Julie A. Davis

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

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.

Role of increased cytosolic free calcium in the pathogenesis of rabbit proximal tubule cell injury and protection by glycine or acidosis.

To assess the role of increased cytosolic free calcium (Caf) in the pathogenesis of acute proximal tubule cell injury and the protection afforded by exposure to reduced medium pH or treatment with glycine, fura-2-loaded tubules were studied in suspension and singly in a superfusion system. The Ca2+ ionophore, ionomycin, increased Caf to micromolar levels and rapidly produced lethal cell injury as indicated by loss of lactate dehydrogenase to the medium by suspended tubules and accelerated leak of fura and failure to exclude Trypan blue by superfused tubules. Decreasing medium Ca2+ to 100 nM prevented the ionomycin-induced increases of Caf and the injury. Reducing medium pH from 7.4 to 6.9 or adding 2 mM glycine to the medium also prevented the cell death, but did not prevent the increase of Caf to micromolar levels. Cells treated with 1799, an uncoupler of oxidative phosphorylation which produced severe adenosine triphosphate (ATP) depletion, did not develop increases of Caf until just before loss of viability. Preventing these increases of Caf with 100 nM Ca2+ medium did not protect 1799-treated cells. Reduced pH and glycine protected 1799-treated cells without ameliorating the increases of Caf. These data demonstrate the toxic potential of increased Caf in the proximal tubule and show that Caf does sharply increase prior to loss of viability in an ATP depletion model of injury, but this increase does not necessarily contribute to the outcome. The potent protective actions of decreased pH and glycine allow the cells to sustain increases of Caf to micromolar levels in spite of severe, accompanying cellular ATP depletion without developing lethal cell injury.

Structural requirements for protection by small amino acids against hypoxic injury in kidney proximal tubules.

Kidney proximal tubules are resistant to hypoxic injury if glycine or L‐alanine is present in their incubation medium. Protection does not depend on the concentration or turnover of ATP in cells. We have investigated structure‐function relationships that govern this protective activity. Among more than 45 amino acids and analogs examined, only glycine, L‐alanine, d‐alanine, β‐alanine, and the neuronal glycine binding site agonist, 1‐aminocyclopropane‐1‐carboxylic acid, were active. The protective effect could not be explained by amino acid metabolism. Ultrastructural features in protected cells were preserved to a degree which suggested that processes responsible for degradation during hypoxia were retarded. These results are consistent with stringent requirements of amino acid molecular structure for protection against hypoxia, and suggest the involvement of highly specific, acceptor‐ligand effects on a process critical for maintaining cellular integrity.—Weinberg, J. M.; Venkatachalam, M. A.; Garza‐Quintero, R.; Roeser, N. F.; Davis, J. A. Structural requirements for protection by small amino acids against hypoxic injury in kidney proximal tubules. FASEB J. 4: 3347–3354; 1990.

Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.

Previous studies have shown that cytosolic-free Ca2+ (Caf) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Caf increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lytic plasma membrane damage, Caf measured with mag-fura-2 exceeded 10 microM in 91% of tubules studied and 68% had increases to greater than 100 microM. Caf increases of similar magnitude that were dependent on influx of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2+-insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca2+ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Caf during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and Ca2+-mediated structural damage, the remarkable elevations of Caf demonstrated here suggest an unexpected resistance to the deleterious effects of increased Caf during energy deprivation in the presence of glycine.

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.

Inner ear changes in the ferret model for Reye's syndrome.

The acute effects of influenza B, aspirin, and hyperammonemia on the inner ear were examined using the ferret model for Reyes syndrome. Histopathologic examination revealed varying degrees of vacuolization in both the sensory and nonsensory endolymphatic tissues of the cochlear and vestibular membranous labyrinth. The secretory epithelial cells of the stria vascularis and the vestibular dark cells appeared to be more severely affected, as demonstrated by the presence of an extensive number of intracytoplasmic vacuoles. The cells of the perilymphatic tissues appeared to be unaltered. Although all neuroepithelial regions of the inner ear appeared to be altered, an unexpected observation was that only the inner hair cells of the organ of Corti were pathologically affected. These results suggest that metabolic and virus-induced derangements may alter inner ear microhomeostasis in patients with Reyes syndrome and may potentially result in loss of hearing in such patients.

Inner ear pathology associated with Reye's syndrome

Severe pathological changes were observed in the inner ear tissues of a 2-month-old patient who died of Reyes syndrome after 5 days of hospitalization. In the organ of Corti, the inner hair cells appeared to be more severely damaged than the outer hair cells. Various degrees of degeneration were observed in all non-sensory epithelial cells lining the cochlear duct. In most turns of the cochlear duct, Reissners membrane was ruptured and/or collapsed onto the organ of Corti. Likewise, both sensory and non-sensory cells of the vestibular end organs were markedly degenerated. These observations suggest that the inner ear tissues are acutely affected in patients with Reyes syndrome, and that the changes may cause impairment of hearing and/or equilibrium in patients who recover.

Effects of Influenza Infection, Aspirin, and an Arginine-Deficient Diet on the Inner Ear in Reye's Syndrome

The individual effects of an influenza B viral infection, aspirin, and an arginine-deficient diet on the inner ear were assessed in the ferret model for Reyes syndrome using both functional and morphological parameters. Auditory brainstem evoked responses recorded from inoculated ferrets revealed threshold elevations and increased latencies during the first 72 hours, but approximated those of control animals by 96 hours. Although there was a mild distention of Reissners membrane, no pronounced structural alterations in sensory or supporting cells were observed in cochleas from inoculated ferrets. The administration of aspirin appeared to alter neither the functional nor the structural integrity of the cochlea. The presentation of an arginine-deficient diet, creating a hyperammonemic condition, led to both altered auditory evoked responses and vacuolization of cochlear tissues after treated animals had undergone seizures and coma. These data demonstrated that both influenza B and the arginine-deficient diet individually affected the hearing of treated animals. The individual agents did not alter the cochlea as severely as when they were presented in combination. These results suggest that hearing impairment in patients with Reyes syndrome may be a result of potentiation of certain metabolic-altering agents.