT) by superoxide anion produced with xanthine and xanthine oxidase. One unit of SOD is defined as the amount of protein that inhibited the rate of NBT reduction by half. GSH-Px activity was measured according to Paglia and Valentine [13], by monitoring the oxidation of reduced NADPH at 340 nm. Enzyme units were defined as the number of micromoles of NADPH oxidized per minute and calculated using the extinction coefficient of NADPH at 340 nm (6.22 × 106/mole/cm). Results were reported as units per gram protein. Protein concentrations in all samples were measured using the method of Lowry et al.[14]. The Mann–Whitney U-test was used for statistical analysis, with significance accepted at P < 0.05.

Results

There was no significant differences in BUN, serum creatinine, K+ and Na+ levels among the three groups, but serum γ-GT and urinary protein levels were higher in group 2 than in group 1 and 3 (P < 0.05; Table 1). Figure 1a shows the normal appearance of the kidney in the control group; in group 2, the tubular necrosis was grade 2–4 ( Fig. 1b), but in group 3 there was a marked reduction in the extent of tubular damage ( Fig. 1c). The semiquantitive analysis of renal histology is also shown in Table 1.

Table 1.  The effect of gentamicin and melatonin on the biochemical values and renal histology in rats
 Group*
Variable123
  • *

    Group 1, control; group, 2, gentamicin; group 3, gentamicin + melatonin.

Urea (mg/L)140150150
Creatinine, (mg/L)  8  7  9
Na+ (mmol/L) 138137138
K+ (mmol/L)   3.8  3.9  3.7
γ-GT IU/L5441325452
Protein (mg/L)490850540
Grade of necrosis
 0  8  –  6
 1  –  –  –
 2  –  1  1
 3  –  3  1
 4  –  4  –
Mean ( sd)
Catalase (k/s/mg protein) 27.6 (10.96) 21.2 (9.73) 29.7 (12.27)
GSH-Px (IU/mg protein)  8.57 (2.70)  6.91 (2.53)  9.22 (2.60)
MDA (µg/g tissue)  1.06 (0.58)  2.41 (0.33)  1.94 (0.48)
SOD (U/mg protein)160.3 (8.18)120.8 (16.54) 50.8 (17.99)
Figure 1.

a, The normal appearance of the kidney in the control group; b, widespread tubular necrosis in group 1; and c, sparse necrotic tubules in group 3. All haematoxylin and eosin × 50.

Table 1 shows that SOD, catalase and GSH-Px activities were lower, in contrast to MDA which was higher, in the kidney tissues from rats in group 2 than in the control (P < 0.05). Melatonin given concomitantly with gentamicin (group 3) caused significantly higher SOD, catalase and GSH-Px activities, and lower MDA levels, in the kidney tissues than in rats treated with gentamicin alone.

Discussion

The understanding of aminoglycoside nephrotoxicity is clinically important; such nephrotoxicity is typically associated with anoliguric acute renal failure, i.e. azotaemia in the presence of a urine output of 1–2 L/day. A decline in GFR and increase in serum creatinine is usually not apparent until 7–10 days of aminoglycoside treatment [1]. Guidet and Shah [3] reported that in rats receiving gentamicin (100 mg/kg/day) for 5 consecutive days there was no increase in blood urea nitrogen or serum creatinine level, as in the present study.

Much evidence from in vitro and in vivo studies support the concept that reactive oxygen metabolites, including free radical species (e.g. superoxide and hydroxyl radical) and others (e.g. hydrogen peroxide, hypochlorous acid) are important mediators of tissue injury [15–18]. Hydroxyl radical scavengers and/or iron chelators can be protective in several models of tissue injury [18,19]. Walker and Shah [20] showed that gentamicin in vitro enhances the generation of hydrogen peroxide by renal cortical mitochondria, and that iron chelators and hydroxyl-radical scavengers protect against gentamicin-induced acute renal failure. In two separate studies, dimethylthiourea and deferroxamin, an iron chelator, provided functional and histological protection against acute renal failure in rats treated with gentamicin for 8 days [2]. Most of the hydrogen peroxide generated by mitochondria is derived from superoxide anion [21]. Superoxide anion and hydrogen peroxide may interact to generate the hydroxyl radical [22,23].

The present study indicates that gentamicin induces oxidative stress, as shown by significant increases in lipoperoxides in renal tissue and two other changes related to stress, i.e. decreases in GSH-Px and catalase activity. However, there were no significant differences in SOD activity between control and the gentamicin-treated rats, although there was a significant difference in SOD activity between groups 2 and 3. On light microscopy, there was a marked reduction in the extent of tubular necrosis in group 3 ( Fig. 1c).

An association between nephrotoxicity and oxidative stress has been confirmed in many experimental models. The administration of either SOD or α-tocopherol significantly reduced nephrotoxicity symptoms caused by adriamycin [24,25]. The renal depletion of GSH favours both the cephaloridine toxicity and tubular necrosis produced by paracetamol [26,27]. The relationship between gentamicin-oxidative stress and nephropathy was clear in the present study, where an increase in lipoperoxides and tubular necrosis, and a decrease of GSH-Px and catalase levels in the kidney, were positively correlated with biochemical signs characteristic of this nephropathy, i.e. proteinuria and increased serum γ-GT. There was no significant different in BUN, creatinine, K+ and Na+; this contrasts with other reports showing significant changes in these variables [4].

The association of melatonin with gentamicin substantially reduced hyperproteinuria and restored the levels of markers of oxidative stress nephrotoxicity. Melatonin not only prevented lipoperoxidation, GSH-Px depletion and catalase activity, but also decreased lipoperoxide levels to control values, whereas those of catalase and GSH-Px were significantly higher. Because Walker and Shah [2] reported that the protective effect of hydroxyl radical scavengers and iron chelators was unrelated to the uptake of gentamicin by renal cortical tissue, gentamicin levels in the kidney cortices were not measured in the present study. The preventive effect of melatonin on the nephrotoxicity induced by gentamicin possibly depends on its ability to neutralise the increase in free radicals caused by gentamicin. Montilla et al.[28] recently reported that melatonin prevents the nephrotoxicity caused by adriamycin. Their interpretation is supported by the presence of melatonin in the metabolism of unicellular organisms and its synergism with GSH and other antioxidants in the removal of free radicals derived from oxygen. Melatonin is strongly lipophilic and passes easily through biological membranes; because it has no requirement for specific receptors, it is especially useful for scavenging free radicals. Melatonin selectively neutralizes OH radicals, the most reactive and toxic free radicals derived from oxygen [29,30]. Antolin et al.[31] showed that melatonin could prevent cell damage in brain tissue induced by porphyrins, by increasing the mRNA levels of the antioxidant enzymes. Barlow-Walden et al.[32] reported an increase of GSH-Px activity in the brain of rats after acute administration of melatonin, supporting the double mechanism by which this neurohormone may protect cells against oxidative damage. Giusti et al.[8,33] showed that melatonin inhibits kainate (kainic acid)-induced brain injury, proposing that the such neuroprotection might be a results of the antioxidative properties of melatonin. Kotler et al.[34] indicated that melatonin is important in providing indirect protection against free radical injury, by stimulating gene expression for antioxidant enzymes. Princ et al.[35] reported that the administration of pharmacological doses of melatonin reduced and/or prevented α-aminolaevulinic acid-induced lipid peroxidation in both the cerebral cortex and cerebellum, providing further evidence of melatonin’s action as a free radical scavenger.

These results from brain tissue support the role of melatonin in renal tissue; we propose that melatonin acts in the kidney as a potent scavenger of free radicals, so preventing the toxic effect of gentamicin, biochemical changes and tubular necrosis accompanying this nephropathy. These results also reinforce the significant role of free radicals in the pathogenesis of nephropathies induced by gentamicin, particularly as melatonin is probably the most potent and specific antioxidant for hydroxyl radicals. Further studies are needed to confirm the ability of melatonin to increase the mRNA levels for antioxidant enzymes in the kidney.

Authors

E. Özbek, MD, Assistant Professor.

Y. Turkoz.

E. Sahna.

F. Ozugurlu.

B. Mizrak, MD, Associate Professor.

M. Ozbek, MD, Resident.

Ancillary