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Cyclosporin A (CsA) is one of the most efficient immunosuppressive drugs that are widely used in organ transplantation. However, its clinical use is often limited by acute and chronic nephrotoxicity. One of the possible mechanisms of CsA-induced nephrotoxicity is thought to be reactive oxygen species (ROS) formation. The present work was designed to investigate the potential protective effect of wheat germ oil (WGO) as antioxidant on CsAinduced nephrotoxicity in rats. It was found that treatment of animals with CsA alone significantly increased the levels of blood urea nitrogen and serum creatinine. In addition, CsA has also reduced the renal content of reduced glutathione, as well as the enzymatic activities of superoxide dismutase and catalase. Furthermore, these effects were associated with an increase in lipid peroxidation, inducible NO-synthase (iNOS), and NF-B expression. Moreover, histopathological examination showed severe damage of the renal tissue in animals treated with CsA alone. The present work demonstrates that administration of WGO 5 days before and concurrently during CsA administration ameliorated all these negative effects and improved renal function via inhibition of ROS, iNOS and NF-B expression. Most interestingly, the immunosuppressive effect of CsA has not been affected by WGO. INTRODUCTION Organ transplantation is one of the most miraculous achievements in modern medicine due to its contribution in decreasing the mortality rate of patients with organs failure. However, the risk of transplant rejection threatens the success of the transplantation process. Transplant rejection can be prevented by the use of an immunosuppressive agent to suppress the reaction of the immune system to the transplanted tissue. Cyclosporin A (CsA) is one of the calcineurin inhibitors that suppress T-cell activation by inhibiting the cellular phosphatase calcineurin (Schreiber and Crabtree, 1992). Calcineurin inhibitors (CNI) are among the most efficient immunosuppressive drugs and therefore are widely used in transplantation and for the treatment of many inflammatory diseases including psoriasis, and rheumatoid arthritis. However, the clinical use of the calcineurin inhibitor CsA is strongly limited by acute and chronic nephrotoxicity which remain a major clinical problem (Mihatsch et al., 1998; Fellstrom, 2004). The mechanisms of CsA-induced renal injury are not fully elucidated. However, one of the possible mechanisms of CsA-induced nephrotoxicity is thought to be over production of reactive oxygen species (ROS) and a consequent imbalance between oxidants and endogenously produced antioxidants (Ichikawa et al., 1994; Serino et al., 1994; Az. J. Pharm Sci. Vol. 50, September, 2014. 56 Akool et al., 2008). The harmful effects induced by CsA can be antagonized by using a powerful antioxidant agent (Tariq et al., 1999; Uz et al., 2011). Wheat germ oil (WGO) is the richest known natural source of vitamin E (Shurpalekar and Rao, 1977). In addition to the most powerful natural antioxidant vitamin E (Wolf et al., 1998; Ohkatsu et al., 2001), WGO is also rich in unsaturated fatty acids, mainly oleic, and α-linoleic acids that may exert inhibition of oxidative stress (Sjovall et al., 2000; Alessandri et al., 2006). Furthermore, WGO is rich in functional phytochemicals, mainly flavonoids, sterols, octacosanols and glutathione (Zhu et al., 2006). Recently, it has been reported that WGO intake results in a rapid increase in the content of vitamin E in different rat tissues and exerts high protection against oxidative damage (Mehranjani et al., 2007; Field et al., 2008). Thus it was interesting to investigate the potential protective effect of WGO against CsA-induced nephrotoxicity in rats. MATERIALS AND METHODS Animals Twenty-four male Wistar albino rats weighing 200-250 g were housed in a 12 h dark/light cycle animal facility with controlled humidity and constant temperature. The animals were fed a standard diet and water was supplied ad libitum. The animals were kept under observation for one week before the treatments for adaptation. The experimental protocol used in this study was approved by the Institutional Animal Ethics Committee. Drugs and Chemicals CsA was purchased from Sandoz Ltd, Basel, Switzerland. WGO was purchased from SEDICO Pharmaceutical Co., 6 October City, Egypt. Inducible NO-synthase (iNOS), Nuclear factor kappa-B (NF-B), and catalase (CAT) ELISA kits were purchased from EIAab Science Co., Ltd., China. Reduced glutathione (GSH), Superoxide dismutase (SOD), and thiobarbituric acid reactive substances (TBARS) assay kits were purchased from Oxford biomedical research (Oxford MI, USA), Trevigen (USA), and Cell Biolabs (USA) respectively. Interleukin-2 (IL-2) ELISA kit was purchased from Uscn Life Science Inc, Wuhan, China. Blood urea nitrogen (BUN) and serum creatinine assay kits were purchased from Bioassay system (USA). Experimental Design Twenty-four male Wistar albino rats were used in this study. The animals were randomly divided into four groups, 6 animals in each. The first group (Control) was administered the vehicle (castor oil) of CsA intraperitoneal (i.p.) daily for 21 days. The second group received CsA [25 mg/kg/day i.p.] daily for 21 days (Pari and Sivasankari, 2008). The third group was administered WGO [900mg/kg/day by oral gavage] (Abd El-Hameed et al., 2013) 5 days before and concurrently during CsA administration daily for 21 days. The fourth group received WGO daily for 21 days. Twenty-four hours after the last treatment, blood samples were collected for the determination of serum levels of creatinine, BUN as well as IL-2. After terminal bleeding, animals were sacrificed by cervical dislocation. The left kidney was dissected immediately after death, washed with ice cold phosphate buffered saline (PBS) and kept at -20oC till used. The right kidney was fixed in 10% neutral-buffered formal saline for histopathological investigation. Az. J. Pharm Sci. Vol. 50, September, 2014. 57 Assessment of BUN and Serum creatinine BUN and serum creatinine levels were determined by commercial kits according to the manufacturer’s instructions (Bioassay system, USA). Assessment of Serum IL-2 The level of IL-2 in serum was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Uscn Life Science Inc, Wuhan, China). Determination of oxidative damage markers Determination of Lipid Peroxides The level of malondialdehyde (MDA) [the by-product of lipid peroxidation] in renal tissues was measured using a TBARS assay kit according to the manufacturer’s instructions (Cell Biolabs, Inc., USA). Briefly, the unknown MDA containing samples or MDA standards are first reacted with thiobarbituric acid (TBA) at 95°C. After a brief incubation, the samples and standards can be read spectrophotometrically. Determination of iNOS expression The protein level of iNOS in renal tissues was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (EIAab Science Co., Ltd., China). Determination of NF-B expression The level of Total NF-B in renal tissues was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (EIAab Science Co., Ltd., China). Determination of endogenous antioxidants Determination of GSH GSH in renal tissues was determined by colorimetric assay according to the manufacturer’s instructions (Oxford biomedical research, Oxford MI, USA). Briefly, the method is based on the reaction of GSH with Ellman’s reagent (5, 5 ́-dithiobis-2-nitrobenzoic acid (DTNB)) which gives rise to a product that can be quantified spectrophotometrically at 412 nm. Determination of SOD SOD activity in renal tissues was determined by assay kit according to the manufacturer’s instructions (Trevigen, USA). In Trevigen’s superoxide dismutase assay, superoxide radicals generated from the conversion of xanthine to uric acid and hydrogen peroxide by xanthine oxidase (XOD), converts nitroblue tetrazolium (NBT) to NBT-diformazan. NBT-diformazan absorbs light at 550 nm. SODs reduce superoxide radical concentrations and thereby lower the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBTdiformazan is a measure of SOD activity. Determination of CAT activity CAT activity in renal tissues was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (EIAab Science Co., Ltd., China). Az. J. Pharm Sci. Vol. 50, September, 2014. 58 Histopathological Examination Kidney specimens from all animals were dissected immediately after death, and fixed in 10% neutral-buffered formal saline for at least 72 hours. All the specimens were washed in tap water for half an hour and then dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin. Serial sections of 6μm thick were cut and stained with Haematoxylin and eosin (Drury and Walligton, 1980) for histopathological investigation. Statistical Analysis Results are expressed as means ± SD. Statistical analysis was performed using Student’s t test and for multiple comparisons the ANOVA test for significance. P-values below 0.05 were considered as indication for statistically significant differences between conditions compared. RESULTS & DISCUSSION CsA is one of the most important immunosuppressive agents that are commonly used in organ transplantation and in the treatment of autoimmune diseases. However, the clinical use of CsA is strongly limited by acute and chronic nephrotoxicity which remain a major clinical problem. The mechanisms of CsA-induced nephrotoxicity are still not fully elucidated. It has been demonstrated that over production of ROS and consequently oxidative stress plays a major role in CsA-induced renal damage (Ichikawa et al., 1994; Serino et al., 1994; Akool et al., 2008). Previously, it has been reported that antioxidants have the ability to attenuate nephroto

WHEAT GERM OIL ATTENUATES CYCLOSPORIN A-INDUCED RENAL INJURY IN RATS VIA INHIBITION OF ROS, INOS, AND NF-B EXPRESSION