THE ROLE OF CYSTEINE PROTEASES IN HYPOXIA-INDUCED RAT RENAL PROXIMAL TUBULAR INJURY

The role of the lysosomal proteases cathepsins B and L and the calcium -dependent cytosolic protease calpain in hypoxia-induced renal proxima l tubular injury was investigated. As compared to normoxic tubules, ca thepsin B and L activity, evaluated by the specific fluorescent substr ate -phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was hot increas ed in hypoxic tubules or the medium used for incubation of hypoxic tub ules in spite of high lactate dehydrogenase (LDH) release into the med ium during hypoxia. These data in rat proximal tubules suggest that ca thepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated prox imal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amid o-4-methylcoumarin was developed. The calcium ionophore ionomycin indu ced a dose-dependent increase in calpain activity. This increase in ca lpain activity occurred prior to cell membrane damage as assessed by L DH release. Tubular calpain activity increased significantly by 7.5 mi n of hypoxia, before there was significant LDH release, and further in creased during 20 min of hypoxia. The cysteine protease inhibitor N-be nzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH rele ase after 20 min of hypoxia and completely prevented the increase in c alpain activity during hypoxia. The increase in calpain activity durin g hypoxia and the inhibitor studies with CBZ therefore supported a rol e for calpain as a mediator of hypoxia-induced proximal tubular injury .