Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity

Abstract

Chemotherapy drug-induced nephrotoxicity limits clinical applications for treating cancers. Pyroptosis, a newly discovered programmed cell death, was recently reported to be associated with kidney diseases. However, the role of pyroptosis in chemotherapeutic drug-induced nephrotoxicity has not been fully clarified. Herein, we demonstrate that the chemotherapeutic drug cisplatin or doxorubicin, induces the cleavage of gasdermin E (GSDME) in cultured human renal tubular epithelial cells, in a time- and concentration-dependent manner. Morphologically, cisplatin- or doxorubicin-treated renal tubular epithelial cells exhibit large bubbles emerging from the cell membrane. Furthermore, activation of caspase 3, not caspase 9, is associated with GSDME cleavage in cisplatin- or doxorubicin-treated renal tubular epithelial cells. Meanwhile, silencing GSDME alleviates cisplatin- or doxorubicin-induced HK-2 cell pyroptosis by increasing cell viability and decreasing LDH release. In addition, treatment with Ac-DMLD-CMK, a polypeptide targeting mouse caspase 3-Gsdme signaling, inhibits caspase 3 and Gsdme activation, alleviates the deterioration of kidney function, attenuates renal tubular epithelial cell injury, and reduces inflammatory cytokine secretion in vivo. Specifically, GSDME cleavage depends on ERK and JNK signaling. NAC, a reactive oxygen species (ROS) inhibitor, reduces GSDME cleavage through JNK signaling in human renal tubular epithelial cells. Thus, we speculate that renal tubular epithelial cell pyroptosis induced by chemotherapy drugs is mediated by ROS-JNK-caspase 3-GSDME signaling, implying that therapies targeting GSDME may prove efficacious in overcoming chemotherapeutic drug-induced nephrotoxicity.

Fig. 1. Cisplatin or doxorubicin induces cleavage of GSDME in renal tubular epithelial cells in vitro and in vivo.

A, B Western blot analysis of GSDME in HK-2 cells treated with various concentrations of cisplatin (0, 5, 10, and 20 μM) for 48 h, and with 20 μM cisplatin for different times (0, 3, 6, 12, 24, and 48 h). C, D Western blot analysis of GSDME in HK-2 cells incubated with various concentrations of doxorubicin (0, 1, 2, and 4 μg/ml) for 48 h and 4 μg/ml doxorubicin for different times (0, 3, 6, 12, 24, and 48 h). E, F Serum creatinine and BUN detection of normal and cisplatin-treated mice after 72 h. G Hematoxylin–Eosin (HE) staining of control and cisplatin-treated mice. Scale bar, 50 μm. H–J Western blot analysis of Gsdme in mice and caspase 3 in control and cisplatin-treated mice. All data are presented as means ± SD from at least three independent experiments (n = 3 for in vitro experiment; n = 6 for in vivo experiment). ***p < 0.001 versus control group, ****p < 0.0001 using two-tailed Student’s t tests.

Fig. 2. Z-DEVD-FMK decreases cisplatin- or doxorubicin-induced pyroptosis in HK-2 cells.

A, E Representative light microscopy images of HK-2 cells treated with cisplatin (20 μM) or doxorubicin (doxorubicin, 4 μg/ml) before or after Z-DEVD-FMK (100 μM) intervention. The red arrow shows bubbles emerging from the plasma membrane. Scale bar, 50 μm. Cytotoxicity and cell viability were detected using the LDH assay (B, F) and CCK-8 detection (C, G) in HK-2 cells induced by cisplatin (20 μM) or doxorubicin (4 μg/ml) in the presence or absence of Z-DEVD-FMK (100 μM). Western blot analysis of GSDME and caspase 3 (CASP 3) cleavage in cisplatin-treated (20 μM) (D) and doxorubicin-treated (4 μg/ml) (H) HK-2 cells in the presence or absence of Z-DEVD-FMK (100 μM). All data are presented as mean ± SD from three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 3. Caspase 3, rather than caspase 9, contributes to cisplatin- or doxorubicin-induced pyroptosis in HK-2 cells.

A, E Representative light microscopy images of HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) in the presence or absence of CASP 3 or CASP 9 siRNA. A red arrow indicates bubbles emerging from the plasma membrane. Scale bar, 50 μm. Cytotoxicity and cell viability were determined using LDH assay (B, F) and CCK-8 detection (C, G) for HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) in the presence or absence of CASP 3 or CASP 9 siRNA. D, H Western blot analysis of cleavage of GSDME in cisplatin- (20 μM) or doxorubicin- (4 μg/ml) treated HK-2 cells in the presence or absence of CASP 3 or CASP 9 siRNA. All data are presented as mean ± SD from three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 4. GSDME knockout alleviates cisplatin- and doxorubicin-induced pyroptosis in HK-2 cells.

A Western blot analysis of GSDME expression in HK-2 cells treated with px459-GSDME-KO plasmid. B, C Percentage of PI⁺ HK-2 cells were detected by flow cytometry in the normal group (NC) and GSDME knockout group (GSDME-KO) treated with cisplatin (20 μM) or doxorubicin (4 μg/ml). D, G Representative light microscopy images of HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) in NC and GSDME-KO group. The red arrow indicates bubbles emerging from the plasma membrane. Scale bar, 50 μm. Cytotoxicity and cell viability were determined using the LDH assay (E, H) and CCK-8 detection (F, I) in NC and GSDME-KO HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml). J, K Western blot analysis of cleavage of GSDME and caspase 3 in cisplatin- (20 μM) or doxorubicin- (4 μg/ml) treated HK-2 cells in NC and GSDME-KO group. All data are presented as mean ± SD from three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 5. Ac-DMLD-CMK decreases cisplatin-induced renal pyroptosis in vivo.

A, B Serum creatinine and BUN detected in cisplatin-treated mice before or after Ac-DMLD-CMK pre-treatment. C HE staining of cisplatin-treated mice before or after Ac-DMLD-CMK pre-treatment. Scale bar, 50 μm. D–F Western blot analysis of GSDME and caspase 3 in cisplatin-treated mice before, or after, Ac-DMLD-CMK pretreatment. G, H mRNA expression of kidney injury-related genes and inflammatory-related genes in cisplatin-treated mice before, or after, Ac-DMLD-CMK pretreatment. All data are shown as means ± SD from six independent experiments (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 6. GSDME cleavage is induced by ERK and JNK signaling in HK-2 cells.

A, F Western blot analysis of ERK and JNK phosphorylation in HK-2 cells incubated with cisplatin (20 μM) or doxorubicin (4 μg/ml) for 3 h. B, G Western blot analysis of the cleavage of GSDME and caspase 3 in cisplatin- (20 μM) or doxorubicin- (4 μg/ml) treated HK-2 cells for 48 h with or without pretreatment of U0126 (10 μM) and SP600125 (10 μM). Cytotoxicity and cell viability were determined using the LDH assay (C, H) and CCK-8 detection (D, I) in HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) for 48 h with or without pre-treatment of U0126 (10 μM) and SP600125 (10 μM). E, J Representative light microscopy images of HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) for 48 h with or without pre-treatment of U0126 (10 μM) and SP600125 (10 μM). The red arrow indicates bubbles emerging from the plasma membrane. Scale bar, 50 μm. All data are presented as mean ± SD from three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 7. U0126 and SP600125 alleviate cisplatin-induced renal pyroptosis in vivo.

A, B Western blot analysis of ERK and JNK in kidney tissues from different groups. C, D Serum creatinine and BUN detected in cisplatin-treated mice before or after U0126 and SP600125 pretreatment. E, F mRNA expression of renal injury-related genes in kidney tissues from different groups. G Representative images of HE staining in kidney tissues from different groups. Scale bar, 50 μm. H–J Western blot analysis of GSDME and caspase 3 in cisplatin-treated mice before, or after, Ac-DMLD-CMK pretreatment. All data are presented as mean ± SD from six independent experiments (n = 6). **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.

Fig. 8. ROS induce cleavage of GSDME through JNK signaling in HK-2 cells.

A, F Representative light microscopy images of HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) with or without pretreatment of N-acetylcysteine (NAC, 5 mM) for 48 h. The red arrow indicates bubbles emerging from the plasma membrane. Scale bar, 50 μm. Cell viability and cytotoxicity were detected using the LDH (B, G) and CCK-8 detection (C, H) in HK-2 cells. D, I The ROS level of cisplatin (20 μM) or doxorubicin (4 μg/ml) treated HK-2 cells for 48 h in the presence or absence of NAC (5 mM). E, J Western blot analysis of cleavage of GSDME and caspase 3 in cisplatin- (20 μM) or doxorubicin- (4 μg/ml) treated HK-2 cells for 48 h with or without pretreatment of NAC (5 mM). K–N Western blot analysis of ERK and JNK phosphorylation in HK-2 cells treated with cisplatin (20 μM) or doxorubicin (4 μg/ml) for 3 h in the presence or absence of NAC (5 mM). All data are presented as mean ± SD from three independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA followed by the Tukey’s method.