Effects of silymarin on methotrexate-induced nephrotoxicity in rats

Abstract

Methotrexate (MTX) is widely used in the treatment of various malignancies and nononcological diseases but its use has been limited by its nephrotoxicity. Silymarin (SLY), a natural flavonoid, has been reported to have antioxidant, anti-inflammatory and anti-apoptotic effects. This study was carried out to determine whether SLY exerts a protective effect against MTX-induced nephrotoxicity. Rats were divided into six groups: Group 1 (saline, i.p., single injection), Group 2 (0.5% carboxymethyl cellulose (CMC), by gavage once daily for five consecutive days), Group 3 (SLY, 300 mg/kg per day, i.p. for five consecutive days), Group 4 (MTX, 20 mg/kg, i.p., single injection), Group 5 (MTX + CMC similarly as groups 2 and 4) and Group 6 (MTX + CMC + SLY similarly as groups 2, 3 and 4). Histopathologic alterations including apoptotic changes of the kidney were evaluated. MTX injection exhibited dilated Bowman’s space, inflammatory cell infiltration, glomerular and peritubular vascular congestion and swelling of renal tubular epithelium cells. Apoptotic cell death was also markedly increased in renal tubules after MTX administration. SLY treatment resulted in statistically significant amelioration in the histological alterations and reduced the number of TUNEL-positive cells as compared with the MTX treated rats (p < 0.05). In conclusion, SLY treatment leads to a reduction on MTX-induced renal damage in rats. Since SLY is safe and acceptable for human consumption, further studies to define the exact mechanism of the protecting effect of SLY on MTX-induced nephrotoxicity and the optimum dosage of this compound would be useful.

• Apoptosis
• methotrexate
• nephrotoxicity
• rat
• silymarin

Introduction

Methotrexate (MTX), a cytotoxic chemotherapeutic agent, is widely used to treat certain types of malignancies such as leukemia,1 lymphoma,2 breast cancer,3 osteosarcoma,4 head and neck cancers.5 It is also used in the treatment of non-cancerous diseases, including rheumatoid arthritis,6 psoriasis7 and inflammatory bowel diseases.8 Since more than 90% of MTX is excreted unchanged via the kidneys,9 MTX treatment, particularly at high doses, may cause renal failure.4 The MTX-induced nephrotoxicity is believed to be mediated by two primary mechanisms.10 The first is MTX-induced crystal nephropathy, which occurs when MTX and its metabolites precipitate within the renal tubules.11^,12 The second mechanism of MTX-induced renal injury is direct tubular toxicity; MTX causes the reactive oxygen radical overproduction in the kidney.10^,13^,14 Several studies have revealed that MTX administration causes increased malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity and decreased catalase activity, glutathione (GSH) levels and superoxide dismutase activity in the blood and kidney.15–17 It has recently been reported that in addition to oxidative stress, abnormal generation of inflammatory mediators and neutrophil infiltration contribute to MTX-induced renal damage.16

Silymarin (SLY) is a purified flavonoid extract isolated from the seeds of Silybum marinum (milk thistle).18 SLY has antioxidative, anti-inflammatory, immunomodulatory and liver-regenerating properties.19 It is acceptable for human consumption and widely used as a hepatoprotection and as a supportive therapy of liver disorders for more than three decades.20 SLY has also hypolipidemic properties21 and inhibitory effect on low density lipoprotein (LDL) oxidation in vitro22 indicating it may be beneficial on the prevention of atherosclerosis. SLY has also antiviral properties; it has been shown that SLY displays both prophylactic and therapeutic effects against hepatitis C virus infection.23 Anticarcinogenic effects of SLY have been shown in human breast cancer cells.24 SLY has also antiulcer activity against experimentally-induced peptic ulcers25 and induces pancreatic function recovery after alloxan induced pancreatic damage in rats.26 SLY also exerts protective effects against nephrotoxicity induced by cisplatin,27 adriamycin28 and gentamicin.29 However, as far as we know there are no reports on the estimation of the effects of silymarin against MTX-induced renal injury. Therefore, the present study aimed to evaluate the scavenging activity of SLY on MTX-induced nephrotoxicity in rat model.

Materials and methods

Experimental design

The study protocol was approved by the local ethics committee of the Firat University, Elazig, Turkey. Experiments involving the animals were conducted according to the policy of the European convention for the protection of vertebrate animals used for experimental and other scientific purposes in accordance with “Recommendations on the Establishment of Animal Experimental Guidelines”, and ethical procedures were conducted under Reduction, Replacement and Refinement. The study was carried out at the Experimental Research Unit of Firat University (FUDAM). Thirty six male Sprague–Dawley rats aged 8 weeks, weighing between 180 and 200 g were used in this study. Rats were fed with standard pellets and tap water, and 12 h light and dark cycles, standard temperature and humidity conditions were maintained. The rats were randomly divided into six groups, consisting of six animals per group. During 5 days of experimental stage, the rats treated as follows:

• Group 1 (Control): Rats received saline intraperitoneally (i.p.) as a single dose on the first day and served as a control.

• Group 2 (CMC): Rats received 0.5% carboxymethylcellulose (CMC) (Sigma, St. Louis, MO) by gavage daily for 5 days.

• Group 3 (SLY): Rat received SLY (Sigma) (300 mg/kg per day, i.p.) for 5 days.

• Group 4 (MTX): Rats received MTX at the dose of 20 mg/kg, i.p. as a single dose on the first day.

• Group 5 (MTX + CMC): Rats received MTX at the dose of 20 mg/kg, i.p. as a single dose on the first day and then received 0.5% CMC by gavage daily for 5 days.

• Group 6 (MTX + CMC + SLY): Rats received MTX at the dose of 20 mg/kg, i.p. as a single dose on the first day and then received SLY (300 mg/kg per day, i.p.) in 0.5% CMC by gavage for 5 days.

At the end of the experiment, the rats were decapitated under xylazine (10 mg/kg)–ketamine (75 mg/kg) anesthesia. Kidney tissues were removed and fixed with 10% neutralized formalin solution for histological examination and TUNEL assay.

Histological evaluation

Formalin-fixed tissues were embedded in paraffin blocks and the blocks were cut into 5 -μm thick sections, deparaffinized and hydrated in descending series of ethyl alcohol and distilled water and then stained with hematoxylin and eosin (H&E), dehydrated, cleared in xylene and mounted using Canada balsam. The slides were then examined using (NovelN-800 M, Ningbo, China) light microscope and microscopic scoring was done by an observer who was blinded to the treatment condition. Severity of kidney injury was semiquantitatively evaluated using the following alterations: (a) vascular congestion in glomerular and peritubular area, (b) inflammatory cell infiltration, (c) dilatation of Bowman’s space and (d) swelling of renal tubular epithelium cells. Scores were given as 0 = absent, 1 = weak, 2 = moderate and 3 = strong for each criteria. The microscopic score of each tissue was calculated as the sum of the scores given to each criterion, and at least five microscopic areas were examined under a 20× objective to score each specimen.

TUNEL assay

Parafin blocks were sectioned 5 µm and the sections were taken to slides with poly-l-lysine. ApopTag plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Cat no: S7101, USA) was used for detection of apoptotic cells. According to the instructions of the manufacturer sections were deparaffinized in xylene, dehydrated through graded alcohol and washed in PBS. Tissues were incubated in a 0.05% proteinase K solution for 10 min. Then tissues were incubated with 3% hydrogen peroxide for five minutes to prevent endogenous peroxidase activity. Later washing with PBS, the tissues were placed in equilibration buffer for six minutes and in working solution (70% reaction buffer plus 30% TdT enzyme) at 37 °C under moist conditions for one hour. Stop/wash buffer were applied during for 10 min and then anti-digoxigenin-peroxidase for 30 min. Diaminobenzidine (DAB) substrate was used to show apoptotic cells. Cross-sections contrast-stained with Harrishematoxylin was sealed using a proper covering solution. Preparations were observed and evaluated using a research microscope (NovelN-800 M, Ningbo, China) and photographed. Cells with blue nuclei after TUNEL staining using Harrishematoxylin were considered normal, whereas cells with brown nuclei were considered apoptotic. At least 500 cells were counted on each field and the apoptotic index was calculated as a ratio of the TUNEL-positive cell number to the total cell number.

Statistical analyses

Results were expressed as the means ± standard error of the mean. Statistical significant difference was determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Probability values (p) less than 0.05 were considered to be statistically significant. All analyses were performed using SPSS version 22.0 software.

Results

Renal histology

The results of the histological scoring of the renal tissue are shown in Figure 1. Microscopic examination of kidney sections of control group showed normal morphology (Figure 2A). There were no differences in the CMC (Figure 2B) and SLY (Figure 2C) groups when compared with the control group (p > 0.05). MTX group revealed dilated Bowman’s space (Figure 2D), inflammatory cell infiltration (Figure 2D), glomerular and peritubular vascular congestion (Figure 2E) and swelling of renal tubular epithelium cells (Figure 2F) (p < 0.05). The findings of MTX + CMC group were similar to those of MTX group including dilatation of Bowman’s space (Figure 2H), inflammatory cell infiltration (Figure 2G), glomerular and peritubular vascular congestion (Figure 2H) and swelling of renal tubular epithelium cells (Figure 2I) (p > 0.05). SLY treatment resulted in significant amelioration of dilated Bowman’s space (Figure 2K), inflammatory cell infiltration (Figure 2J), glomerular and peritubular vascular congestion (Figure 2K) and swelling of renal tubular epithelium cells (Figure 2L) in the MTX + CMC + SLY group compared with MTX group (p < 0.05).

Figure 1. The histological scores of all the groups. Values are mean ± SD for six rats in each group. (a,b,c): Values with common superscripts are not statistically different, whereas values without common superscripts are statistically significantly different (p value <0.05).

Figure 2. Photomicrographs of kidney sections stained with hematoxylin and eosin (scale bars = 20 μm), showing: (A) Group 1 (control), (B) Group 2 (CMC) and (C) Group 3 (SLY) show similarly undamaged kidney; (D) Group 4 (MTX) dilatation of Bowman’s space (arrow) and inflammatory cell infiltration (asterisk); (E) Group 4 (MTX) glomerular (arrows) and peritubular (asterisks) vascular congestion; (F) Group 4 (MTX) swelling of renal tubular epithelium cells (arrows); (G) Group 5 (MTX + CMC) inflammatory cell infiltration (asterisk); (H) Group 5 (MTX + CMC) dilatation of Bowman’s space (thick arrow) and glomerular (thin arrow), peritubular (asterisks) vascular congestion; (I) Group 5 (MTX + CMC) swelling of renal tubular epithelium cells (arrows); (J) Group 6 (MTX + CMC + SLY) inflammatory cell infiltration (asterisk); (K) Group 6 (MTX + CMC + SLY) glomerular (thin arrow) and peritubular (asterisk) vascular congestion, Bowman’s space (thick arrow); (L) Group 6 (MTX + CMC + SLY) swelling of renal tubular epithelium cells (arrows).

Evaluation of apoptosis in kidney tissues

The results of the apoptotic index are shown in Figure 3. Using TUNEL assay to detect apoptotic renal tubular cells in the kidney sections, control (Figure 4A), CMC (Figure 4B) and SLY (Figure 4C) groups were similar and showed only a few TUNEL-positive cells. The number of TUNEL-positive cells markedly increased in the MTX (Figure 4D) and the MTX + CMC (Figure 4E) groups compared with the control group (p < 0.05). Treatment with SLY (MTX + CMC + SLY group) (Figure 4F) reduced the number of TUNEL-positive cells as compared with the MTX group (p < 0.05).

Figure 3. The apoptotic index of all the groups. Values are mean ± SD for six rats in each group. (a,b,c): Bars with common superscripts are not statistically different, whereas values without common superscripts are statistically significantly different (p value <0.05).

Figure 4. Representative photomicrographs of TUNEL staining in all six groups (scale bars=20 μm), showing: (A) Group 1 (control), (B) Group 2 (CMC) and (C) Group 3 (SLY) similarly only few TUNEL-positive cells (arrow); (D) Group 4 (MTX) and (E) Group 5 (MTX + CMC) similarly a lot of TUNEL-positive cells (arrows); (F) Group 6 (MTX + CMC + SLY) rare TUNEL-positive cells (arrows).

Discussion

Drugs used for cancer chemotherapy produce acute toxic side effects in multiple organ systems. MTX is one of the most widely used anticancer drugs and administration of high-dose MTX is an important component in the treatment of a variety of childhood and adult cancers.30^,31 MTX-induced nephrotoxicity continues to occur despite the preventive measures such as administration of pharmacokinetically guided leucovorin, urinary alkalinization and intravenous hydration.31^,32 MTX-induced renal toxicity can be life threatening because, it further leads to delayed elimination of MTX, and the resulting sustained, elevated circulating MTX level may lead to the development of other MTX-related toxicities.30^,32 Treatment approaches including dialysis-based methods to remove MTX have limited effectiveness.30

Several experimental therapeutic studies for the prevention of MTX-induced nephrotoxicity have been conducted. Abdel-Raheem and Khedr16 reported that montelukast ameliorated MTX-induced histopathological alterations in the kidney tissues such as glomerular atrophy, tubular cystic dilatation, tubular degeneration, brush border disintegration and leucocyte cell infiltration. Abraham et al.33 revealed that MTX treatment lead to severe glomerular and tubular damage in the kidney tissues and pretreatment with melatonin reduced MTX-induced damage to the kidney. Uzkeser et al.34 indicated that MTX-induced histopathological changes including interstitial inflammation, swelling in the tubular epithelial cells and desquamated cells within the tubule lumen were healed with mirtazapine treatment. Sener et al.35 reported that although severe glomerular congestion and degeneration, dilatation in Bowman’s space, inflammatory cell infiltration and tubular degeneration were determined in MTX-treated group, these changes were mild in MTX plus L-carnitine treated rats. In another study,36 while severe glomerular congestion and degeneration, dilatation in Bowman’s space, inflammatory cell infiltration in interstitium and tubular degeneration were determined in the MTX-treated rats, there were mild glomerular and tubular degeneration and mild inflammatory cell infiltration in the interstitium in MTX plus pentoxifylline treated rats. In addition, pentoxifylline administration decreased the number of TUNEL-positive cells which were increased in MTX-treated rats. In the present study, the histopathological evidence of kidney injury such as dilated Bowman’s space, inflammatory cell infiltration, glomerular and peritubular vascular congestion and swelling of renal tubular epithelium cells has been observed in rats treated with MTX. In consistence with the previous reports, our findings indicate that SLY exerts a renoprotective effect against MTX-induced kidney damage in rats. In addition, the histopathological examination of kidneys of MTX-treated rats revealed increased apoptotic cells number in renal tubular epithelium cells. SLY treatment reduced the number of apoptotic cells as compared with the MTX group.

Several experimental studies have also highlighted the renoprotective properties of SLY against nephrotoxicity induced by different drugs such as cisplatin,27 doxorubicin,28 gentamicin,29 cyclosporine37 and chemicals such as arsenic,38 manganese.39 Similarly, mild nephroprotective action of SLY against MTX-induced renal injury was determined in the present study. The renoprotective effects of SLY have been mainly attributable to its antioxidant and free radical scavenging properties. Besides its antioxidant actions, SLY has also anti-inflammatory and immunomodulatory properties.19 More recently, Manna et al.40 reported that SLY has anti-inflammatory and cytoprotective effects via blocking TNF-induced activation of NF-kB and the kinases. They also indicate that SLY suppresses caspase activation leading to the inhibition of apoptosis and finally cytoprotection. Consequently, it seems likely that SLY could have diminished the detrimental effects of MTX by balancing oxidant-antioxidant status, regulation of immunomodulatory functions and inhibition of inflammation in the present study.

In conclusion, SLY treatment leads to a reduction on MTX-induced renal damage in rats. Since SLY is safe and acceptable for human consumption, further studies to define the exact mechanism of the protecting effect of SLY on MTX-induced nephrotoxicity and the optimum dosage of this compound would be useful.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This research was supported by Firat University Scientific Research Projects Management Unit (FUBAP) project number TF1152.