Role of Caspases in Hypoxia-Induced Necrosis of Rat Renal Proximal Tubules

Effect of the specific caspase inhibitor Z-Asp-2,6-dichloro-benzoyloxymethylketone (Z-D-DCB) on caspase activity (top panel) and lactate dehydrogenase (LDH) release (bottom panel) after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the caspase inhibitor Z-D-DCB for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of both LDH release and caspase activity. Caspase activity was determined using the fluorescent substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.01 versus normoxia; ^***P < 0.01 versus hypoxia; ^****P < 0.001 versus hypoxia (n = 5).

Effect of the specific caspase inhibitor Z-D-DCB on calpain activity after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the caspase inhibitor Z-D-DCB for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of calpain activity using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-AMC, as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.001 versus hypoxia (n = 4).

Effect of the specific caspase inhibitor Z-D-DCB and the specific calpain inhibitors PD150606 and calpain inhibitor 1 (CI1) on the activity of purified calpain in vitro. Purified μ-calpain (20 μg) was preincubated with the inhibitors or the vehicle in the calpain assay buffer in the presence of 5 mM CaCl₂ for 10 min at 37°C. The fluorescent substrate N-succinyl-Leu-Tyr-AMC (final concentration, 50 μM), was then added. After an additional 30-min incubation, calpain activity was determined as described in Materials and Methods. ^*P < 0.001 versus purified calpain alone (n = 4).

Effect of the specific calpain inhibitor PD150606 (PD) on caspase activity after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the calpain inhibitor PD150606 for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of caspase activity using the fluorescent substrate Ac-YVAD-AMC, as described in Materials and Methods. ^*P < 0.01 versus normoxia; ^**P < 0.01 versus hypoxia (n = 5).

Effect of the calpain inhibitors PD150606 and calpain inhibitor 1 (CI1) on the activity of purified caspase 1 in vitro. Purified caspase 1 (57 ng) was preincubated with the inhibitors or the vehicle for the inhibitors. The fluorescent substrate Ac-YVAD-AMC was then added. After an additional 30-min incubation, caspase activity was determined as described in Materials and Methods. The caspase inhibitor Z-D-DCB was used as a positive control. ^*P < 0.001 versus purified caspase alone (n = 3).

Effect of the combined caspase and calpain inhibition on LDH release after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with either the caspase inhibitor Z-D-DCB (100 μM) or the calpain inhibitor PD150606 (100 μM), or a combination of both Z-D-DCB (100 μM) and PD150606 (100 μM) for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of LDH release as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.05 versus hypoxia; ^***P < 0.01 versus hypoxia; ^****P < 0.001 versus hypoxia (n = 5).

Caspase activity was measured in proximal tubules using the fluorescent substrate Ac-DEVD-AMC (DEVD) in addition to Ac-YVAD-AMC (YVAD). The caspase assay was performed as described in Materials and Methods. Both total fluorescence and Z-D-DCB-inhibitable fluorescence (caspase activity) was measured in normoxic (N) and hypoxic (H) tubules. The fluorescent substrate Ac-YVAD-AMC (YVAD) is preferentially cleaved by caspases 1, 4, and 5, and Ac-DEVD-AMC (DEVD) is cleaved by caspases 2, 3, and 7 and CED-3. ^**P < 0.01 versus normoxia (n = 4).