Pharmacological Inhibition of Caspase-1 Ameliorates Cisplatin-Induced Nephrotoxicity through Suppression of Apoptosis, Oxidative Stress, and Inflammation in Mice

Abstract

Caspase-1 is a proinflammatory caspase responsible for the proteolytic conversion of the precursor forms of interleukin-1β to its active form and plays an important role in the pathogenesis of various inflammatory diseases. It was reported that genetic deficiency of caspase-1 prevented cisplatin-induced nephrotoxicity. However, whether pharmacological inhibition of caspase-1 also has a preventive effect against cisplatin-induced kidney injury has not been evaluated. In this study, we examined the effect of Ac-YVAD-cmk, a potent caspase-1-specific inhibitor, on renal function and histology in cisplatin-treated mice and explored its underlying mechanisms. We found that administration of Ac-YVAD-cmk effectively attenuated cisplatin-induced renal dysfunction, as evidenced by reduced plasma levels of blood urea nitrogen and creatinine, and histological abnormalities, such as tubular cell death, dilatation, and cast formation. Administration of Ac-YVAD-cmk inhibited caspase-3 activation as well as caspase-1 activation and attenuated apoptotic cell death, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, in the kidneys of cisplatin-treated mice. Cisplatin-induced G2/M arrest of renal tubular cells was also reduced by caspase-1 inhibition. In addition, administration of Ac-YVAD-cmk reversed increased oxidative stress and depleted antioxidant capacity after cisplatin treatment. Moreover, increased macrophage accumulation and elevated expression of cytokines and chemokines were attenuated by caspase-1 inhibition. Taken together, these results suggest that caspase-1 inhibition by Ac-YVAD-cmk protects against cisplatin-induced nephrotoxicity through inhibition of renal tubular cell apoptosis, oxidative stress, and inflammatory responses. Our findings support the idea that caspase-1 may be a promising pharmacological target for the prevention of cisplatin-induced kidney injury.

Figure 1

Effects of Ac-YVAD-cmk on body weight, renal function, and renal histology in cisplatin-treated mice. Mice were injected intraperitoneally with 10 mg/kg AC-YYAD-cmk for 3 days, starting 1 h prior to a single dose of cisplatin (15 mg/kg). (a) Percent change in body weight. (b) Plasma blood urea nitrogen (BUN). (c) Plasma creatinine. (d) Representative images of hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining on kidney sections. Scale bar: 25 μm. (e) Tubular injury score. Con: control, CP: cisplatin, and CP + YVAD: cisplatin plus Ac-YVAD-cmk. n = 8 per group. All data are expressed as the mean ± SEM. ^∗∗∗P < 0.001 vs. Con. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. CP.

Figure 2

Effects of Ac-YVAD-cmk on cisplatin-induced apoptotic cell death in the kidneys. (a) Western blot analysis of the expression of cleaved caspase-1 and cleaved caspase-3 in the kidneys. Graphs show the results of quantitative analysis of cleaved caspase-1 (b) and cleaved caspase-3 (c). (d) Representative images of TUNEL staining on kidney sections. Nuclei were counterstained with DAPI. Scale bar: 50 μm. (e) The number of TUNEL-positive cells in 5 random fields for each kidney. (f) Representative immunofluorescence staining of Ki67 (green) and p-H3 (red) on kidney sections. Nuclei were counterstained with DAPI. Scale bar: 50 μm. (g) Percentage of cells in the G2/M phase among all proliferative (Ki67-positive) tubular epithelial cells. Con: control, CP: cisplatin, and CP + YVAD: cisplatin plus Ac-YVAD-cmk. n = 8 per group. All data are expressed as the mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. Con. ^#P < 0.05 and ^###P < 0.001 vs. CP.

Figure 3

Effects of Ac-YVAD-cmk on cisplatin-induced G2/M cell cycle arrest in the kidneys. (a) Representative immunofluorescence staining of Ki67 (green) and p-H3 (red) on kidney sections. Nuclei were counterstained with DAPI. Scale bar: 50 μm. (b) Percentage of cells in the G2/M phase among all proliferative (Ki67-positive) tubular epithelial cells. Con: control, CP: cisplatin, and CP + YVAD: cisplatin plus Ac-YVAD-cmk. n = 8 per group. All data are expressed as the mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. Con. ^#P < 0.05 and ^###P < 0.001 vs. CP.

Figure 4

Effects of Ac-YVAD-cmk on cisplatin-induced oxidative damage in the kidneys. Representative images of immunohistochemical staining using anti-4-HNE antibody in the cortex (a) and glomerulus (b). Scale bar: 25 μm. Graphs show the percentage of 4-HNE-stained area per field in the cortex (c) and glomerulus (d). (e) Level of malondialdehyde (MDA). (f) GSH/GSSG ratio. Real-time RT-PCR analysis of CYP2E1 (g), SOD2 (h), catalase (i), and glutathione synthetase (j) in the kidneys. Con: control, CP: cisplatin, and CP + YVAD: cisplatin plus Ac-YVAD-cmk. n = 8 per group. All data are expressed as the mean ± SEM. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. Con. ^#P < 0.05 and ^##P < 0.01 vs. CP.

Figure 5

Effects of Ac-YVAD-cmk on cisplatin-induced inflammatory responses. (a) Representative images of immunohistochemical staining using anti-Mac-2 antibody in the glomerulus. Black arrows indicate Mac-2 positive cells. Scale bar: 25 μm. (b) Percentage of Mac-2 positive cells per glomerulus. Real-time RT-PCR analysis of TNF-α (c), IL-6 (d), MCP-1 (e), and CXCL1 (f) in the kidneys. Con: control, CP: cisplatin, and CP + YVAD: cisplatin plus Ac-YVAD-cmk. n = 8 per group. All data are expressed as the mean ± SEM. ^∗∗∗P < 0.001 vs. Con. ^#P < 0.05 and ^##P < 0.01 vs. CP.