Roles for both the tripeptide, GSH, and individual amino acids in modifying the cellular response to oxygen deprivation-induced injury have been suggested by prior work in kidney and other tissues, but the precise interrelationships have not been clearly defined. We have studied the effects of GSH, its component amino acids, and related compounds on the behavior of isolated renal proximal tubules in a well characterized model of hypoxic injury in vitro. GSH, the combination of cysteine, glutamate, and glycine and glycine alone, when present in the medium during 30 min hypoxia, a duration sufficient to produce extensive irreversible injury in untreated tubules, were protective. Significant effects were detected at 0.25 mM concentrations of the reagents, and protection was nearly complete at concentrations of 1 mM and above. Glutamate and cysteine alone were not protective. The exogenous GSH added to the tubule suspensions was rapidly degraded to its component amino acids. Treatment of tubules with GSH or cysteine, but not glycine, increased intracellular GSH levels. Oxidized GSH was protective. Serine, N-(2-mercaptopropionyl)-glycine, and a panel of agents known to modify injury produced by reactive oxygen metabolites were without benefit. These observations identify a novel and potent action of glycine to modify the course of hypoxic renal tubular cell injury. This effect is independent of changes in cellular GSH metabolism and appears to be unrelated to alterations of cell thiols or reactive oxygen metabolites. Further elucidation of its mechanism may provide insight into both the basic pathophysiology of oxygen deprivation-induced cell injury and a practical way to ameliorate it.

The mechanisms responsible for the large increases of intracellular ATP levels seen after isolated rabbit proximal tubules are treated with exogenous adenine nucleotides were studied. Exogenous ATP was rapidly degraded via adenosine as far as hypoxanthine. Degradation of AMP to adenosine was substantially inhibited by beta-glycerol phosphate. In studies of the ability of individual exogenous purines to increase intracellular ATP levels, single large doses of adenosine were less effective than equimolar doses of exogenous ATP but were substantially more effective than exogenous inosine or hypoxanthine. Exogenous guanine derived compounds increased only cell GTP. Incremental delivery of smaller doses of adenosine to maintain medium levels greater than 5 microM or inhibition of adenosine deaminase with erythro-9-[3-(2-hydroxynonyl)]adenine or 2'-deoxycoformicin enhanced the nucleoside's effectiveness. However, the initial increase of cell ATP was still greater after treatment with exogenous ATP than after adenosine and, in the presence of adenosine deaminase inhibition, larger increases of cell ATP were produced by 50 microM adenosine than by 250 microM adenosine. These observations are most consistent with substrate inhibition of adenosine kinase by adenosine. Furthermore, the adenosine kinase inhibitor, 5-iodotubercidin, prevented the increases of cell ATP resulting from exogenous adenosine or exogenous ATP. These studies demonstrate how the differential uptake and utilization characteristics of nucleosides and bases can fully account for the increases of intracellular nucleotides produced in isolated tubules by exogenous purines.

We have determined whether glycine or glutathione can protect rabbit proximal tubules damaged by chemical inhibitors of oxidative phosphorylation: antimycin A, rotenone, cyanide, oligomycin, or carbonyl cyanide m-chlorophenylhdrazone (CCCP). All the agents severely depleted cell ATP levels within 15 min and caused lethal cell injury, as quantified by lactate dehydrogenase (LDH) release. Glycine and glutathione largely prevented this injury without altering the primary effects of the inhibitors on tubule respiration or the depletion of ATP. Buthionine sulfoximine and 1,3-bis(2-chloroethyl)-1-nitrosourea decreased cell glutathione but did not prevent the protective effects of either glycine or glutathione in tubules treated with rotenone. Protection was sustained during both a 15-min exposure and a 45-min postwash period irrespective of whether the wash removed the agent or mitochondrial function recovered. Cysteine uniquely induced a dramatic recovery of mitochondrial function in tubules washed after treatment with CCCP. These data 1) demonstrate that the cytoprotective effects of glycine previously seen during hypoxia extend to other tubule lesions characterized by severe ATP depletion, 2) emphasize the actions of glycine to preserve cell structural integrity in spite of sustained severe impairment of ATP-generating processes in proximal tubules, and 3) indicate that it is glycine rather than intracellular or extracellular glutathione which mediates protection.

Exposure to 1 mM ouabain for greater than 30 min caused lethal cell injury to isolated rabbit proximal tubules as measured by increased lactate dehydrogenase release. Addition of 2 mM glycine or glutathione to the incubation medium prevented this injury and a sharp fall of cell ATP that accompanied it. Glycine and glutathione did not alter rapid, early effects of ouabain to deplete cell K+ and inhibit respiration. Preservation of cellular glutathione was not required for protection. Glycine did not ameliorate ouabain-induced increases of cell water and did not prevent lethal cell injury associated with cell swelling produced by incubation in a high K+ concentration medium. In contrast, 100 mM mannitol, which at least partially ameliorated swelling in both ouabain and high-K+ medium, prevented lethal injury in high-K+ medium and decreased it in the presence of ouabain. The combination of glycine and mannitol completely prevented ouabain-induced lethal injury and cell water increases. These observations indicate that glycine, unlike mannitol, does not protect against primary volume-induced insults. Ouabain-induced lethal cell injury results from a process that includes both a volume component ameliorated by mannitol and a volume-independent component that is prevented by glycine and is closely associated with accelerated ATP depletion.

To clarify the roles of butyrate and acylglycine formation in hypoxic proximal tubule cell injury and protection by glycine and to test the contribution of iodoacetate-suppressible metabolism to protection, (1) it was determined whether protection by glycine is fully expressed when glucose, lactate, alanine, and butyrate are replaced by alpha-ketoglutarate as the sole substrate for the tubules, (2) butyrate metabolism and acylglycine formation were directly measured in control and hypoxic preparations, and (3) it was assessed whether injury produced by iodoacetate, a potent inhibitor of glycolytic metabolism, is subject to protection by glycine. Susceptibility to hypoxic injury in medium with alpha-ketoglutarate as the sole substrate was similar to that seen in medium containing glucose, lactate, alanine, and butyrate. Tubules in alpha-ketoglutarate medium showed high degrees of protection by glycine against injury produced by 30-min of hypoxia, by iodoacetate alone, and by iodoacetate combined with hypoxia. Protection did not require preservation of cell ATP or glutathione. In glucose-lactate-alanine-butyrate medium, butyrate, measured by gas chromatography, was rapidly metabolized by oxygenated tubules and fully accounted for basal rates of oxygen consumption. Butyrate utilization stopped during hypoxia. Neither aspect of butyrate metabolism was altered by glycine. Formation of acylglycines was assessed by gas chromatography/mass spectroscopy. In preparations treated with glycine, butyrylglycine was detected under both oxygenated and hypoxic conditions; the quantities, however, were small and no other acylglycines were found. These observations indicate that protective effects of glycine are independent of short-chain acylglycine formation and glycolytic metabolism.