The diminution and modulation role of water-soluble gallic acid-carboxymethyl chitosan conjugates against the induced nephrotoxicity with cisplatin

Abstract

The toxicity of cisplatin (CDDP) toward the renal tubules and its severe effects on the proximal tubules limits its further use in cancer therapy. The current study was undertaken to evaluate the protective effects of gallic acid-grafted O-carboxymethyl chitosan (GA@CMCS) against nephrotoxicity induced by CDDP in rats. Renal injury was assessed in the GA@CMCS/CDDP-treated rats using kidney injury molecule-1 (KIM-1). Moreover, the levels of reduced glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) were measured. The comet assay was performed to measure the DNA damage. The renoprotective activity of GA@CMCS was supported by histo- and immuno-pathological studies of the kidney. GA@CMCS significantly normalized the increases in kidney homogenate of KIM-1, MDA, and NO-induced by CDDP and significantly increased GSH as compared with the CDDP group. GA@CMCS also significantly protects rat kidneys from CDDP-induced histo- and immuno-pathological changes. Both biochemical findings and histo- and immuno-pathological evidence showed the renoprotective potential of GA@CMCS against CDDP-induced oxidative stress, inflammation, and renal dysfunction in rats. In conclusion, GA@CMCS has been shown to mitigate the nephrotoxicity impact of CDDP in cancer therapy.

Figure 1

Schematic diagram for the extraction of crab chitosan, its partial degradation, carboxymethylation, and grafting of GA for the preparation of GA@CMCS conjugate.

Figure 2

(A) Collective FTIR spectra of GA, LMWCS, CMCS, and the conjugate (GA@CMCS) for comparison of their respective characteristic vibration bands. (B) UV–Vis spectra of the GA, LMWCS, CMCS, and (GA@CMCS) in aqueous acetic acid (0.5%, v/v) (0.5 mg/mL).

Figure 3

The effect of different treatments in CDDP-induced nephrotoxicity on (A) kidney weight of rats (B) body weight change (%) of rats, and (C) renal index of rats in experimental groups. G1, Control; G2, GA; G3, CMCS; G4, GA@CMCS; G5, CDDP; G6, CDDP + GA; G7, CDDP + CMCS; G8, CDDP + GA@CMCS. Values are means ± SE (n = 8). Data were analyzed by a one-way ANOVA test followed by Tukey’s post hoc test for multiple comparisons. ^ap < 0.001 versus control group (G1), ^bp < 0.001 vs. GA (G2), ^cp < 0.001 vs. CMLMWC group (G3), ^d p < 0.001 vs. GA@CMCS group (G4). ^ep < 0.001 versus the CDDP group (G5), ^f p < 0.001 versus the (CDDP + GA) group (G6).

Figure 4

The effect of different treatments on KIM-1 levels in CDDP-induced nephrotoxicity in all the studied groups. Values are means ± SE (n = 8). Data were analyzed by a one-way ANOVA test followed by Tukey’s post hoc test for multiple comparisons. ^ap < 0.001 versus control group (G1), ^bp < 0.001 vs. GA (G2), ^cp < 0.001 vs. CMCS group (G3), ^dp < 0.001 vs. GA@CMCS group (G4). ^ep < 0.001 versus the CDDP group (G5), ^fp < 0.001 versus the (CDDP + GA) group (G6).

Figure 5

Comet assay showing the percentage of DNA damage in rats subjected to different treatments in CDDP-induced nephrotoxicity. (A) the control group showed a small percentage of DNA damage (2%—5%). (B) the GA group showed (6–15%) DNA damage. (C) the CMCS group showed (7–13%) DNA damage. (D) the GA@CMCS group showed (5–11%) DNA damage. (E) high DNA damage (70–80%) was observed in the CDDP group compared with the control group. (F) the (CDDP + GA) group revealed reduced DNA damage compared with the CDDP group (30–40%). (G) the (CDDP + CMCS) group revealed reduced DNA damage compared with the CDDP group (20–30%). (H) the (CDDP + GA@CMCS) group revealed reduced DNA damage compared with the CDDP group (27–30%).

Figure 6

Histological evaluations of kidney sections in all studied groups. The control group (A) showed a normal structure of the renal corpuscle (asterisk) and renal tubules, proximal convoluted tubules (yellow arrow), and distal convoluted tubules (arrowhead). Notice the glomerulus (asterisk), urinary space (red arrow), and Bowman's capsule (black arrow). The GA group (B), the CMCS group (C), and the GA@CMCS group (D) all show a normal structure of the renal corpuscle (asterisk) and renal tubules. The CDDP group (E) showed the cortical tubules have become shrunken and atrophic and have relatively expanded interstitial spaces. Fibrosis and atrophy of the glomerulus are seen. Notice the glomerular basement membrane is diffusely and fairly uniformly thickened. The (CDDP + GA) group (F), the (CDDP + CMCS) group (G), and the (CDDP + GA@CMCS) group (H) show the structure of the renal corpuscle (asterisk) and renal tubules appear more or less normal. (H & E stain, scale bar: 5 µm).

Figure 7

The effect of different treatments on COX-2 immunoreactivity and densitometry in CDDP-induced nephrotoxicity in all the studied groups. (A–H) COX-2 immunoreactivity evaluations in the kidney of rats. The control group (A), the GA group (B), the CMCS group (C), and the GA@CMCS group (D) showed a pale stain in the proximal tubular cells and a pale stain in the distal convoluted tubules and glomeruli. The CDDP group (E) showed intense COX-2 immunoreactivity in the proximal tubular cells as compared with the control. The (CDDP + GA) group (F), and the (CDDP + CMCS) group (G) showed moderately decreased COX-2 immunoreactivity in the proximal convoluted tubules as compared with the CDDP group. The (CDDP + GA@CMCS) group (H) showed a pale stain in the proximal convoluted tubules as compared with the CDDP group. (COX-2 expression, scale bar: 5 µm). All data have been presented as mean ± SE. **p < 0.01, and ***p < 0.001. (I) COX-2 densitometry immunohistochemistry expression analysis in the medulla in all studied groups.

Figure 8

The effect of different treatments on caspase-3 immunoreactivity and densitometry in CDDP-induced nephrotoxicity in all the studied groups. Caspase-3 immunoreactivity evaluations in the cortex (I) and the medulla (II) of the kidney of rats. (III) caspase-3 densitometry immunohistochemistry expression analysis in both cortex and medulla in all studied groups. Caspase-3 expression using immunohistochemical staining in kidney sections: (A) the control group, (B) the GA group, (C) the CMCS group, (D) the GA@CMCS group, (E) the CDDP group, (F) the (CDDP + GA) group, (G) the (CDDP + CMCS) group, and (H) the (CDDP + GA@CMCS) group. (Caspase-3 expression, scale bar: 50 um). All data have been presented as mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 9

The effect of different treatments on Na⁺/K⁺-ATPase immunoreactivity and densitometry in CDDP-induced nephrotoxicity in all the studied groups. (I) a micrograph of a section of the kidney showing Na⁺/K⁺-ATPase immunoreactivity in the cortex. (II) a micrograph of a section of the kidney showing Na⁺/K⁺-ATPase immunoreactivity in the medulla. (III) Na⁺/K⁺-ATPase densitometry immunohistochemistry protein expression analysis in both cortex and medulla. Micrographs of the kidney section of (A) the control group, (B) the GA group, (C) the CMCS group, (D) the GA@CMCS group, (E) the CDDP group, (F) the (CDDP + GA) group, (G) the (CDDP + CMCS) group, and (H) the (CDDP + GA@CMCS) group. (Na⁺/K⁺-ATPase expression, scale bar: 50 μm). All data have been presented as mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001.