The role of the
caspases, a newly discovered group of
cysteine proteases, was investigated in a model of
hypoxia-induced necrotic injury of rat renal proximal tubules. An assay for
caspases in freshly isolated rat proximal tubules was developed. There was a 40% increase in tubular
caspase activity after 15 min of
hypoxia in association with increased cell membrane damage as indicated by a threefold increase in
lactate dehydrogenase release. The specific
caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (
Z-D-DCB) attenuated the increase in
caspase activity during 15 min of
hypoxia and markedly decreased
lactate dehydrogenase release in a dose-dependent manner. In the proximal tubules,
Z-D-DCB also inhibited the
hypoxia-induced increase in
calpain activity, another
cysteine protease. In contrast, when
Z-D-DCB was added to purified
calpain in vitro, there was no inhibition of
calpain activity. The
calpain inhibitor (2)-3-(4-iodophenyl)-2-mercapto-2-propenoic
acid (
PD150606) also inhibited the
hypoxia-induced increase in
caspase activity in proximal tubules, but did not inhibit the activity of purified
caspase 1 in vitro. In these experiments,
caspase activity was detected with the fluorescence substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl
coumarin (Ac-YVAD-AMC), which is preferentially cleaved by
caspase 1. However, minimal
caspase activity was detected with the fluorescence substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methyl
coumarin (
Ac-DEVD-AMC), which is cleaved by
caspases 2, 3, and 7. The present study in proximal tubules demonstrates that (1)
caspase inhibition protects against necrotic injury by inhibition of
hypoxia-induced
caspase activity; and (2)
caspase 1 may be the
caspase involved. Thus, although the role of
caspases in apoptotic cell death is well established, this study provides new evidence that
caspases contribute to necrotic cell death as well.