Panduratin A Derivative Protects against Cisplatin-Induced Apoptosis of Renal Proximal Tubular Cells and Kidney Injury in Mice

Figures

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  Scheme 1. Semi-synthesis of DD-217.

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  Scheme 2. Semi-synthesis of DD-218 and DD-218.

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  Figure 1. Chemical structures of panduratin A and its derivatives.

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  Figure 2. Effects of panduratin A and its derivatives on the cytotoxicity of CDDP in human renal proximal tubular cells. (A) RPTEC/TERT1 cell viability was measured following treatment for 72 h with: 0.1% DMSO (control): 50 µM CDDP alone; with co-incubation of panduratin A (PanA), DD-217, DD-218, DD-219 each at concentration of 1, 5, and 10 µM. (B) Viability of RPTEC/TERT1 cells was measured following treatment for 72 h with: 0.1% DMSO (control), panduratin A, DD-217, DD-218, DD-219 each at concentration of 1, 2.5, 5, and 10 µM. (C) Apoptosis of RPTEC/TERT1 cells was measured following treatment for 48 h with: 0.1% DMSO (control); 5 µM DD-218; 5 µM DD-219; 50 µM CDDP; 50 µM CDDP combined with 5 µM DD-218 (CDDP+DD-218); 50 µM CDDP combined with 5 µM DD-219 (CDDP+DD-219). Percentage of cell viability and apoptosis is shown as mean ± SD from at least 3 independent experiments: * p < 0.05 compared to control; # p < 0.05 compared to CDDP treatment.

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  Figure 3. Effects of panduratin A (PanA), DD-218, and DD-219 on the cytotoxicity of CDDP on cancer cell lines. Viability of A549 cells and HCT116 cells was measured following treatment for 72 h with: 0.1% DMSO (control); 5 µM PanA; 5 µM DD-218; 5 µM DD-219; 50 µM CDDP; 50 µM CDDP in the presence of 5 µM PanA (CDDP+PanA); 5 µM DD-218 (CDDP+DD-218); 5 µM DD-219 (CDDP+DD-219). Percentage of cell viability is shown as mean + SD from at least 3 independent experiments: * p < 0.05 compared to control; # p < 0.05 compared to CDDP treatment; NS indicates non-significance compared to CDDP treatment.

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  Figure 4. (A) Effect of DD-218 on AMPK expression. RPTEC/TERT1 cells were incubated for 24 h with 0.1% DMSO and DD-218 (1, 2.5, and 5 µM). (B) Effect of AMPK inhibitor on the protective of panduratin A (PanA) and DD-218. RPTEC/TERT1 cells were incubated with: 0.1% DMSO (control); 10 µM compound (Comp-C); 5 µM PanA; 5 µM DD-218; 50 µM CDDP; combinations as indicated. (C) Effects of PanA and DD-218 on expression of p-ERK1/2, Bcl-2, and cleaved-caspase 3 and cleaved-caspase 7 in renal tubular cells. RPTEC/TERT1 cells were incubated for 24 h with: 0.1% DMSO (control); 5 µM PanA; 5 µM DD-218; 50 µM CDDP in the presence of 5 µM PanA (CDDP+PanA); 50 µM CDDP in the presence of 5 µM DD-218 (CDDP+DD-218). Values are shown as mean ± SD of at least 3 independent experiments: * p < 0.05 compared to control, ** p < 0.01; # p < 0.05 compared to CDDP treatment.

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  Figure 5. Effects of DD-218 on CDDP-induced intracellular ROS accumulation and mitochondria function in renal tubular cells. (A) Intracellular ROS levels of RPTEC/TERT1 cells were measured following treatment for 30 min with: 0.1% DMSO (control); 5 µM WR-1065; 5 µM DD-218; 50 µM CDDP; 50 µM CDDP combined with 5 µM WR-1065 (CDDP+WR-1065); 50 µM CDDP combined with 5 µM DD-218 (CDDP+DD-218); 1 mM H2O2; 1 mM H2O2 combined with 5 µM WR-1065. (B) RPTEC/TERT1 cell viability was measured following incubation for 72 h with: 0.1% DMSO (control); 5 µM WR-1065; µM DD-218; 50 µM CDDP; 50 µM CDDP combined with 5 µM WR-1065 (CDDP+WR-1065); 50 µM CDDP combined with 5 µM DD-218 (CDDP+DD-218). (C) Mitochondrial membrane potential was determined by measurement of red/green fluorescence ratio. Data are expressed as mean ± SD from at least 3 independent experiments: * p < 0.05 compared to control; # p < 0.05 compared to CDDP treatment.

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  Figure 6. Effects of DD-218 on OCT2 function in renal tubular cells. 3H-MPP+ uptake in RPTEC/TERT1 cells was measured following incubation for 10 min and 24 h with: 0.1% DMSO (control) or 5 µM DD-218. Percentage of 3H-MPP+ uptake relative to control is shown as mean ± SD from at least 3 independent experiments: NS indicates non-significance compared to control.

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  Figure 7. The protective effect of DD-218 on CDDP-induced nephrotoxicity. C57BL/6 mice were treated with: saline for 3 days (control) n = 5; 50 mg/kg/day of DD-218 for 6 days, n = 5; a single dose 20 mg/kg of CDDP n = 6; 50 mg/kg/day of DD-218 for 3 days followed by co-treatment of a single dose of 20 mg/kg of CDDP and 50 mg/kg/day of DD-218 for 3 days, n = 7. (A) Body weight of mice after treatments. (B) Serum creatinine. (C) Renal platinum accumulation. Values are expressed as means ± SD: * p < 0.05 compared to control; NS indicates non-significance compared to control; # p < 0.05 compared to CDDP treatment.

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  Figure 8. Protein expression of kidney injury markers. Representative blots (A) and quantitative renal expression of cleaved-caspase 3 (B), NGAL (C), and p-ERK1/2 (D). Mice were treated with saline for 72 h, n = 5; 50 mg/kg/day for 6 days of DD-218, n = 5; a single dose of 20 mg/kg of CDDP, n = 6; co-treatment with 50 mg/kg/day for 3 days of DD-218 followed by a single dose of 20 mg/kg of CDDP, n = 7. Values are expressed as means ± SD: * p < 0.05 compared to control; # p < 0.05 compared to CDDP treatment.