MATERIALS AND METHODS

A total of 18 healthy male New Zealand white rabbits, weighing 2.5-3.0 kg, were used throughout this study. The animals purchased from the Veterinary Control and Research Institute, Elazig, Turkey, were kept under standard laboratory conditions (12 h light/12 h dark and 24±3°C) and fed with standard commercial rabbit chow (pellet form, in the sack, Elazig Food Company). Food and water were provided ad libitum. The experimental protocol was approved by the Veterinary Control and Research Institute Ethics Committee.

The rabbits were randomly divided into 3 equal groups; the first group served as control and received a single intraperitoneal dose of 0.9% saline. Rabbits from the second group were intraperitoneally injected with a single 5 mg kg^-1 body weight cisplatin dose. The CP dosage was selected on the basis of its effectiveness in inducing nephrotoxicity (Ozer et al., 2011). The third group of rabbits was treated with GSE (proanthocyanidins dissolved in water) by oral gavage at the dose of 250 mg kg^-1 body weight (Yildirim et al., 2011) for 6 consecutive days before and 6 consecutive days after the CP injection.

After CP GSE treatments, blood samples were collected from all animals by puncture of the jugular vein into sterile microtubes. After clotting at room temperature for 1 h, samples were centrifuged at 3000 g for 10 min at room temperature and sera were carefully harvested and stored at -20°C until analysed. Then, all rabbits were decapitated under slight ether anaesthesia and kidney samples were immediately collected for biochemical and histopathological examinations. The kidney tissues were homogenized in glass-glass homogenizer with a buffer containing 1.5% potassium chloride to obtain 1:10 (w/v) whole homogenates.

Malondialdehide (MDA) levels: The concentrations of malondialdehyde, reflecting the lipid peroxidation intensity were directly measured in the homogenates using the thiobarbituric-acid reaction described by Placer et al. (1966) and values were expressed as nmol g^-1 tissue.

Reduced GSH levels: The glutathione contents in kidney were measured at 412 nm using the method of Sedlak and Lindsay (1968) and expressed as nmol g^-1 tissue for kidney tissue.

Catalase activity: The kidney catalase activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm, according to the method of Aebi (1983) and was expressed as katal g^-1 protein.

Glutathione peroxidase activity: The glutathione peroxidase activity was determined according to the method of Lawrence and Burk (1976) and expressed as U g^-1 protein for kidney tissue.

Protein determination: The protein concentration was also measured in the supernatants by the method of Lowry et al. (1951).

Kidney function tests: Serum urea, creatinine, Blood Urea Nitrogen (BUN) levels were measured using auto analyzer (Olympus AU 600, Japan).

Histopathology: At the end of the experiment, necropsy of the rabbits was performed and kidney tissue samples were fixed in 10% buffered neutral formalin. Paraffin-embedded blocks were routinely processed and 5 μm thick sections were stained with haematoxylin-eosin and examined under an optical microscope. (Olympus BX51) and 10 microscopic fields were examined in 20X magnification. Every fields were evaluated as severe (+++), moderate (++), mild (+) and none (-).

Statistical analysis: Data are presented as Mean±Standard error of means (SEM). One-way analysis of variance and post hoc Duncan's test were used to determine the differences between groups in terms of all studied parameters using the SPSS/PC computer program (version 12.0; SPSS, Chicago, IL, USA). The Kruskal Wallis test was used for the comparison between the control and experimental groups for histopathological examination. Differences were considered as significant when p-value was less than 0.05.

RESULTS

As reported in Table 1, the serum urea, BUN, creatinine were dramatically increased in rabbits from the CP group compared to the not treated controls (p<0.001). by contrast, when animals received the oral GSE treatment, the enzyme activities remained similar to the control values.

The antioxidant/oxidant balance in kidney from rabbits treated with CP alone or coupled to GSE was summarized in Table 2.

It was observed that CP increase MDA levels (p<0.001), the most important indicator of oxidative damage and decreased the activity of CAT (p<0.05). Administration of CP did not caused statically a change in GSH levels and GSH-P_X activity (p>0.05). Furthermore, it was found that the mean MDA content in kidney from GSE treated rabbits (CP/GSE group) was dramatically lowered compared to the animals treated with CP alone although it was remained significantly higher than in the healthy controls (p<0.001).

The histopathological changes observed in the kidney are summarized in Table 3. Whereas no lesion was found in the healthy controls (Fig. 1c).

Table 1:	Effects of cisplatin (5 mg kg-1, intraperitoneally) and grape seed extract (GSE, per os, 250 mg kg-1 for 6 days before and 6 days after)
Values with different letters with in column are significant different at p<0.01

Table 2:	Effects of cisplatin (5 mg kg-1, intraperitoneally) and grape seed extract (GSE, per os, 250 mg kg-1 for 6 days before and 6 days after cisplatin injection) treatments on antioxidant/oxidant equilibrium in kidney homogenates in rabbits
Results are expressed as Mean±standard error of mean (SEM). Different superscripts in the same column indicate significant difference (p<0.05 or more) between groups

The most significant changes in CP group are severe acute tubular necrosis in proximal tubular epithelial cells, desquamation, cloudy swelling, tubular dilation, tubular epithelium karyomegali with formation of hyaline cylinders in tubular lumina (Fig. 1a). In addition to this view, mononuclear cell infiltration of the intertubular spaces, thickening of the basal membrane of glomerulus and postnecrotic fibrosis are the other changes.

Fig. 1(a-c):

Kidney histopathology in rabbits treated with cisplatin (5 mg kg-1, intraperitoneally) and with cisplatin and grape seed extract (per os, 250 mg kg-1 for 6 days before and 6 days after cisplatin injection). Picture a- Severe acute tubular necrosis with hyaline cylindrical formations (arrow heads). Cisplatin group. X 20 Picture b- Mild interstitial nephritis. Cisplatin+GSE group X 20 Picture c- Normal histological appearance of kidney (control group)

Table 3:	Effects of cisplatin (5 mg kg-1, intraperitoneally) and grape seed extract (GSE, per os, 250 mg kg-1 for 6 days before and 6 days after cisplatin injection) treatments on kidney histology in rabbits
Different superscripts in the same column indicate significant difference (p<0.05 or more) between groups. SH: Standard error

In the CP+GSE treatment group degenerative changes were observed instead of severe necrotic changes in the proximal tubules (Fig. 1b). Tubular dilation was not observed in this group but intraluminal hyaline cylinders was found milder compared to the CP group.

DISCUSSION

Cisplatin (CP) is a potent chemotherapeutic agent that has gained widespread use against various malignant tumors in different experimental animals and in a variety of human malignancies (Kociba et al., 1970; Prasad and Giri, 1994). However, high-dose therapy with CP is limited by its cumulative nephrotoxicity and neurotoxicity (Dwyer et al., 1999). CP is toxic to the renal proximal tubules (Yao et al., 2007). Recently, it has been postulated that oxidative stress and reactive oxygen species (ROS) are involved in the pathogenesis of CP-induced nephrotoxicity. It has been shown that superoxide anion (O₂), H₂O₂ and hydroxyl radical (OH^-) are involved in CP-induced nephrotoxicity (Yao et al., 2007).

The ROS generated during normal cellular processes are immediately detoxified by endogenous antioxidants like GSH, CAT and so on but excessive ROS accumulation by CP causes an antioxidant status imbalance, leads to LPO and GSH depletion (Kim et al., 2006). Oxidative stress resulting from cisplatin is defined by increased production of oxidative stress markers and decreased concentrations of antioxidants (Davis et al., 2001; Ajith et al., 2007). As a marker of oxidative stress, we evaluated CAT and GSH-P_X activity with GSH and MDA levels in kidneys. Our results in rabbit renal tissue demonstrated GSH depletion, enhanced GSH-Px and increased LPO after CP injection, which is in accordance with earlier findings (Sugihara et al., 1987). GSH protects the cell against the toxic effects of hydroxyl radicals and singlet oxygen. The decrease in GSH levels is associated with lower activity of glutathione related enzymes. Hence, more tissue damage due to the poor control on free radicals (Kandeel et al., 2011). The decrease in the enzymatic antioxidant, CAT, activities in the kidney tissue may be responsible for increased levels of MDA (Ige et al., 2011). The histological evaluation of the kidney preparations in pretreatment group also revealed a decrease CP-induced tubular necrosis. CP-induced renal damage is associated with increased renal vascular resistance and histologic damage to proximal tubular cells. In concordance with the results of earlier studies, our study on histological examination revealed major disruption in architecture of straight portion of proximal tubules due to CP toxicity. The toxicity was characterized by widespread degeneration of tubular architecture, tubular congestion, swelling and necrosis, luminal congestion and an infiltration of polymorphonuclear neutrophils. CP intoxication showed severe atrophy of glomerulus, which was apparent due to the reduction in its size. Marked dilation of proximal convoluted tubules with slogging of almost entire epithelium due to desquamation of tubular epithelium was evident. Cellular debris in the tubular lumen and increased tissue in the interstitium is also an indication of CP-induced renal necrosis. The changes obtained in the present study run parallel with the earlier report documented (Ravindra et al., 2010). Histopathological inspection robustly supported the results of biochemical assays, henceforth confirming the GSE as ROS scavenger.

A second important parameter to take into account in the evaluation of the kidney damage produced by CP treatments is related to serum creatinine, urea and BUN levels. Creatinine is a metabolite of protein and is excreted in the urine via glomerular filtration and elevation of its levels in the blood is thus an indication of impaired kidney function (Bashandy and Al-Wasel, 2011). The CP injections reduced dramatically the renal function and produced an important increase in the level of creatinine, urea and BUN levels. However, the pretreatment with GSE reduces the observed damage in both cases and to an almost normal (control) level for urea, BUN and creatinine.

Free radical scavengers and antioxidants can ameliorate cisplatin-induced nephrotoxicity (Koyner et al., 2008). During the recent past, a lot of focus has been given to dietary components. Majority of them being without side effects, thus taking the leading role in the fight against oxidative stress caused by different xenobiotics in humans. One such class of dietary components are the polyphenols found in various plant-derived foods. Polyphenols have been recently recognized as functionally active molecules, possessing antioxidant, anticancer, antimutagenic properties, as well as exerting protective effects against several other diseases (Nakamura et al., 2001). Polyphenolic compounds are present in grape, which are powerful antioxidant properties, i.e. free radical scavenging activity (Monagas et al., 2006).

In the present study, GSE treatment efficiently attenuated acute nephrotoxicity induced by a single injection of CP in rabbits. This was evidenced by significant improvement in the disturbed biochemical parameters (elevated BUN and serum creatinine levels and MDA level, with reduced kidney GSH level, increased CP and GSH-Px activities) and reduction of necrotic damage assessed by renal histopathological examination and scoring. The beneficial effects of GSE are well documented in earlier studies. Safa et al. (2010) demonstrated that pretreatment with red GSE protects against gentamicin-induced acute kidney injury as evident on tissue histology. Chis et al. (2009) found that long-term daily administration of GSE offers enhanced antioxidant potential and protection against tissue LPO and protein oxidation. Yildirim et al. (2011) indicate that the antioxidant GSE might have a protective effect against cisplatin-induced testicular damage and oxidative stress in rabbit. The mechanisms by which GSE ameliorate toxicity remains to be elucidated. We supposed they may inhibit LPO by scavenging free radicals and increasing intracellular concentration of glutathione.

In conclusion, this study is the first to report the effects of supplemental GSE in the rabbit kidney after cisplatin exposure. The present finding suggest that GSE protects against cisplatin nephrotoxicity and may be considered as a potentially useful candidate in the combination chemotherapy with cisplatin.