The role of the lysosomal
proteases cathepsins B and L and the
calcium-dependent cytosolic
protease calpain in
hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules,
cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high
lactate dehydrogenase (LDH) release into the medium during
hypoxia. These data in rat proximal tubules suggest that
cathepsins are not released from lysosomes and do not gain access to the medium during
hypoxia. An assay for
calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The
calcium ionophore ionomycin induced a dose-dependent increase in
calpain activity. This increase in
calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular
calpain activity increased significantly by 7.5 min of
hypoxia, before there was significant LDH release, and further increased during 20 min of
hypoxia. The
cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of
hypoxia and completely prevented the increase in
calpain activity during
hypoxia. The increase in
calpain activity during
hypoxia and the inhibitor studies with CBZ therefore supported a role for
calpain as a mediator of
hypoxia-induced proximal tubular injury.