http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2014; 36(2): 130–137 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2014.884135
RESEARCH ARTICLE
Mohamed A. Ibrahim, Azza A. K. El-Sheikh, Hanaa M. Khalaf, and Aly M. Abdelrahman Department of Pharmacology, Faculty of Medicine, Minia University, Minia, Egypt
Abstract
Keywords
Context: The anticancer drug methotrexate (MTX) may cause multi-organ toxicities, including nephrotoxicity. Objective: To investigate effects of peroxisome proliferator activator receptor (PPAR)-a and -g agonists; fenofibrate (FEN) and pioglitazone (PIO), in MTX-induced nephrotoxicity in rats. Methods: Rats were given FEN or PIO (150 or 5 mg/kg/day, respectively) orally for 15 days. MTX was injected as a single dose of 20 mg/kg, i.p. at day 11 of experiment, with or without either PPAR agonists. Results: MTX induced renal toxicity, assessed by increase in serum urea and creatinine as well as histopathological alterations. MTX caused renal oxidative/nitrosative stress, indicated by decrease in GSH and catalase with increase in malondialdehyde and nitric oxide (NOx) levels. In addition, MTX increased renal level of the pro-inflammatory cytokine; tumor necrosis factor (TNF)-a and up-regulated the expression of both the inflammatory and apoptotic markers; NF-kB and caspase 3. Pre-administration of FEN or PIO to MTX-treated rats improved renal function and reversed oxidative/nitrosative parameters. Interestingly, pre-administration of PIO, but not FEN, decreased renal TNF-a level and NF-kB expression compared to MTX alone. Furthermore, PIO had more significant effect than FEN on reversing MTX-induced renal caspase 3 expression. Discussion: Both FEN and PIO conferred protection against MTX-induced nephrotoxicity through comparable amelioration of oxidative/nitrosative stress. FEN lacked any effect on TNF-a/NF-kB, which was reflected on its less improvement on renal histopathology and apoptosis. Conclusion: At indicated dosage, PPAR-g ligand; PIO shows better improvement of MTX-induced nephrotoxicity compared to PPAR-a ligand; FEN due to differential effect on TNF-a/NF-kB inflammatory pathway.
Fnofibrate, nitric oxide, oxidative stress, pioglitazone, TNF-a
Introduction Methotrexate (MTX), a folic acid antagonist, is widely used as anticancer drug in treatment of leukemia and other malignancies as well as used as in the treatment of autoimmune diseases such as rheumatoid arthritis and psoriasis1. Unfortunately, its use is associated with various forms of toxicities, including nephrotoxicity. The exact mechanisms of MTX-induced renal toxicity have not yet been elucidated. However, several hypotheses have been put forward, including direct toxic effect of MTX on the renal tubules2 and increased oxidative stress3. Although several agents with antioxidant properties have been examined as a potential therapy against MTX related toxicity3,4, the results of previous studies did not achieve satisfying nephro-protection. Consequently, the door is still open for further studies, Address for correspondence: Azza A.K. El-Sheikh, PhD, Assistant Professor of Pharmacology, Department of Pharmacology, Faculty of Medicine, Minia University, 61511 Minia, Egypt. Tel: (+2) 01006756740. Fax: (+2) 086 234 2813. E-mail: azza.elsheikh@ mu.edu.eg
History Received 27 September 2013 Revised 23 November 2013 Accepted 13 January 2014 Published online 13 February 2014
exploring a potential adjuvant therapy against renal MTXinduced toxicity. Peroxisome proliferator activator receptors (PPARs) are members of steroid nuclear receptor family expressed in many tissues of the body including the kidney5. There are three types of PPAR receptors; PPAR-a, PPAR-b, and PPAR-g6,7. Among PPAR agonists are hypolipidaemic drugs as fibrates (PPAR-a agonists) and insulin sensitizers agents such as thiazolidinediones or glitazones (PPAR-g agonists); whereas the former activate lipid metabolism, the latter produce several biological effects on adipogenesis, carbohydrate metabolism, inflammatory processes, and cellular proliferation5. The PPAR-a agonists, fibrates, have been shown to have beneficial effects in drug-induced nephropathy in animals8. Recent systematic review and metanalysis data suggested that fibrates have a role in reducing cardiovascular risk in people with mild-to-moderate chronic kidney disease9. Similarly, PPAR-g agonists have shown to exert renal protection via haemodynamic, vascular, anti-inflammatory, anti-oxidant, and metabolic effects10,11. These renal protective effects of
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Protective effect of peroxisome proliferator activator receptor (PPAR)-a and -g ligands against methotrexate-induced nephrotoxicity
DOI: 10.3109/08923973.2014.884135
PPAR agonists suggest that they may provide a novel intervention strategy to prevent MTX-induced renal damage. The aim of the current work was to investigate the effects of administration PPAR-a and -g agonists; fenofibrate (FEN) and pioglitazone (PIO), respectively, on MTX-induced nephrotoxicity in rats and to explore the mechanisms involved.
Materials and methods
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Chemicals Methotrexate (25 mg/ml ampoules) was obtained from Ebewe Co. (Unterach, Austria). Fenofibrate and pioglitazone powders were generous gifts from SigmaPharm Co. and Medical Union Pharmaceuticals Co.; MUP (Cairo, Egypt), respectively. Kits for examining serum urea and creatinine, reduced glutathione (GSH) and catalase were purchased from Biodiagnostic (Egypt). Nuclear factor-kB (NF-kB/p65) and caspase 3 rabbit polyclonal antibodies were purchased from Thermo Fisher Scientific Inc/Lab Vision (Fremont, CA). Tumor necrosis factor (TNF)-a enzyme-linked immuno sorbent assay (ELISA) kit was purchased from WKEA-Med supplies Corp. (Changchun, Jilin, China). Experimental animal design Adult male albino rats weighing 170–200 g were obtained from the National Research Centre (Giza, Egypt). They were housed four rats/cage and fed a standard diet of commercial rat chow (El-Nasr Company, Cairo, Egypt). Tap water was supplied ad libitum. Animals were left to acclimatize for one week before the start of the experiments. All experimental procedures were approved by the Faculty of Medicine board Committee of Minia University. Rats were weighed and divided into six groups (n ¼ 8). FEN and PIO groups were treated with 150 and 5 mg/kg/day, respectively, orally for 15 days12,13. MTX group was injected with a single dose of 20 mg/kg, i.p. at day 11 of the experiment and received no PPAR agonist to serve as positive control14. Combined MTX/FEN and MTX/PIO groups received MTX with the respective PPAR agonist as indicated. FEN and PIO were freshly prepared daily and administered orally by stomach tube, suspended in 0.5% carboxymethylcellulose. Control group received the same volume of vehicles of drugs used by the same route of administration and served as the untreated negative control group. Sample preparation and histological examination At the end of the experimental period, rats were anaesthetized with ether, weighed and sacrificed. Blood samples were collected from neck vessels by decapitation and left for few minutes to clot and then centrifuged for 10 min at 3000g for separation of serum. Kidneys were rapidly excised and weighed. Renal weight to total body weight ratio (renal index) was calculated according to the formula (renal weight/body weight) 100. The right kidney was used to prepare tissue homogenate. Homogenization was done by Glas-Col homogenizer and a 20% w/v homogenate was prepared in ice-cold phosphate buffer (0.01 M, pH 7.4). The homogenate was centrifuged at 1100g for 20 min. The supernatant was then
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divided over several containers to avoid sample thawing and refreezing, and was kept at 80 C till used. For histological examination, the left kidney from each rat was put in 10% formalin, embedded in paraffin, sectioned by a microtome at 5 mm thickness and stained with hematoxylin and eosin for routine histopathological assessment using light microscopy (Olympus CX41, Tokyo, Japan). Histological scoring was performed using five fields from each rat, regarding several criteria, as tubular degeneration and dilatation, as well as dilatation of Bowman’s space and presence of intra-tubular protein casts. All slides were randomly numbered and blind assessment of each slide was performed three times, each in a different setting scoring different microscopic fields. The score given was either absent, mild, moderate, or severe changes. Immunohistochemical examination of NF-kB and caspase 3 expression For immunohistochemical staining, sections were cut into 5 mm then fixed at 65 C for 1 h. De-paraffinization, re-hydration and antigen unmasking was performed as previously described15. The rabbit polyclonal NF-kB and caspase 3 antibodies were employed as they are (ready to use) according to their manufacturer’s specifications. After applying the antibodies, slides were incubated overnight at 4 C. Poly HRP enzyme conjugate was applied for 20 minutes, after which DAB chromogen was applied for 2 min. After rinsing DAB, counterstaining with Mayer Hematoxylin and cover slipping were performed as the final steps before slides were examined under the light microscope (400). Using ImageJ 1.41 (National Institute of Health, Bethesda, MD), total number of cells/field was calculated by counting cells stained with hematoxylin using ImageJ particle count command. Then, immuno-positive cells were counted by the same means after performing color deconvolution command. Results were the average of counting sections from each rat, three sections/ rat. Results were expressed as percent of immuno-positive cells compared to total number of cells. Measurements of kidney function tests and oxidative stress markers Kidney function was assessed via evaluating serum urea and creatinine using enzymatic colorimetric kits (Biodiagnostic, Egypt) according to the manufacturer’s instructions. Assessment of the oxidative stress markers in the kidney was done via evaluating renal homogenate GSH, catalase and the lipid peroxidation marker; malondialdehyde (MDA). GSH was measured using colorimetric kit (Biodiagnostic, Egypt) according to kit instructions. The method was based on the reduction of the 5,50 dithiobis (2-nitrobenzoic acid) (DTNP) with glutathione to produce a yellow compound. The reduced chromogen directly proportional to GSH concentration and its absorption can be measured at 405 nm. Catalase enzyme was measured using colorimetric kit (Biodiagnostic, Egypt). The method depends on the conversion of H2O2 by catalase to H2O and O2. In the presence of peroxidase, remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore with a color intensity inversely proportional to the amount of catalase in
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the original sample. MDA is a reactive aldehyde that reflects lipid peroxidation. Renal content of MDA were determined using the thiobarbituric acid method described by the Mihara and Uchiyama method16. The method depends on measuring the MDA equivalent substances which are breakdown products of lipid peroxides. The thiobarbituric-MDA adduct forms colored complexes when extracted with n-butanol/pyridine; the absorbance of which is read at 532 nm using Bausch & Lomb Spectronic 2000 spectrophotometer (Rochester, NY). Evaluation of nitric oxide end products and TNF-a level Total nitrite (NOx), the stable end product of nitric oxide (NO) was determined spectrophotometrically using the Greiss reagent system. NOx was measured after the reduction of nitrate to nitrite by copperized cadmium granules in glycine buffer at pH 9.7. Quantification of NO 2 was based on the Griess reaction, in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of naphthylethylenediamine and sulfanilamide. TNF-a was measured by the ELISA Kit according to manufacture’s instructions. Briefly, samples containing rat TNF-a antigen were incubated with polyclonal TNF-a antibody. Several washing steps were performed to remove the unbound enzyme, and then a substrate solution was added to induce a colored reaction product, whose intensity was directly proportional to the concentration of rat TNF-a present in the samples. The values were read at 450 nm via an ELISA reader. Statistical analysis Results were expressed as means ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by the Dunnett Multiple Comparison post analysis test was used to analyze the results for statistically significant difference. p Values less than 0.05 were considered significant. Graph Pad Prism was used for statistical calculations (version 5.03 for Windows, Graphpad Software, San Diego, CA).
Results Effect of FEN and PIO on MTX-induced alterations in the change of total body weight, kidney/body wt ratio and kidney function parameters Comparing initial with final animal weights, administration of a single i.p. dose of MTX of 20 mg/kg caused significant decrease in the change of total body weight, with increase in renal weight to total body weight ratio; renal index (Table 1). Prior treatment with either FEN (150 mg/kg/day) or PIO (5 mg/kg/day) 11 days before MTX challenge caused significant improvement in the change of total body weight, which was more pronounced in MTX/PIO group that showed levels not statistically significant from control. In addition, both FEN and PIO pre-treatments significantly improved renal index to levels comparable to control. MTX group showed nephrotoxicity manifested by significant increase in serum urea and creatinine compared to the control
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Table 1. Effect of fenofibrate (FEN) and pioglitazone (PIO) on change (D) of total body weight (wt), kidney/body wt ratio and kidney function parameters in methotrexate (MTX)-treated rats. D Body wt (%)
Kidney/wt ratio
Urea (mg/dl)
Creatinine (mg/dl)
Control 100 ± 3.5 0.66 ± 0.01 27.1 ± 1.1 1.2 ± 0.1 FEN 111.9 ± 7.4 0.6 ± 0.03 26.4 ± 2.2 1.3 ± 0.1 PIO 125.9 ± 8.4 0.7 ± 0.05 26.02 ± 2.4 1.36 ± 0.03 MTX 79.5 ± 4.3a 0.8 ± 0.03a 45.2 ± 7.4a 2.4 ± 0.3a b c b,c MTX/FEN 94.3 ± 3.2 0.6 ± 0.03 26.5 ± 1.9 1.3 ± 0.1b,c b,c c b,c MTX/PIO 99.8 ± 5.7 0.7 ± 0.05 26.3 ± 1.6 1.09 ± 0.02b,c Kidney/wt is ratio of weight of both kidneys divided by total body wt * 100 ratio. Values are representation of 8 observations as means ± SEM. Results are considered significantly different when p50.05. a Significant difference compared to control. b Significant difference compared to the MTX group. c No significant difference compared to control.
group. Pre-administration of either FEN or PIO prior to MTX administration resulted in a significant decrease in serum urea and creatinine when compared to MTX-group, reaching levels statistically comparable to control, with no significant differences between FEN- and PIO-treated groups. Effect of FEN and PIO on MTX-induced alterations in renal histopathological findings Renal tissue showed normal histological findings regarding the structure of renal glomeruli and cortical tubules in the control group (Figure 1A) as well as in FEN- and PIO-treated groups (Figure 1B and C, respectively). In MTX group (Figure 1D), histological sections revealed dilated Bowman’s space with atrophied renal glomeruli. Some tubules were filled with protein casts and inflammatory cell infiltrations. These histological renal changes were improved in MTX groups pre-treated with either FEN or PIO (Figure 1E and F, respectively). Histological scoring of five fields of each rat was done (Table 2), and revealed that the histological improvement was more pronounced in the MTX/PIO than MTX/FEN group, as the latter retained moderate levels of renal tubular degeneration and protein casts with mild tubular and Bowman’s space dilation. Effect of FEN and PIO on MTX-induced renal oxidative markers Rats receiving MTX showed significant decrease in renal GSH levels compared to control group (Table 3). Preadministration of either FEN or PIO in MTX/FEN- and MTX/PIO-treated groups, respectively, caused significant increase in renal GSH compared to MTX alone. Rats treated with MTX alone also showed significant increase in renal MDA levels compared to the control group, which was significantly decreased by pre-treatment with either FEN or PIO. In addition, MTX group showed significant decrease in renal catalase levels compared to control group, whereas MTX/FEN and MTX/PIO groups showed significant increase in renal catalase level compared to the MTX group. There were no significant differences between FENand PIO-treated groups regarding the measured oxidative stress parameters.
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DOI: 10.3109/08923973.2014.884135
Figure 1. Effect of fenofibrate (FEN) and pioglitazone (PIO) on histopathological changes in methotrexate (MTX)-treated rat renal tissues. Photomicrographs of sections in rat kidney (H&E, 200x) of: (A, B and C) control, FEN and PIO groups, respectively, showing normal structure of renal glomeruli (black stars) and cortical tubules (black arrows). (D) MTX-treated group showing dilated Bowman’s space with atrophied renal glomeruli (white stars), tubules filled with protein casts (white arrows) and inflammatory cellular infiltrations (black arrowhead). (E and F) MTX/FEN and MTX/PIO groups, respectively, show improvement of the morphology of the renal cortex and glomeruli, with only mild tubular degeneration (black arrows). Histological scoring is shown in Table 2.
(A)
(B)
(C)
(D)
(E)
(F)
Table 2. Histological scoring of the effect of fenofibrate (FEN) and pioglitazone (PIO) on renal damage in methotrexate (MTX)-treated rats.
Control FEN PIO MTX MTX/FEN MTX/PIO
Tubular degeneration
Tubular dilatation
Dilated Bowman’s space
Protein casts
+ +++ ++ +
++ +
++ +
+ +++ ++
From each animal section, five fields were examined and scored according to the following criteria: () ¼ absent, (+) ¼ mild, (++) ¼ moderate, and (+++) severe changes.
Table 3. Effect of methotrexate (MTX), fenofibrate (FEN) and pioglitazone (PIO) on renal oxidative, nitrosative and inflammatory markers.
Control FEN PIO MTX MTX/FEN MTX/PIO
GSH (mM/g.tissue)
MDA (nM/g.tissue)
Catalase (U/g.tissue)
NOx (nM/g.tissue)
TNF-a (pg/g.tissue)
3.2 ± 0.3 2.5 ± 0.5 2.2 ± 0.4 0.5 ± 0.1a 3.2 ± 0.3b 2.5 ± 0.3b
57.6 ± 1.6 58.9 ± 2.4 56.8 ± 3.1 76.1 ± 1.7a 61 ± 1.3b 61.8 ± 1.8b
21 ± 1.7 18 ± 2.3 16 ± 1.8 6 ± 1.3a 20 ± 1.5b 20 ± 2.7b
99.6 ± 11.2 101.7 ± 6.9 132 ± 11 174.8 ± 5.4a 142.7 ± 7.1b 117.8 ± 4.3b
4.1 ± 1.7 4.6 ± 1.5 4.4 ± 1.1 16.1 ± 1.3a 12.1 ± 3.1a 5.2 ± 2.4b
GSH; reduced glutathione, MDA; malondialdehyde, NOx; nitric oxide end product, and TNF-a; tumor necrosis factor-a. Values represent the means ± S.E.M. n ¼ 8. a Significant difference from control. b Significant difference from MTX-treated groups (p50.05).
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Effect of MTX, FEN and PIO on renal nitrosative stress marker and tissue levels of TNF-a
Effect of FEN and PIO on renal expression of NF-kB and caspase 3 in MTX-treated rats Immuno-staining of rat kidney was done using the inflammatory and apoptotic markers; NF-kB and caspase 3 antibodies (Figures 2 and 3), respectively. Kidneys from
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Figure 2. Effect of fenofibrate (FEN) and pioglitazone (PIO) on nuclear factor-kappa B (NF-kB) immunohistochemical staining in methotrexate (MTX)-treated and untreated rat kidney. Localization of NF-kB immunoreactivity (400) in the kidney of (A) control group, (B and C) sole FEN and PIO-treated groups, respectively, (D) sole MTX-treated group, and (E and F) concomitant MTX with either FEN or PIO pre-treated groups, respectively. Black arrows point at nuclear expression of NF-kB in renal proximal tubule cells. (G) Semiquantitative analysis of NF-kB immunohistochemical staining results. Values are represented as means ± SE of the percent of NF-kB immuno-positive cells in sections of each animal of each group, three fields/section. aSignificant difference compared with control, bsignificant difference compared from MTX group, cno significant difference compared with control. Significant difference is reported when p50.05.
% of NF-κB immunopositive cells
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MTX group showed significant increase in renal content of NOx as compared to control group (Table 3). Preadministration with either FEN or PIO before MTX resulted in a significant decrease in renal NOx levels compared to sole MTX-treated groups. No statistically significant difference was observed between MTX/FEN and MTX/PIO groups in NOx renal level. In the MTX group, there was also a significant increase in the renal level of the inflammatory marker; TNF-a, compared to the control group.
Group treated with PIO before MTX showed significant decrease in the renal level of TNF-a compared to the MTX group. Interestingly, pre-treatment with FEN prior to MTX challenge did not result in any statistical change in renal TNF-a compared to MTX alone.
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Figure 3. Effect of fenofibrate (FEN) and pioglitazone (PIO) on caspase 3 immunohistochemical staining of methotrexate (MTX)treated rat kidney. Immuno-localization of caspase 3 reactivity (400) in the kidney of (A) control, (B) FEN, (C) PIO, (D) MTX, (E) combined MTX/FEN, and (F) combined MTX/PIO-treated groups. (G) semiquantitative analysis of caspase 3 immunohistochemical staining results in kidney. Values are represented as means ± SE of number of caspase 3 immuno-positive cells in sections of each animals, 3 fields/section. aSignificant difference compared with control, bsignificant difference compared from MTX group, c no significant difference compared with control. Significant difference is reported when p50.05.
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control, FEN- and PIO-treated groups (Figure 2A–C, respectively) showed mild cytoplasmic expression of NF-kB in renal tubules, indicating that NF-kB was present in its inactive form. Only mild nuclear expression in the FEN-treated group was noticed, which was proven by semiquantitative analysis insignificant from control levels (Figure 2G). In kidneys of rats treated with MTX alone, NF-kB was observed to be highly expressed in renal nuclei (Figure 2D), indicating nuclear translocation, up-regulation and activation of NF-kB. Interestingly, administration of PIO, but not FEN, prior to MTX caused less staining of NF-kB in renal proximal tubule
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DOI: 10.3109/08923973.2014.884135
nuclei (Figure 2E and F, respectively). For caspase 3 immunostaining, very mild expression was observed in kidneys of control, FEN- and PIO-treated groups (Figure 3A–C, respectively). MTX treatment caused significant increase in caspase 3 expression in renal tubules (Figure 3D), whereas combined MTX/FEN and MTX/PIO treatments significantly decreased caspase 3 expression (Figure 3E and F, respectively) compared to MTX alone. It is noteworthy that MTX/FEN group showed statistically lower levels of improvement than MTX/PIO, where the latter group reached levels statistically comparable to control (Figure 3G).
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Discussion MTX is a widely used indispensable anticancer and immunomodulating agent. However, its clinical use is hampered by serious toxicities; namely nephrotoxicity. The mechanisms underlying MTX toxicity are complex. Several studies have confirmed the contribution of oxidative stress in the pathogenesis of MTX-induced renal damage3,4,14. MTX increases the production of reactive oxygen radicals by stimulated polymorphonuclear neutrophils and impairs the anti-oxidant defense status3. Moreover, precipitation of MTX and its metabolites in the renal tubules17 and blockage in folate synthesis14 in kidneys have been suggested. The present results showed that a single dose of MTX (20 mg/kg) in rats induced marked nephrotoxicity manifested by elevation in serum levels of urea and creatinine. MTXinduced nephrotoxicity was confirmed by histopathological changes; namely dilated Bowman’s space with atrophied renal glomeruli and the presence of protein casts in some renal tubules with inflammatory cellular infiltrations. Similar findings have been previously reported3,18. The data of the present study revealed that MTX-induced nephrotoxicity was associated with enhancement of oxidative stress in renal tissue. MTX-nephrotoxicity increased lipid peroxidation product (MDA) and nitric oxide end product (NOx), as well as decreased the antioxidant status of kidney reflected by decreasing GSH level and catalase activity. These results were in consistence with previous studies18–21. The present study is the first that investigates the role of PPAR receptor agonists in MTX-nephrotoxicity. The results of the current study revealed that the administration of either FEN or PIO prior to MTX attenuated MTX-induced nephrotoxicity. This was evidenced by significant decrease in serum urea and creatinine and confirmed by improvement of the histological changes. Interestingly, pre treatment with the PPAR-g agonist; PIO, before MTX challenge caused better histological improvement compared to the PPAR-a agonist; FEN. In trial to explain the nephro-protective mechanisms of the investigated drugs, we evaluated the oxidative and nitrosative stress parameters as well as measured the pro-inflammatory cytokine; TNF-a, in renal tissue. The data of the present study revealed that the protective effect of PIO was accomplished through significant reversal of oxidative and nitrosative stress parameters, as well as via reduction of TNF-a. On the other hand, FEN-induced nephro-protective effect was through reversal of oxidative and nitrosative stress only, with no effect on renal TNF-a. The combined antioxidant/anti-nitrosative/ anti-inflammatory effects of PIO can explain its superior effect on histopathological improvement compared to FEN. Still, it is possible that FEN may possess anti-inflammatory properties that would be revealed at a different administration dose and/or duration. The reno-protective effects of PPAR-g agonists were previously reported in different models of renal injuries22. The effect of PPAR-g agonist; PIO, on the progression of diabetic nephropathy was studied, where it has been reported that PIO ameliorated many of the physiological, cellular, and molecular processes associated with the disease23. These effects were reported to be caused by modulating the
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inflammatory process and by antioxidant properties independently of the insulin-sensitizing effect24. Similarly, the protective effect of PIO against cisplatin-induced renal damage has been reported, where the protection was suggested to be mediated by antioxidant effect25. At the molecular level, PPAR agonists inhibit the nuclear transcription factor-kB (NF-kB), leading to down-regulation of downstream gene transcription, such as for genes encoding pro-inflammatory cytokines26. PPAR-a agonists have also been reported to possess nephro-protective effects in different animal models10. The mechanism of nephro-protection is still, however, controversial. Despite that the PPAR-a agonist; FEN, was reported to attenuate diabetic nephropathy via several mechanisms including reduction of renal lipotoxicity, inflammation, and oxidative stress, it might still cause renal dysfunction in the presence of other renal disorders27. Similar controversy exists regarding the effect of FEN on TNF-a/NF-kB pathway. While some studies reported that FEN ameliorated the level of this pro-inflammatory cytokine28, others reported no effect of FEN on TNF-a level29, and some studies even reported an increase in production of TNF-a30. In the present study, both FEN and PIO reversed MTXinduced apoptosis, as indicated by their significant effect on renal caspase 3 expression levels compared to MTX alone. However, only PIO succeeded in reverting caspase 3 expression to levels statistically comparable to control, indicating better antiapoptotic effect then FEN at tested dosages. This is in concurrent with previous studies reporting that PIO normalized the expression of caspase 3 in diabetic rat kidney31 and that FEN attenuated the same apoptotic marker in cultured podocytes after doxorubicin challenge8.
Conclusion The PPAR-a and PPAR-g agonists: FEN and PIO, respectively, conferred protection against MTX-induced nephrotoxicity. Their renal protective effects were significantly comparable regarding their effect on oxidative and nitrosative stress markers. However, unlike PIO, FEN did not attenuate the anti-inflammatory pathway, indicated by lacking any significant effect on renal TNF-a level and NF-kB expression. In addition, PIO had significantly higher effect in preventing MTX-induced apoptosis compared to FEN. These findings suggest that PPAR-g agonist; PIO, may be preferred over PPAR-a agonist; FEN, as an adjuvant drug during MTX chemotherapy, to protect against MTX-induced nephrotoxic effects.
Declaration of interest Authors have no conflict of interest to declare.
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oxidative damage and apoptosis in rat kidney. J Physiol Biochem 2013;69:371–381. Cag˘lar Y, Ozgu¨r H, Matur I, et al. Ultrastructural evaluation of the effect of N-acetylcysteine on methotrexate nephrotoxicity in rats. Histol Histopathol 2013;28:865–874. Guan Y, Breyer MD. Peroxisome proliferator-activated receptors (PPARs): novel therapeutic targets in renal disease. Kidney Int 2001;60:14–30. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990;347:645–650. Dreyer C, Krey G, Keller H, et al. Control of the peroxisomal betaoxidation pathway by a novel family of nuclear hormone receptors. Cell 1992;68:879–887. Zhou Y, Kong X, Zhao P, et al. Peroxisome proliferator-activated receptor-a is renoprotective in doxorubicin-induced glomerular injury. Kidney Int 2011;79:1302–1311. Jun M, Zhu B, Tonelli M, et al. Effects of fibrates in kidney disease: a systematic review and meta-analysis. J Am Coll Cardiol 2012;60: 2061–2071. Cheng CF, Chen HH, Lin H. Role of PPARa and its agonist in renal diseases. PPAR Res 2010;2010:345098. doi: 10.1155/2010/345098. Iglesias P, Dı´ez JJ. Peroxisome proliferator-activated receptors agonists in renal disease. Euro J Endocrinol 2006;154:613–621. Gelosa P, Banfi C, Gianella A, et al. Peroxisome proliferatoractivated receptor. Agonism prevents renal damage and the oxidative stress and inflammatory processes affecting the brains of stroke-prone rats. J Pharmacol Exp Ther 2010;335: 324–331. Pereira MG, Caˆmara NO, Campaholle G, et al. Pioglitazone limits cyclosporine nephrotoxicity in rats. Int Immunopharmacol 2006;6: 1943–1951. Asvadi I, Hajipour B, Asvadi A, et al. Protective effect of pentoxyfilline in renal toxicity after methotrexate administration. Eur Rev Med Pharmacol Sci 2011;15:1003–1009. El-Sheikh AA, Morsy MA, Mahmoud MM, et al. Effect of coenzyme-Q10 on doxorubicin-induced nephrotoxicity in rats. Adv Pharmacol Sci 2012;2012:981461. doi: 10.1155/2012/981461. Mihara M, Uchiyama M. Properties of thiobarbituric acid-reactive materials obtained from lipid peroxide and tissue homogenate. Chem Pharm Bull (Tokyo) 1983;31:605–611. Fendler C, Braun J. Use of methotrexate in inflammatory myopathies. Clin Exp Rheumatol 2010;28:S164–S167. Abraham P, Kolli VK, Rabi S. Melatonin attenuates methotrexateinduced oxidative stress and renal damage in rats. Cell Biochem Funct 2010;28:426–433. Uz E, Oktem F, Yilmaz HR, et al. The activities of purinecatabolizing enzymes and the level of nitric oxide in rat kidneys
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RESEARCH ARTICLE
Mohamed A. Ibrahim, Azza A. K. El-Sheikh, Hanaa M. Khalaf, and Aly M. Abdelrahman Department of Pharmacology, Faculty of Medicine, Minia University, Minia, Egypt
Abstract
Keywords
Context: The anticancer drug methotrexate (MTX) may cause multi-organ toxicities, including nephrotoxicity. Objective: To investigate effects of peroxisome proliferator activator receptor (PPAR)-a and -g agonists; fenofibrate (FEN) and pioglitazone (PIO), in MTX-induced nephrotoxicity in rats. Methods: Rats were given FEN or PIO (150 or 5 mg/kg/day, respectively) orally for 15 days. MTX was injected as a single dose of 20 mg/kg, i.p. at day 11 of experiment, with or without either PPAR agonists. Results: MTX induced renal toxicity, assessed by increase in serum urea and creatinine as well as histopathological alterations. MTX caused renal oxidative/nitrosative stress, indicated by decrease in GSH and catalase with increase in malondialdehyde and nitric oxide (NOx) levels. In addition, MTX increased renal level of the pro-inflammatory cytokine; tumor necrosis factor (TNF)-a and up-regulated the expression of both the inflammatory and apoptotic markers; NF-kB and caspase 3. Pre-administration of FEN or PIO to MTX-treated rats improved renal function and reversed oxidative/nitrosative parameters. Interestingly, pre-administration of PIO, but not FEN, decreased renal TNF-a level and NF-kB expression compared to MTX alone. Furthermore, PIO had more significant effect than FEN on reversing MTX-induced renal caspase 3 expression. Discussion: Both FEN and PIO conferred protection against MTX-induced nephrotoxicity through comparable amelioration of oxidative/nitrosative stress. FEN lacked any effect on TNF-a/NF-kB, which was reflected on its less improvement on renal histopathology and apoptosis. Conclusion: At indicated dosage, PPAR-g ligand; PIO shows better improvement of MTX-induced nephrotoxicity compared to PPAR-a ligand; FEN due to differential effect on TNF-a/NF-kB inflammatory pathway.
Fnofibrate, nitric oxide, oxidative stress, pioglitazone, TNF-a
Introduction Methotrexate (MTX), a folic acid antagonist, is widely used as anticancer drug in treatment of leukemia and other malignancies as well as used as in the treatment of autoimmune diseases such as rheumatoid arthritis and psoriasis1. Unfortunately, its use is associated with various forms of toxicities, including nephrotoxicity. The exact mechanisms of MTX-induced renal toxicity have not yet been elucidated. However, several hypotheses have been put forward, including direct toxic effect of MTX on the renal tubules2 and increased oxidative stress3. Although several agents with antioxidant properties have been examined as a potential therapy against MTX related toxicity3,4, the results of previous studies did not achieve satisfying nephro-protection. Consequently, the door is still open for further studies, Address for correspondence: Azza A.K. El-Sheikh, PhD, Assistant Professor of Pharmacology, Department of Pharmacology, Faculty of Medicine, Minia University, 61511 Minia, Egypt. Tel: (+2) 01006756740. Fax: (+2) 086 234 2813. E-mail: azza.elsheikh@ mu.edu.eg
History Received 27 September 2013 Revised 23 November 2013 Accepted 13 January 2014 Published online 13 February 2014
exploring a potential adjuvant therapy against renal MTXinduced toxicity. Peroxisome proliferator activator receptors (PPARs) are members of steroid nuclear receptor family expressed in many tissues of the body including the kidney5. There are three types of PPAR receptors; PPAR-a, PPAR-b, and PPAR-g6,7. Among PPAR agonists are hypolipidaemic drugs as fibrates (PPAR-a agonists) and insulin sensitizers agents such as thiazolidinediones or glitazones (PPAR-g agonists); whereas the former activate lipid metabolism, the latter produce several biological effects on adipogenesis, carbohydrate metabolism, inflammatory processes, and cellular proliferation5. The PPAR-a agonists, fibrates, have been shown to have beneficial effects in drug-induced nephropathy in animals8. Recent systematic review and metanalysis data suggested that fibrates have a role in reducing cardiovascular risk in people with mild-to-moderate chronic kidney disease9. Similarly, PPAR-g agonists have shown to exert renal protection via haemodynamic, vascular, anti-inflammatory, anti-oxidant, and metabolic effects10,11. These renal protective effects of
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Protective effect of peroxisome proliferator activator receptor (PPAR)-a and -g ligands against methotrexate-induced nephrotoxicity
DOI: 10.3109/08923973.2014.884135
PPAR agonists suggest that they may provide a novel intervention strategy to prevent MTX-induced renal damage. The aim of the current work was to investigate the effects of administration PPAR-a and -g agonists; fenofibrate (FEN) and pioglitazone (PIO), respectively, on MTX-induced nephrotoxicity in rats and to explore the mechanisms involved.
Materials and methods
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Chemicals Methotrexate (25 mg/ml ampoules) was obtained from Ebewe Co. (Unterach, Austria). Fenofibrate and pioglitazone powders were generous gifts from SigmaPharm Co. and Medical Union Pharmaceuticals Co.; MUP (Cairo, Egypt), respectively. Kits for examining serum urea and creatinine, reduced glutathione (GSH) and catalase were purchased from Biodiagnostic (Egypt). Nuclear factor-kB (NF-kB/p65) and caspase 3 rabbit polyclonal antibodies were purchased from Thermo Fisher Scientific Inc/Lab Vision (Fremont, CA). Tumor necrosis factor (TNF)-a enzyme-linked immuno sorbent assay (ELISA) kit was purchased from WKEA-Med supplies Corp. (Changchun, Jilin, China). Experimental animal design Adult male albino rats weighing 170–200 g were obtained from the National Research Centre (Giza, Egypt). They were housed four rats/cage and fed a standard diet of commercial rat chow (El-Nasr Company, Cairo, Egypt). Tap water was supplied ad libitum. Animals were left to acclimatize for one week before the start of the experiments. All experimental procedures were approved by the Faculty of Medicine board Committee of Minia University. Rats were weighed and divided into six groups (n ¼ 8). FEN and PIO groups were treated with 150 and 5 mg/kg/day, respectively, orally for 15 days12,13. MTX group was injected with a single dose of 20 mg/kg, i.p. at day 11 of the experiment and received no PPAR agonist to serve as positive control14. Combined MTX/FEN and MTX/PIO groups received MTX with the respective PPAR agonist as indicated. FEN and PIO were freshly prepared daily and administered orally by stomach tube, suspended in 0.5% carboxymethylcellulose. Control group received the same volume of vehicles of drugs used by the same route of administration and served as the untreated negative control group. Sample preparation and histological examination At the end of the experimental period, rats were anaesthetized with ether, weighed and sacrificed. Blood samples were collected from neck vessels by decapitation and left for few minutes to clot and then centrifuged for 10 min at 3000g for separation of serum. Kidneys were rapidly excised and weighed. Renal weight to total body weight ratio (renal index) was calculated according to the formula (renal weight/body weight) 100. The right kidney was used to prepare tissue homogenate. Homogenization was done by Glas-Col homogenizer and a 20% w/v homogenate was prepared in ice-cold phosphate buffer (0.01 M, pH 7.4). The homogenate was centrifuged at 1100g for 20 min. The supernatant was then
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divided over several containers to avoid sample thawing and refreezing, and was kept at 80 C till used. For histological examination, the left kidney from each rat was put in 10% formalin, embedded in paraffin, sectioned by a microtome at 5 mm thickness and stained with hematoxylin and eosin for routine histopathological assessment using light microscopy (Olympus CX41, Tokyo, Japan). Histological scoring was performed using five fields from each rat, regarding several criteria, as tubular degeneration and dilatation, as well as dilatation of Bowman’s space and presence of intra-tubular protein casts. All slides were randomly numbered and blind assessment of each slide was performed three times, each in a different setting scoring different microscopic fields. The score given was either absent, mild, moderate, or severe changes. Immunohistochemical examination of NF-kB and caspase 3 expression For immunohistochemical staining, sections were cut into 5 mm then fixed at 65 C for 1 h. De-paraffinization, re-hydration and antigen unmasking was performed as previously described15. The rabbit polyclonal NF-kB and caspase 3 antibodies were employed as they are (ready to use) according to their manufacturer’s specifications. After applying the antibodies, slides were incubated overnight at 4 C. Poly HRP enzyme conjugate was applied for 20 minutes, after which DAB chromogen was applied for 2 min. After rinsing DAB, counterstaining with Mayer Hematoxylin and cover slipping were performed as the final steps before slides were examined under the light microscope (400). Using ImageJ 1.41 (National Institute of Health, Bethesda, MD), total number of cells/field was calculated by counting cells stained with hematoxylin using ImageJ particle count command. Then, immuno-positive cells were counted by the same means after performing color deconvolution command. Results were the average of counting sections from each rat, three sections/ rat. Results were expressed as percent of immuno-positive cells compared to total number of cells. Measurements of kidney function tests and oxidative stress markers Kidney function was assessed via evaluating serum urea and creatinine using enzymatic colorimetric kits (Biodiagnostic, Egypt) according to the manufacturer’s instructions. Assessment of the oxidative stress markers in the kidney was done via evaluating renal homogenate GSH, catalase and the lipid peroxidation marker; malondialdehyde (MDA). GSH was measured using colorimetric kit (Biodiagnostic, Egypt) according to kit instructions. The method was based on the reduction of the 5,50 dithiobis (2-nitrobenzoic acid) (DTNP) with glutathione to produce a yellow compound. The reduced chromogen directly proportional to GSH concentration and its absorption can be measured at 405 nm. Catalase enzyme was measured using colorimetric kit (Biodiagnostic, Egypt). The method depends on the conversion of H2O2 by catalase to H2O and O2. In the presence of peroxidase, remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore with a color intensity inversely proportional to the amount of catalase in
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the original sample. MDA is a reactive aldehyde that reflects lipid peroxidation. Renal content of MDA were determined using the thiobarbituric acid method described by the Mihara and Uchiyama method16. The method depends on measuring the MDA equivalent substances which are breakdown products of lipid peroxides. The thiobarbituric-MDA adduct forms colored complexes when extracted with n-butanol/pyridine; the absorbance of which is read at 532 nm using Bausch & Lomb Spectronic 2000 spectrophotometer (Rochester, NY). Evaluation of nitric oxide end products and TNF-a level Total nitrite (NOx), the stable end product of nitric oxide (NO) was determined spectrophotometrically using the Greiss reagent system. NOx was measured after the reduction of nitrate to nitrite by copperized cadmium granules in glycine buffer at pH 9.7. Quantification of NO 2 was based on the Griess reaction, in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of naphthylethylenediamine and sulfanilamide. TNF-a was measured by the ELISA Kit according to manufacture’s instructions. Briefly, samples containing rat TNF-a antigen were incubated with polyclonal TNF-a antibody. Several washing steps were performed to remove the unbound enzyme, and then a substrate solution was added to induce a colored reaction product, whose intensity was directly proportional to the concentration of rat TNF-a present in the samples. The values were read at 450 nm via an ELISA reader. Statistical analysis Results were expressed as means ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by the Dunnett Multiple Comparison post analysis test was used to analyze the results for statistically significant difference. p Values less than 0.05 were considered significant. Graph Pad Prism was used for statistical calculations (version 5.03 for Windows, Graphpad Software, San Diego, CA).
Results Effect of FEN and PIO on MTX-induced alterations in the change of total body weight, kidney/body wt ratio and kidney function parameters Comparing initial with final animal weights, administration of a single i.p. dose of MTX of 20 mg/kg caused significant decrease in the change of total body weight, with increase in renal weight to total body weight ratio; renal index (Table 1). Prior treatment with either FEN (150 mg/kg/day) or PIO (5 mg/kg/day) 11 days before MTX challenge caused significant improvement in the change of total body weight, which was more pronounced in MTX/PIO group that showed levels not statistically significant from control. In addition, both FEN and PIO pre-treatments significantly improved renal index to levels comparable to control. MTX group showed nephrotoxicity manifested by significant increase in serum urea and creatinine compared to the control
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Table 1. Effect of fenofibrate (FEN) and pioglitazone (PIO) on change (D) of total body weight (wt), kidney/body wt ratio and kidney function parameters in methotrexate (MTX)-treated rats. D Body wt (%)
Kidney/wt ratio
Urea (mg/dl)
Creatinine (mg/dl)
Control 100 ± 3.5 0.66 ± 0.01 27.1 ± 1.1 1.2 ± 0.1 FEN 111.9 ± 7.4 0.6 ± 0.03 26.4 ± 2.2 1.3 ± 0.1 PIO 125.9 ± 8.4 0.7 ± 0.05 26.02 ± 2.4 1.36 ± 0.03 MTX 79.5 ± 4.3a 0.8 ± 0.03a 45.2 ± 7.4a 2.4 ± 0.3a b c b,c MTX/FEN 94.3 ± 3.2 0.6 ± 0.03 26.5 ± 1.9 1.3 ± 0.1b,c b,c c b,c MTX/PIO 99.8 ± 5.7 0.7 ± 0.05 26.3 ± 1.6 1.09 ± 0.02b,c Kidney/wt is ratio of weight of both kidneys divided by total body wt * 100 ratio. Values are representation of 8 observations as means ± SEM. Results are considered significantly different when p50.05. a Significant difference compared to control. b Significant difference compared to the MTX group. c No significant difference compared to control.
group. Pre-administration of either FEN or PIO prior to MTX administration resulted in a significant decrease in serum urea and creatinine when compared to MTX-group, reaching levels statistically comparable to control, with no significant differences between FEN- and PIO-treated groups. Effect of FEN and PIO on MTX-induced alterations in renal histopathological findings Renal tissue showed normal histological findings regarding the structure of renal glomeruli and cortical tubules in the control group (Figure 1A) as well as in FEN- and PIO-treated groups (Figure 1B and C, respectively). In MTX group (Figure 1D), histological sections revealed dilated Bowman’s space with atrophied renal glomeruli. Some tubules were filled with protein casts and inflammatory cell infiltrations. These histological renal changes were improved in MTX groups pre-treated with either FEN or PIO (Figure 1E and F, respectively). Histological scoring of five fields of each rat was done (Table 2), and revealed that the histological improvement was more pronounced in the MTX/PIO than MTX/FEN group, as the latter retained moderate levels of renal tubular degeneration and protein casts with mild tubular and Bowman’s space dilation. Effect of FEN and PIO on MTX-induced renal oxidative markers Rats receiving MTX showed significant decrease in renal GSH levels compared to control group (Table 3). Preadministration of either FEN or PIO in MTX/FEN- and MTX/PIO-treated groups, respectively, caused significant increase in renal GSH compared to MTX alone. Rats treated with MTX alone also showed significant increase in renal MDA levels compared to the control group, which was significantly decreased by pre-treatment with either FEN or PIO. In addition, MTX group showed significant decrease in renal catalase levels compared to control group, whereas MTX/FEN and MTX/PIO groups showed significant increase in renal catalase level compared to the MTX group. There were no significant differences between FENand PIO-treated groups regarding the measured oxidative stress parameters.
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DOI: 10.3109/08923973.2014.884135
Figure 1. Effect of fenofibrate (FEN) and pioglitazone (PIO) on histopathological changes in methotrexate (MTX)-treated rat renal tissues. Photomicrographs of sections in rat kidney (H&E, 200x) of: (A, B and C) control, FEN and PIO groups, respectively, showing normal structure of renal glomeruli (black stars) and cortical tubules (black arrows). (D) MTX-treated group showing dilated Bowman’s space with atrophied renal glomeruli (white stars), tubules filled with protein casts (white arrows) and inflammatory cellular infiltrations (black arrowhead). (E and F) MTX/FEN and MTX/PIO groups, respectively, show improvement of the morphology of the renal cortex and glomeruli, with only mild tubular degeneration (black arrows). Histological scoring is shown in Table 2.
(A)
(B)
(C)
(D)
(E)
(F)
Table 2. Histological scoring of the effect of fenofibrate (FEN) and pioglitazone (PIO) on renal damage in methotrexate (MTX)-treated rats.
Control FEN PIO MTX MTX/FEN MTX/PIO
Tubular degeneration
Tubular dilatation
Dilated Bowman’s space
Protein casts
+ +++ ++ +
++ +
++ +
+ +++ ++
From each animal section, five fields were examined and scored according to the following criteria: () ¼ absent, (+) ¼ mild, (++) ¼ moderate, and (+++) severe changes.
Table 3. Effect of methotrexate (MTX), fenofibrate (FEN) and pioglitazone (PIO) on renal oxidative, nitrosative and inflammatory markers.
Control FEN PIO MTX MTX/FEN MTX/PIO
GSH (mM/g.tissue)
MDA (nM/g.tissue)
Catalase (U/g.tissue)
NOx (nM/g.tissue)
TNF-a (pg/g.tissue)
3.2 ± 0.3 2.5 ± 0.5 2.2 ± 0.4 0.5 ± 0.1a 3.2 ± 0.3b 2.5 ± 0.3b
57.6 ± 1.6 58.9 ± 2.4 56.8 ± 3.1 76.1 ± 1.7a 61 ± 1.3b 61.8 ± 1.8b
21 ± 1.7 18 ± 2.3 16 ± 1.8 6 ± 1.3a 20 ± 1.5b 20 ± 2.7b
99.6 ± 11.2 101.7 ± 6.9 132 ± 11 174.8 ± 5.4a 142.7 ± 7.1b 117.8 ± 4.3b
4.1 ± 1.7 4.6 ± 1.5 4.4 ± 1.1 16.1 ± 1.3a 12.1 ± 3.1a 5.2 ± 2.4b
GSH; reduced glutathione, MDA; malondialdehyde, NOx; nitric oxide end product, and TNF-a; tumor necrosis factor-a. Values represent the means ± S.E.M. n ¼ 8. a Significant difference from control. b Significant difference from MTX-treated groups (p50.05).
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Effect of MTX, FEN and PIO on renal nitrosative stress marker and tissue levels of TNF-a
Effect of FEN and PIO on renal expression of NF-kB and caspase 3 in MTX-treated rats Immuno-staining of rat kidney was done using the inflammatory and apoptotic markers; NF-kB and caspase 3 antibodies (Figures 2 and 3), respectively. Kidneys from
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Figure 2. Effect of fenofibrate (FEN) and pioglitazone (PIO) on nuclear factor-kappa B (NF-kB) immunohistochemical staining in methotrexate (MTX)-treated and untreated rat kidney. Localization of NF-kB immunoreactivity (400) in the kidney of (A) control group, (B and C) sole FEN and PIO-treated groups, respectively, (D) sole MTX-treated group, and (E and F) concomitant MTX with either FEN or PIO pre-treated groups, respectively. Black arrows point at nuclear expression of NF-kB in renal proximal tubule cells. (G) Semiquantitative analysis of NF-kB immunohistochemical staining results. Values are represented as means ± SE of the percent of NF-kB immuno-positive cells in sections of each animal of each group, three fields/section. aSignificant difference compared with control, bsignificant difference compared from MTX group, cno significant difference compared with control. Significant difference is reported when p50.05.
% of NF-κB immunopositive cells
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MTX group showed significant increase in renal content of NOx as compared to control group (Table 3). Preadministration with either FEN or PIO before MTX resulted in a significant decrease in renal NOx levels compared to sole MTX-treated groups. No statistically significant difference was observed between MTX/FEN and MTX/PIO groups in NOx renal level. In the MTX group, there was also a significant increase in the renal level of the inflammatory marker; TNF-a, compared to the control group.
Group treated with PIO before MTX showed significant decrease in the renal level of TNF-a compared to the MTX group. Interestingly, pre-treatment with FEN prior to MTX challenge did not result in any statistical change in renal TNF-a compared to MTX alone.
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Figure 3. Effect of fenofibrate (FEN) and pioglitazone (PIO) on caspase 3 immunohistochemical staining of methotrexate (MTX)treated rat kidney. Immuno-localization of caspase 3 reactivity (400) in the kidney of (A) control, (B) FEN, (C) PIO, (D) MTX, (E) combined MTX/FEN, and (F) combined MTX/PIO-treated groups. (G) semiquantitative analysis of caspase 3 immunohistochemical staining results in kidney. Values are represented as means ± SE of number of caspase 3 immuno-positive cells in sections of each animals, 3 fields/section. aSignificant difference compared with control, bsignificant difference compared from MTX group, c no significant difference compared with control. Significant difference is reported when p50.05.
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control, FEN- and PIO-treated groups (Figure 2A–C, respectively) showed mild cytoplasmic expression of NF-kB in renal tubules, indicating that NF-kB was present in its inactive form. Only mild nuclear expression in the FEN-treated group was noticed, which was proven by semiquantitative analysis insignificant from control levels (Figure 2G). In kidneys of rats treated with MTX alone, NF-kB was observed to be highly expressed in renal nuclei (Figure 2D), indicating nuclear translocation, up-regulation and activation of NF-kB. Interestingly, administration of PIO, but not FEN, prior to MTX caused less staining of NF-kB in renal proximal tubule
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DOI: 10.3109/08923973.2014.884135
nuclei (Figure 2E and F, respectively). For caspase 3 immunostaining, very mild expression was observed in kidneys of control, FEN- and PIO-treated groups (Figure 3A–C, respectively). MTX treatment caused significant increase in caspase 3 expression in renal tubules (Figure 3D), whereas combined MTX/FEN and MTX/PIO treatments significantly decreased caspase 3 expression (Figure 3E and F, respectively) compared to MTX alone. It is noteworthy that MTX/FEN group showed statistically lower levels of improvement than MTX/PIO, where the latter group reached levels statistically comparable to control (Figure 3G).
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Discussion MTX is a widely used indispensable anticancer and immunomodulating agent. However, its clinical use is hampered by serious toxicities; namely nephrotoxicity. The mechanisms underlying MTX toxicity are complex. Several studies have confirmed the contribution of oxidative stress in the pathogenesis of MTX-induced renal damage3,4,14. MTX increases the production of reactive oxygen radicals by stimulated polymorphonuclear neutrophils and impairs the anti-oxidant defense status3. Moreover, precipitation of MTX and its metabolites in the renal tubules17 and blockage in folate synthesis14 in kidneys have been suggested. The present results showed that a single dose of MTX (20 mg/kg) in rats induced marked nephrotoxicity manifested by elevation in serum levels of urea and creatinine. MTXinduced nephrotoxicity was confirmed by histopathological changes; namely dilated Bowman’s space with atrophied renal glomeruli and the presence of protein casts in some renal tubules with inflammatory cellular infiltrations. Similar findings have been previously reported3,18. The data of the present study revealed that MTX-induced nephrotoxicity was associated with enhancement of oxidative stress in renal tissue. MTX-nephrotoxicity increased lipid peroxidation product (MDA) and nitric oxide end product (NOx), as well as decreased the antioxidant status of kidney reflected by decreasing GSH level and catalase activity. These results were in consistence with previous studies18–21. The present study is the first that investigates the role of PPAR receptor agonists in MTX-nephrotoxicity. The results of the current study revealed that the administration of either FEN or PIO prior to MTX attenuated MTX-induced nephrotoxicity. This was evidenced by significant decrease in serum urea and creatinine and confirmed by improvement of the histological changes. Interestingly, pre treatment with the PPAR-g agonist; PIO, before MTX challenge caused better histological improvement compared to the PPAR-a agonist; FEN. In trial to explain the nephro-protective mechanisms of the investigated drugs, we evaluated the oxidative and nitrosative stress parameters as well as measured the pro-inflammatory cytokine; TNF-a, in renal tissue. The data of the present study revealed that the protective effect of PIO was accomplished through significant reversal of oxidative and nitrosative stress parameters, as well as via reduction of TNF-a. On the other hand, FEN-induced nephro-protective effect was through reversal of oxidative and nitrosative stress only, with no effect on renal TNF-a. The combined antioxidant/anti-nitrosative/ anti-inflammatory effects of PIO can explain its superior effect on histopathological improvement compared to FEN. Still, it is possible that FEN may possess anti-inflammatory properties that would be revealed at a different administration dose and/or duration. The reno-protective effects of PPAR-g agonists were previously reported in different models of renal injuries22. The effect of PPAR-g agonist; PIO, on the progression of diabetic nephropathy was studied, where it has been reported that PIO ameliorated many of the physiological, cellular, and molecular processes associated with the disease23. These effects were reported to be caused by modulating the
Immunopharmacol Immunotoxicol, 2014; 36(2): 130–137
inflammatory process and by antioxidant properties independently of the insulin-sensitizing effect24. Similarly, the protective effect of PIO against cisplatin-induced renal damage has been reported, where the protection was suggested to be mediated by antioxidant effect25. At the molecular level, PPAR agonists inhibit the nuclear transcription factor-kB (NF-kB), leading to down-regulation of downstream gene transcription, such as for genes encoding pro-inflammatory cytokines26. PPAR-a agonists have also been reported to possess nephro-protective effects in different animal models10. The mechanism of nephro-protection is still, however, controversial. Despite that the PPAR-a agonist; FEN, was reported to attenuate diabetic nephropathy via several mechanisms including reduction of renal lipotoxicity, inflammation, and oxidative stress, it might still cause renal dysfunction in the presence of other renal disorders27. Similar controversy exists regarding the effect of FEN on TNF-a/NF-kB pathway. While some studies reported that FEN ameliorated the level of this pro-inflammatory cytokine28, others reported no effect of FEN on TNF-a level29, and some studies even reported an increase in production of TNF-a30. In the present study, both FEN and PIO reversed MTXinduced apoptosis, as indicated by their significant effect on renal caspase 3 expression levels compared to MTX alone. However, only PIO succeeded in reverting caspase 3 expression to levels statistically comparable to control, indicating better antiapoptotic effect then FEN at tested dosages. This is in concurrent with previous studies reporting that PIO normalized the expression of caspase 3 in diabetic rat kidney31 and that FEN attenuated the same apoptotic marker in cultured podocytes after doxorubicin challenge8.
Conclusion The PPAR-a and PPAR-g agonists: FEN and PIO, respectively, conferred protection against MTX-induced nephrotoxicity. Their renal protective effects were significantly comparable regarding their effect on oxidative and nitrosative stress markers. However, unlike PIO, FEN did not attenuate the anti-inflammatory pathway, indicated by lacking any significant effect on renal TNF-a level and NF-kB expression. In addition, PIO had significantly higher effect in preventing MTX-induced apoptosis compared to FEN. These findings suggest that PPAR-g agonist; PIO, may be preferred over PPAR-a agonist; FEN, as an adjuvant drug during MTX chemotherapy, to protect against MTX-induced nephrotoxic effects.
Declaration of interest Authors have no conflict of interest to declare.
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