Resveratrol improves ifosfamide-induced Fanconi syndrome in rats

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Abstract

Regarding the mechanisms of ifosfamide (IFO)-induced urinary toxicity, several hypotheses have been put forward, among which oxidative stress and depletion of glutathione are suggested. This investigation elucidates the role of free radicals in IFO-induced toxicity and the protection by resveratrol, a natural phytoalexin. Wistar albino rats were injected intraperioneally with saline (0.9% NaCl; control), saline + resveratrol (RVT; 10 mg/kg/day), ifosfamide (IFO; 50 mg/kg/day) or IFO + RVT for 5 days. Urine was collected for 24 h during the 5th day, and at the 120th h after the first injections, animals were killed by decapitation and trunk blood was collected. Lactate dehydrogenase (LDH) activity, total antioxidant capacity (AOC) and pro-inflammatory cytokines TNF-α, IL-β and IL-6 were assayed in plasma samples. Kidney and bladder tissues were obtained for biochemical and histological analysis. Formation of reactive oxygen species in the tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lucigenin probes. The results demonstrated that IFO induced a Fanconi syndrome characterized by increased urinary sodium, phosphate, glucose and protein, along with increased serum creatinine and urea levels. On the other hand, RVT markedly ameliorated the severity of renal dysfunction induced by IFO. Furthermore IFO caused a significant decrease in plasma AOC, which was accompanied with significant increases in the levels of the pro-inflammatory mediators and LDH activity, while RVT treatment reversed all these biochemical indices. In the saline-treated IFO group, glutathione levels were decreased significantly, while the malondialdehyde levels, myeloperoxidase activity and collagen content were increased in both tissues, which were in parallel with the increases in CL values. In the RVT-treated IFO group, all of these oxidant responses were prevented significantly. Our results suggest that IFO causes oxidative damage in the renal and bladder tissues and resveratrol, via its antioxidant effects, protects these tissues. Therefore, its therapeutic role in preventing the development of chemotherapeutic drug-induced major toxicity in the urinary system requires further elucidation.

Introduction

Ifosfamide, an alkylating antineoplastic drug and a structural analogue of cyclophosphamide, is used to treat a wide range of tumors in both adults and children (Zalupski and Baker, 1988, Schoenike and Dana, 1990, De Kraker, 1991). It is an active drug used in the treatment of germ-cell tumors, rhabdomyosarcoma, some other soft tissue sarcomas, Wilms' tumor, bone sarcomas, lymphomas and neuroblastoma (Straka et al., 2003). However, the clinical efficacy of ifosfamide is greatly limited because of its severe cytotoxic side effects, including hemorrhagic cystitis and nephrotoxicity (Skinner et al., 1993), which are characterized by morphological damage, aminoaciduria, glucosuria, phosphaturia, increased fractional sodium excretion and urine flow (Mimic-Oka and Simic, 1997). Understanding the cytotoxic effects of ifosfamide was further enhanced by its use in the development of an experimental model for the study of Fanconi syndrome in rats (Berliner et al., 1950), which also provided an opportunity to analyze novel protective agents against this toxicity.

Ifosfamide is a prodrug that must first be biotransformed by the cytochrome P450 system before it can exert its therapeutic or toxic effects (Wagner, 1994, Wang and Chan, 1995, Springate et al., 1999). Detailed studies have demonstrated that its hepatic metabolite chloroacetaldehyde (CAA) causes kidney damage, which is associated with a dramatic decrease in the intracellular ATP and a large increase in lactate deydrogenase (LDH) release (Dubourg et al., 2002). These effects are closely related with the disturbance of cellular energy metabolism and depletion of cellular thiol compounds, i.e. glutathione and coenzyme A derivatives (Sood and O'Brien, 1993, Springate, 1997) that ultimately result in cellular oxidative damage. Since GSH is an important molecule in the cellular defense against chemically reactive toxic compounds or oxidative stress, a significant reduction in GSH levels as a consequence of IFO metabolites diminishes the effectiveness of antioxidant enzyme defense systems. Considering the possible role of GSH depletion in the pathogenesis of IFO-induced toxicity, interest has focused on the antioxidant compounds that are capable of stimulating GSH synthesis.

Resveratrol (3,5,4′-trans-trihydroxystilbene), a natural phytoalexin present in grapes, peanuts, mulberries and red wine, when added to cultured cardiomyocytes in low micromolar concentrations, was shown to induce a number of endogenous antioxidants and phase 2 enzymes, including superoxide dismutase (SOD), catalase, glutathione (GSH), glutathione reductase (GR), glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1) (Cao and Li, 2004). This chemically mediated upregulation of cellular defenses is accompanied by a markedly increased resistance to cardiac cell injury elicited by reactive oxygen species and doxorubicin (Gusman et al., 2001). Resveratrol also maintains antioxidant defenses and reduces renal oxidative damage in a rat model of cholestasis, as assessed by tissue levels of malondialdehyde, reduced glutathione and nitric oxide (Ara et al., 2005). On the other hand, when resveratrol was administered to the rats with ovarian ischemia–reperfusion injury, lipid peroxidation and tissue damage were reduced (Hascalik et al., 2004). Resveratrol has additional pharmacological effects including anti-inflammatory properties, modulation of lipid metabolism and prevention of cancer (Fremont, 2000, Gusman et al., 2001, Wallerath et al., 2002, Dong, 2003). The anti-inflammatory effect of resveratrol is related with its inhibitory action on oxidation, leukocyte priming and expression of inflammatory mediators. An immunohistochemical study has shown that resveratrol attenuated the expression of CD86 in the glomerular endothelium and peritubular vessels in rats with ischemia–reperfusion injury (Saito et al., 2005). In our previous studies, we have also demonstrated that resveratrol improves renal damage in ischemia–reperfusion and sepsis-induced oxidative renal injury (Şener et al., 2006a, Kolgazi et al., 2006).

Therefore, in the light of the above mentioned observations, the present study was aimed to investigate the possible beneficial effects of resveratrol on ifosfamide-induced kidney and urinary bladder damage.

Section snippets

Experimental design

All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee. Wistar albino rats (200–250 g) of both sexes were kept at a constant temperature (22 ± 1 °C) with 12 h light and dark cycles and fed a standard rat chow.

The rats were divided into four groups, each consisting of 8 animals. Two groups of rats were administered first intraperitoneally (ip) with ifosfamide (50 mg/kg/day); immediately after either saline (1 ml) or resveratrol (RVT;

Results

In the saline-treated rats, IFO caused significant elevations in serum urea and creatinine levels (p < 0.01–0.001) and significant decreases in serum potassium, phosphate, glucose and albumin levels (p < 0.05–0.001) when compared to control values (Table 1), while resveratrol treatment significantly reduced the elevated serum urea and creatinine levels (p < 0.05–p < 0.001) and prevented the decreases in serum phosphate, glucose and albumin concentrations seen after IFO treatment (p < 0.05). Moreover,

Discussion

In the present study, altered kidney functions, as evidenced by elevations in serum urea and creatinine levels and marked urinary losses of sodium, potassium, phosphate, protein and glucose, indicate the development of Fanconi syndrome. Increases in lipid peroxidation, collagen content, luminol and lucigenin CL levels and myeloperoxidase activity due to toxic effects of IFO were accompanied by significant reductions in glutathione levels of the renal and bladder tissues, implicating the

Acknowledgment

The authors are grateful to Ozgur Goknel, the Medical Director, in MICROGEN Pharmeceutical, for supplying resveratrol.

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