GLYCINE PROTECTION AGAINST HYPOXIC INJURY IN ISOLATED RAT PROXIMAL TUBULES - THE ROLE OF PROTEASES

Background. Isolated rat proximal tubules are frequently used as a mod el to study hypoxic injury. Glycine is a very effective protective age nt against hypoxia-induced cell injury in this model. The mechanisms i nvolved in hypoxic renal injury and glycine protection are still debat ed. We have focused on the role of proteolytic enzymes. Methods. Isola ted rat proximal tubules in suspension were gassed with either 95%O-2/ 5%CO2 or 95%N(2)5%CO2 to create normoxic or hypoxic conditions. Cell i njury was assessed by the release of LDH. Activity of proteolytic enzy mes was measured by quantifying the release of fluorescent 7-amino-4-m ethylcoumarin from specific substrates, which were added to tubules in suspension or to cytosolic fractions of permeabilized tubules. Result s. Fifteen minutes of hypoxia caused cell injury, which was completely prevented by glycine. Activities of serine-, aspartate; and the calci um-dependent cysteine protease calpain were increased in these hypoxic tubules in suspension, but only calpain activity was attenuated by gl ycine. Cytosolic fractions obtained by digitonin-permeabilization of h ypoxic (15 min) tubules showed increased proteolytic activity of all m easured classes of proteases and glycine prevented these increases. In measurements performed at an earlier time point (7.5 min) neither cha nges in calpain activity nor effects of glycine were detected. Calpain activity was not inhibited directly by glycine. Conclusions. Hypoxia increases the activity of several classes of proteases. The effects of glycine on protease activation are equivocal, and may merely reflect the potential of glycine to prevent hypoxia-induced lethal membrane in jury.