Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules.

Abstract

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.

Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

“…28,32,33 Tubules were suspended at 3.0 to 5.0 mg of tubule protein/ml in a 95% O 2 /5% CO 2 5.0 sodium butyrate, 3% dialyzed dextran (T-40, Pharmacia), and 2 glycine. Medium A was also supplemented with 0.5 mg/ml of bovine gelatin (75 bloom) to suppress aggregation of the isolated tubules during the prolonged experimental incubation periods.…”

“…This situation has improved with recognition that much of their sensitivity is because of the formation of pathological plasma membrane pores that can be blocked by glycine at physiological levels. 28,29 By suppressing this type of plasma membrane damage, replacement of glycine allows examination of specific injury mechanisms in vitro, uncomplicated by the plethora of postmortem degenerative changes that would otherwise occur in multiple cellular systems. 30 -32 We have found that, despite glycine cytoprotection, freshly isolated, kidney proximal tubule cells develop a profound …”

“…This situation has improved with recognition that much of their sensitivity is because of the formation of pathological plasma membrane pores that can be blocked by glycine at physiological levels. 28,29 By suppressing this type of plasma membrane damage, replacement of glycine allows examination of specific injury mechanisms in vitro, uncomplicated by the plethora of postmortem degenerative changes that would otherwise occur in multiple cellular systems. 30 -32 We have found that, despite glycine cytoprotection, freshly isolated, kidney proximal tubule cells develop a profound mitochondrial functional deficit during hypoxia/reoxygenation that is characterized by incomplete recovery of energization during reoxygenation, impaired respiration for substrates dependent on function of electron transport complex I, partial de-energization, and persistence of mitochondrial matrix condensation.…”

Immature Renal Tubules Are Resistant to Prolonged Anoxia

“…Recent investigations have indicated that the amino acid content of the suspension solution may have profound effects on the stability of a suspension of tubules and the severity of an injury to renal tubules (4,5). Moreover, the common buffer solutions used for tubules from mature rats are different from those used for immature tubules.…”

“…The standard buffered Ringer's solution used to study adult tubules contains lactate. alanine, and butyrate (5,6), and glutamine and malate are frequently added in studies of immature tubules (7). Therefore, we studied the stability of both mature and immature tubules in three buffer solutions.…”