Figures & data
Figure 1. Propidium iodide staining of nuclei in freshly isolated proximal tubule cells is increased by cisplatin and decreased by the caspase inhibitor. The cells were stained with color combination of DNA-specific dyes Hoechst 33342 and propidium iodide (PI). Vehicle-treated proximal tubule cells (Veh) demonstrated an intact structure; they excluded PI and stained mostly with Hoechst in blue color (A). Cells treated with 50 µM cisplatin (Cis50) showed extensive PI staining in red color, indicative of loss of membrane integrity and necrosis. Nuclear morphology was disrupted and those cells were more brightly stained indicative of chromatin condensation in globular or crescent-shaped figures (B). Cells treated with 50 µM cisplatin in combination with pan caspase inhibitor, 50 µM QVD-OPH (Cis50+QVD) demonstrated a normal morphology compared with the cells treated with 50 µM cisplatin only (C,D). (*p < 0.05 vs. Veh, **p < 0.05 vs. Cis50).
Figure 2. LDH release (%) from freshly isolated proximal tubule cells is increased by cisplatin and decreased by the caspase inhibitor. LDH release was under 10% in vehicle-treated proximal tubule cells (Veh). Cells treated with 50 µM cisplatin (Cis50) showed increased LDH release. Co-treatment of QVD-OPH (Cis50 + QVD) decreased LDH release significantly. (*p < 0.05 vs. Veh, **p < 0.05 vs. Cis50).
Figure 3. Caspase-1 activity and protein of freshly isolated proximal tubule cells treated with cisplatin is reduced by the caspase inhibitor. (A) Caspase-1 activity was increased significantly in 50 µM cisplatin-treated proximal tubule cells (Cis50) compared with vehicle-treated group (Veh) (*p < 0.05 vs. Veh). Co-treatment of QVD-OPH (Cis50 + QVD) decreased caspase-1 activity significantly (**p < 0.05 vs. Cis50). (B) Representative immunoblot of caspase-1 showed active caspase-1 (10 kDa) was increased significantly in Cis50 compared with Veh (#p < 0.05 vs. Veh). Cis50 + QVD decreased the expression of active caspase-1 (##p < 0.05 vs. Cis50 + Veh). Densitometry of caspase-1 immunoblots is shown.
Figure 4. Activation of caspase-1 substrates IL-1α and IL-1β in cisplatin-treated freshly isolated proximal tubule cells. (A) On immunoblot, NLRP3 was strongly expressed in PTs with no significant changes between groups on densitometry. (B) On immunoblot, BID (22 kDa) was unchanged between the groups. (C) IL-1α activity was significantly increased in Cis50 (*p < 0.05 vs. Veh), and was decreased significantly in combination with QVD-OPH (**p < 0.05 vs. Cis50). (D) IL-1β activity showed increases in Cis50 (#p < 0.05 vs. Veh), and was decreased significantly in combination with QVD-OPH (##p < 0.05 vs. Cis50).
