Glycine protection against hypoxic injury in isolated rat proximal tubules : the role of proteases

Isolated rat proximal tubules are frequently used as a model to study hypoxic injury. Glycine is a very effective protective agent against hypoxia-induced cell injury in this model. The mechanisms involved in hypoxic renal injury and glycine protection are still debated. We have focused on the role of proteolytic enzymes. Isolated rat proximal tubules in suspension were gassed with either 95%O2/5%CO2 or 95%N2/5%CO2 to create normoxic or hypoxic conditions. Cell injury was assessed by the release of LDH. Activity of proteolytic enzymes was measured by quantifying the release of fluorescent 7-amino-4-methylcoumarin from specific substrates, which were added to tubules in suspension or to cytosolic fractions of permeabilized tubules. Fifteen minutes of hypoxia caused cell injury, which was completely prevented by glycine. Activities of serine-, aspartate-, and the calcium-dependent cysteine protease calpain were increased in these hypoxic tubules in suspension, but only calpain activity was attenuated by glycine. Cytosolic fractions obtained by digitonin-permeabilization of hypoxic (15 min) tubules showed increased proteolytic activity of all measured classes of proteases and glycine prevented these increases. In measurements performed at an earlier time point (7.5 min) neither changes in calpain activity nor effects of glycine were detected. Calpain activity was not inhibited directly by glycine. 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 injury.