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Role of Ketotifen in Methotrexate-Induced Nephrotoxicity in Rats

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Role of Ketotifen in Methotrexate-Induced Nephrotoxicity in Rats Egyptian Journal of Basic and Clinical Pharmacology December 2017, Vol. 7, No. 2: 70-80 http://www.ejbcp.eg.net/ Original Article Role of Ketotifen in Methotrexate-induced Nephrotoxicity in Rats Marwa M. M. Refaie1*, Salwa A. Ibrahim1, Shaimaa A. Sadek2, and Aly M. Abdelrahman1 1Department of Pharmacology, Faculty of Medicine, Minia University, Minia, Egypt 2Department of pathology, Faculty of Medicine, Minia University, Minia, Egypt. A B S T R A C T Copyright © 2017 Methotrexate (MTX) is a chemotherapeutic agent that interrupts folate metabolism by inhibition of Marwa M. M. Refaie dihydrofolate reductase; a required precursor for co-factors share in macromolecule biosynthesis. et al. This is an open MTX is complicated by marked nephrotoxicity especially in high doses so that in current study the access article possible protective effect of an anti-inflammatory drug (ketotifen) on MTX-induced nephrotoxicity distributed under the and the mechanisms involved were investigated. Two doses of ketotifen (1 and 10mg/kg) were given Creative Commons orally to rats for 14 days, in the presence or absence of nephrotoxicity induced by a single intraperitoneal (ip) injection of MTX (20 mg/kg) at day 11th of the experiment. Serum urea, creatinine, Attribution License, renal reduced glutathione (GSH), malondialdehyde (MDA), total nitrites (NO ), catalase, superoxide which permits x dismutase (SOD) activity, caspase-3, tumor necrosis factor alpha (TNFα), nuclear factor kappa B unrestricted use, (NFκB) immunoexpressions, and renal histopathology were measured. Results showed that ketotifen distribution, and succeeded in reversing MTX-induced nephrotoxicity to levels insignificant from control. reproduction in any medium, provided the Key Words: Methotrexate; Ketotifen; Nephrotoxicity; Antioxidant; Anti-inflammatory. original work is Corresponding Author: Marwa M.M. Refaie Email: [email protected] 1. INTRODUCTION Methotrexate (MTX) is one of the most successful enhanced by MTX induced free radicals (Khurana et al., drugs in cancer chemotherapy. It has been used in 2011; Theoharides et al., 2012). Several antioxidants treatment of various types of cancer, ectopic pregnancy were tried to protect from MTX induced nephrotoxicity and autoimmune disorders such as rheumatoid arthritis. as curcumin (Morsy et al., 2013) and caffeic acid It is an antiproliferative chemotherapeutic that disturbs phenethyl ester (Akyol et al., 2014). Ketotifen has an folate metabolism by inhibition of dihydrofolate anti-inflammatory, histamine receptor antagonist effects, reductase a required precursor for co-factors involved in prevents calcium entry, and inhibits the release of macromolecule biosynthesis (AL-Nailey, 2010; Stika., leukotrienes (El-Haggar et al., 2015). Ketotifen has anti- 2012). inflammatory and anti-oxidant properties so it was reported that ketotifen had protective effect in different The only effective medical intervention for ectopic models of nephrotoxicity (Tong et al., 2016; Reena, pregnancy was surgery until medical management was 2016). introduced in the 1980s. MTX has been used for medical therapy for ectopic pregnancies. Its mechanism of action, In present study the possible protective effect of competitive inhibition of folate-dependent steps in another anti-oxidant and anti-inflammatory (ketotifen) nucleic acid synthesis, effectively kills the rapidly on MTX nephrotoxicity were evaluated. dividing ectopic trophoblast. MTX was used in ectopic 2. MATERIALS AND METHODS pregnancies in the 1960s to aide surgical removal of the placenta from its abdominal implantation sites. In the 2.1. Chemicals 1980s, MTX was used in multidose regimens with intervening folinic acid rescue (Stika, 2012; Skubisz and Ketotifen fumarate powder was from APG Co.USA. Tong, 2012). MTX vial was from Menapharm co., Egypt. The polyclonal rabbit/antirat caspase-3, tumor necrosis factor The efficacy of MTX is limited by severe side effects alpha (TNFα) and nuclear factor kappa B (NFκB) anti- and toxic conditions; nephrotoxicity is one of the major body were from Lab Vision, USA. In addition, side effects of MTX and can cause acute renal failure in biotinylated goat antirabbit secondary antibody was from high doses. There is a great interest in expanding the (Transduction Laboratories, USA), urea, reduced clinical usefulness of MTX by developing new agents in glutathione (GSH), superoxide dismutase (SOD) and order to reduce its nephrotoxicity (Morsy et al., 2013). catalase kits were from Biodiagnostic, Egypt and There is a major role of inflammation and mast cells creatinine from Humen, Germany. stimulation in MTX induced nephropathy which was 70 Marwa M.M. Refaie et al. 2.2. Animals and experimental design kit was used for GSH. Results were expressed as mmol/g tissue (Beutler et al., 1963). Adult male Wistar albino rats weighing about 450– 550 g were purchased from the Animal Research Centre, 2.4.3. Assessment of renal catalase levels. Giza, Egypt. Rats were kept in cages in standard housing Assessment of renal homogenate catalase enzyme conditions and were left to acclimatize for one week. activity was determined from the rate of decomposition Animals were supplied with laboratory chow and tap of H O as described by colorimetric kit. The results were water. This study was conducted in accordance with 2 2 expressed as unit/g tissue (Aebi, 1984). ethical standards and approved by committee of faculty of medicine, Minia University, Egypt. 2.4.4. Assessment of renal SOD levels. Ketotifen powder was dissolved in water. The assessment of SOD levels was based on the ability of the enzyme to inhibit the Rats were randomly divided into 6 groups (n = 6 phenazinemethosulphate-mediated reduction of each); group Ӏ: received vehicle distilled water for 14 nitrobluetetrazolium dye and results were expressed as days and ip saline at day 11; group II was treated with unit/g tissue (Nishikimi et al., 1972). low dose of ketotifen (1mg/kg/d orally) for 14 days and ip saline at day 11; group III was treated with high dose 2.4.5. Assessment of renal MDA Levels. of ketotifen (10 mg/kg/d orally) and ip saline at day 11; group IV was treated with vehicle for 14 days and MTX Kidney lipid peroxidation was determined as (20 mg/kg) at day 11; group V was treated with a low thiobarbituric acid reacting substance and is expressed as equivalents of MDA, using 1, 1, 3, 3- dose of ketotifen (1 mg/kg/d orally) for 14 days and ip injection of MTX (20 mg/kg ) at day 11; group VI was tetramethoxypropane as standard. Results were treated with a high dose of ketotifen (10 mg/kg/d orally) expressed as nmol/g tissue (Mihara and Uchiyama, for 14 days plus ip injection of MTX (20mg/kg ) at day 1983). 11. The dose of MTX and ketotifen were based on 2.4.6. Assessment of NOx Levels. previous studies (Asvadi et al., 2011; Fitzgerald et al., 2013; El-Haggar et al., 2015). The assessment of renal oxidation end products of NOx, nitrite and nitrate served as an index of NOx 2.3. Collection of the samples and storage. production. This method was depended on Griess reaction. Results were expressed as nmol/g tissue (Sogut After 3 days of MTX injection, each rat was weighed then sacrificed. Venous blood were collected from the et al., 2003). jugular vein and centrifuged at 5000 rpm for 15min 2.4.7. Histopathological and immunohistochemical (JanetzkiT30 centrifuge, Germany). measurements. Both kidneys were removed and weighed. One For histopathological assessment; renal tissue was kidney was sectioned longitudinally then fixed in 10% fixed in 10% formalin, embedded in paraffin, sectioned formalin and embedded in paraffin for histopathological by a microtome at 5μm thickness and stained with and immunohistochemical examinations. The remained hematoxylin and eosin. Three slides from each animal part of the kidneys was snap frozen in liquid nitrogen and group; each one with three sections was subjected to a kept at−80◦C. Renal tissue homogenate was prepared for scoring system used in assessing the histopathological biochemical analysis, kidneys were homogenized (Glas- changes using light microscopy (Olympus CX41). Col homogenizer, USA) and a 20% w/v homogenate was prepared in ice-cold phosphate buffer (0.01M, pH 7.4). Caspase-3, TNFα and NFκB immunolabeled cells The kidney homogenate was centrifuged at 3000 rpm for were counted. 3 sections were examined and cells were 20 min and the supernatant was kept at−80◦C till used. counted in 3 adjacent non overlapping fields. Immunohistochemical staining was performed for 2.4. Measurements. caspase-3, TNFα and NFκB using polyclonal rabbit/antirat antibody according to previously published 2.4.1. Assessment of serum urea and creatinine protocol (Shirai et al., 1985; Côté et al., 1993) Urea (Vassault et al., 1999) and serum creatinine respectively. Sections were deparaffinized, hydrated (Bartels et al., 1971) were determined using colorimetric then washed in 0.1M phosphate buffer. These sections diagnostic kits according to the manufacturer’s were treated with 0.01% trypsin for 10 min at 37◦C then instructions for detection of renal function and washed with phosphate buffer for 5 min. Endogenous nephrotoxicity. peroxidases were quenched by treatment with 0.5% H2O2 in methanol and nonspecific binding was inhibited by 2.4. 2. Assessment of renal GSH levels. normal goat serum diluted 1:50 in 0.1M phosphate Assessment of renal tissue GSH, SOD and catalase buffer. Tissue was incubated in the primary antibody enzyme levels were detected for evaluation of renal overnight at 4◦C. Afterwards, tissue was washed and antioxidant defense mechanisms. A spectrophotometric incubated in biotinylated goat antirabbit secondary 71 Role of Ketotifen in Methotrexate-induced Nephrotoxicity in Rats antibody (1:2000) for 30 min. Following 30 min ketotifen plus MTX significantly decreased MDA and incubation in vectastain ABC reagent, the substrate NOX when compared to MTX treated rats. diaminobenzidine was added for 6 min which gives 3.3. Effect of ketotifen on renal GSH, SOD and brown color at the immunoreactive sites. catalase levels in MTX- treated rats.
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