Indole-3-carbinol protects against cisplatin-induced acute nephrotoxicity: role of calcitonin gene-related peptide and insulin-like growth factor-1

Abstract

Nephrotoxicity associated with the clinical use of the anticancer drug cisplatin is a limiting problem. Thus, searching for new protective measures is required. Indole-3-carbinol is a powerful anti-oxidant, anti-inflammatory and anti-tumor agent. The present study aimed to investigate the potential protective effect of indole-3-carbinol against cisplatin-induced acute nephrotoxicity in rats. Rats were pre-treated with 20 mg/kg indole-3-carbinol orally before giving cisplatin (7 mg/kg). Cisplatin-induced acute nephrotoxicity was demonstrated where relative kidney weight, BUN and serum creatinine were significantly increased. Increased oxidative stress was evident in cisplatin group where GSH and SOD tissue levels were significantly depleted. Also, lipid peroxidation and NOX-1 were increased as compared to the control. Additionally, renal expression of pro-inflammatory mediators was induced by cisplatin. Cisplatin-induced cell death was shown by increased caspase-3 and decreased expression of EGF, IGF-1 and IGF-1 receptor. Nephrotoxicity, oxidative stress, inflammation and apoptotic effects induced by cisplatin were significantly ameliorated by indole-3-carbinol pre-treatment. Besides, the role of CGRP in cisplatin-induced nephrotoxicity was explored. Furthermore, cisplatin cytotoxic activity was significantly enhanced by indole-3-carbinol pre-treatment in vitro. In conclusion, indole-3-carbinol provides protection against cisplatin-induced nephrotoxicity. Also, reduced expression of CGRP may play a role in the pathogenesis of cisplatin-induced renal injury.

Figure 1. Expression of Nuclear factor-kappa B by immunohistochemical staining.

(A) Control group shows a minimal expression of NF-kB. (B) Cisplatin-injected group shows an extensive NF-kB expression (brown staining). (C) Indole-3-carbinol pre-treated group shows a limited NF-kB expression. (D) Indole-3-carbinol-only treated group showing a minimal NF-kB expression. (E) Area percentage of immuno-positive reaction. Values are given as mean ± SD for each group. a or b: Statistically significant from the control or the cisplatin group, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test.

Figure 2. Effects of cisplatin and indole-3-carbinol on the expression of caspase-3 in kidney tissues of rats.

a or b: Statistically significant when compared to the control or the cisplatin group, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test.

Figure 3. Effects of cisplatin and indole-3-carbinol on the expression of growth factors and calcitonin gene-related peptide in kidney tissues of rats.

a or b: Statistically significant when compared to the control or the cisplatin group, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test. EGF: epidermal growth factor; IGF-1: insulin-like growth factor-1; IGF-1R: insulin-like growth factor-1 receptor; CGRP: calcitonin gene-related peptide.

Figure 4

Representative photomicrographs of kidney sections stained by H&E: (A,B) Control group showing normal glomeruli (g) and normal histological structure of the tubules (t). (C,D) Cisplatin-injected group showing necrosis (n), degeneration (d) and cystic dilatation of the tubules with focal hemorrhages (h) in between. Besides, homogenous eosinophilic casts in lumen of medullary tubules (c) were shown. (E) Indole-3-carbinol pre-treated rats showing degeneration (d) in the lining epithelium of the tubules. (F) Indole-3-carbinol only-treated rats showing normal histological structures.

Figure 5

Cytotoxicity of various concentrations of cisplatin alone or in combination with indole-3-carbinol in hela cancer cell line (a) and PC3 cancer cell line (b). ^a,bp < 0.05: Statistically significant when compared to the control or the corresponding group treated with cisplatin alone, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test.

Figure 6

(A) Effects of cisplatin and/or indole-3-carbinol treatment on tumor growth in Ehrlich ascites carcinoma solid tumor model in mice. a or b: Statistically significant when compared to the control or the cisplatin group, respectively, P < 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test. (B) Representative photomicrographs of Ehrlich ascites carcinoma solid tumor sections taken from the different treatment group and stained by H&E. T: intact structure of tumor cells. n: necrosis of tumor cells. a: apoptosis of tumor cells. (C) Representative photomicrographs of H&E-stained kidney sections taken from the different groups in Ehrlich ascites carcinoma solid tumor model in mice. A: Untreated mouse bearing Ehrlich ascites carcinoma solid tumor showing normal glomeruli (g) and tubules (t) at the renal cortex. B: Ehrlich ascites carcinoma solid tumor-bearing mouse injected with cisplatin (5 mg/kg) showing degeneration together with focal aggregation of lymphoid cells (red arrow) surrounding the congested blood vessel (v). (C) Ehrlich ascites carcinoma solid tumor-bearing mouse treated with cisplatin and indole-3-carbinol showing degeneration (d) in the epithelial lining of the tubules (t). (D) Ehrlich ascites carcinoma solid tumor-bearing mouse treated with indole-3-carbinol alone showing normal glomeruli (g) and tubules (t).