Inhibitors Directed towards Caspase-1 and -3 Are Less Effective than Pan Caspase Inhibition in Preventing Renal Proximal Tubular Cell Apoptosis

Abstract

Background: Uncontrolled apoptosis contributes to tubular cell deletion in renal scarring. Caspase-3 has a central role in the execution of apoptosis and may provide a target for regulating cell death. Here we evaluate three caspase inhibitors: B-D-FMK (pan caspase inhibitor), Z-DEVDFMK (predominantly Caspase-3 inhibitor) and Z-VAD-FMK (predominantly Caspase-1 and -3 inhibitor) to ameliorate apoptosis induced by cisplatin in rat proximal tubular (RPT) cells. Methods: Caspase-3 activity (substrate cleavage assay) and protein (immunocytochemistry and Western blotting), apoptosis (Annexin V flow cytometry, in situend labelling of fragmented DNA, light/electron microscopy and DNA laddering) and cell survival (trypan blue exclusion and propidium iodide flow cytometry) were determined in RPT cells exposed to cisplatin with and without caspase inhibitors. Results: Cisplatin induced a dose-dependent increase in Caspase-3 activity and 8-fold of increase in apoptosis (p < 0.01) when applied for 24 h at 100 µM. B-D-FMK (40 µM), Z-DEVD-FMK (15 µM) and Z-VAD-FMK (22 µM) almost completely inhibited the 25-fold increase in Caspase-3 activity and decreased apoptosis from 15.9 ± 4.4 to 2.0 ± 0.6% (p < 0.01), 15.0 ± 2.2 and 15.0 ± 2.2% respectively. DNA ladders were visible in cisplatin-treated cells, which disappeared following addition of B-D-FMK and decreased with Z-VAD-FMK and Z-DEVD-FMK. Cisplatin reduced cell survival to 61% by trypan blue exclusion. B-D-FMK and Z-VAD-FMK increased this to 87 and 75%, but Z-DEVD-FMK had no significant effect. Conclusions: Cisplatin causes an increase in RPT apoptosis that is associated with increased Caspase-3 activity. All caspase inhibitors were equally effective at reducing Caspase-3 activity, however the pan caspase inhibitor B-D-FMK was more effective at preventing apoptosis and increasing cell survival. Therefore, pan caspase inhibition offers the greatest potential for the prevention of renal tubular cell deletion by uncontrolled apoptosis.