Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules.

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.

Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.

Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.

Glycine-treated, hypoxic, proximal tubules developed a progressive energetic defect that resulted in failure to restore ATP levels to greater than 10 to 20% of control values during reoxygenation after 60 minutes of hypoxia despite continued cytoprotection by glycine. The defect was not corrected by supplementation with exogenous purines and was not modified by lowering the pH during hypoxia or reoxygenation. In the continued presence of glycine, the failure to restore ATP was associated with impaired recovery of structural changes that developed during hypoxia and, if glycine was withdrawn, lethal membrane damage occurred. The lesion was significantly ameliorated by the presence during hypoxia of two agents known to suppress development of the mitochondrial permeability transition, cyclosporine A and butacaine, which were most effective when used in combination. The data suggest that development of the mitochondrial permeability transition in glycine-protected tubules during hypoxia contributes to continued metabolic and structural impairment and cell death that occur despite glycine replete conditions such as exist frequently during in vivo insults and may be a target for therapeutic maneuvers.

Differential cytoprotection by glycine against oxidant damage to proximal tubule cells.

Tert-butyl hydroperoxide (tBHP) injured freshly isolated proximal tubules in an Fe-dependent fashion that was ameliorated by a lipophilic antioxidant, diphenyl-p-phenylenediamine (DPPD), but was only minimally affected by glycine. Menadione-induced injury was Fe-independent and was unaffected by DPPD, but was strongly blocked by glycine. Fe was highly toxic when intracellular loading was facilitated by concomitant treatment with hydroxyquinoline (HQ). This toxicity was blocked by DPPD or chelating the Fe, but not by glycine. All of the lesions were characterized by severe depletion of glutathione and other soluble thiols. Menadione induced large increases in protein associated with the Triton-insoluble cytoskeleton and decreases in protein thiol content, consistent with extensive cross linking, but did not increase thiobarbituric acid reactive substances (TBARS). tBHP and HQ + Fe had either no effect or only moderate, delayed effects on cytoskeletal proteins, but induced substantial increases of TBARS. Glycine did not the alter changes in cytoskeletal proteins, thiols, or TBARS produced by any of the agents. Protection against tBHP toxicity by deferoxamine and DPPD was accompanied by substantial suppression of TBARS accumulation. Superimposition of hypoxia during tBHP exposure reduced TBARS accumulation and restored cytoprotective activity to glycine. Thus, in contrast to its consistently strong cytoprotection against a number of other insults, glycine is only variably cytoprotective against oxidant lesions in freshly isolated proximal tubules. Extensive oxidative crosslinking of proteins is compatible with maintenance of glycine cytoprotection against lethal membrane damage. Fe-induced injury to proximal tubules associated with lipid peroxidation as manifested by TBARS formation is a relatively glycine-insensitive insult.

Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.

To better define the role of Ca2+ in pathophysiological alterations of the proximal tubule microvillus actin cytoskeleton, we studied freshly isolated tubules in which intracellular free Ca2+ was equilibrated with highly buffered, precisely defined medium Ca2+ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage and resulting nonspecific changes. Increases of Ca2+ to > or = 10 microM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca(2+)-dependent, villin-mediated actin cytoskeletal disruption in tubule cell microvillar damage under conditions conceivably present during pathophysiological states. However, despite prior evidence for cytosolic free Ca2+ increases of the same order of magnitude and similar structural microvillar alterations, Ca(2+)- and villin-mediated events did not appear to account for the initial microvillar damage that occurs during ATP depletion induced by antimycin alone or hypoxia.

Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury.

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.

Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells.

We have examined the dependence of unesterified fatty acid accumulation by intact, freshly isolated proximal tubules on Ca2+, pH, and the cytoprotective amino acid, glycine, during injury induced by hypoxia, antimycin, or antimycin plus ionomycin. In the absence of glycine, similarly high levels of fatty acid accumulation were seen during all three injury conditions irrespective of whether tubules were incubated in normal 1.25 mM Ca2+ medium or in medium where Ca2+ was buffered to 0.1 microM, a maneuver which prevented injury-associated increase of cytosolic-free Ca2+ as measured with fura 2. In the presence of glycine, which strongly suppressed development of lethal membrane damage for at least 60 min and did not have any apparent direct effects on fatty acid accumulation, both Ca(2+)-independent and Ca(2+)-dependent components of fatty acid accumulation were discernible. The Ca(2+)-independent component accounted for approximately 2/3 of fatty acid accumulation and did not vary as Ca2+ ranged from 10 nM to 1 microM. Unequivocal Ca(2+)-dependent accumulation occurred when Ca2+ exceeded 10 microM. Lowering pH to 6.9 had a moderate, generalized suppressive effect on fatty acid accumulation, including the major Ca(2+)-independent component, irrespective of the presence of glycine. These data emphasize the role of Ca(2+)-independent fatty acid accumulation during proximal tubule cell injury, clarify the modulatory actions of the potent, intrinsic cytoprotective factors, glycine and reduced pH, and provide insight into the relationship between fatty acid accumulation and lethal membrane damage.

Role of intracellular pH during cytoprotection of proximal tubule cells by glycine or acidosis.

Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca(2+)-induced injury, but there is little information about alterations of intracellular pH (pHi) in renal tubules during either injury or its modification by decreasing medium pHi or other potent protective factors such as glycine. pHi was assessed with 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein during proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytosolic free Ca2+ induced by treatment with the calcium ionophore ionomycin, alone and in combination with antimycin. Freshly isolated rabbit proximal tubules studied under superfusion conditions in the presence of probenecid were suitable for monitoring pHi during relatively prolonged and severe injury states. Probenecid, used to promote the retention of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pHi during simple ATP depletion, but pHi fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence of antimycin, ionomycin induced intracellular alkalinization in Ca(2+)-replete medium, which may have further enhanced the severity of injury. When medium Ca2+ was buffered to 100 nM, ionomycin induced intracellular acidification, which likely resulted from a combination of Ca2+/H+ exchange activity of the ionophore and H+ uptake during Ca(2+)-ATPase-mediated extrusion of Ca2+ released by ionomycin from intracellular pools. Alterations of pHi did not contribute to glycine cytoprotection because glycine did not affect the behavior of pHi during treatment with antimycin, ionomycin, or both agents in combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of human umbilical vein endothelial cells by glycine and structurally similar amino acids against calcium and hydrogen peroxide-induced lethal cell injury.

Cultured human umbilical vein endothelial cells treated with either the calcium ionophore, ionomycin, or ionomycin plus cyanide-m-chlorophenylhydrazone had immediate severe depletion of adenosine triphosphate, (ATP) and increases of cytosolic free calcium (Caf) and then sustained lethal cell injury as manifested by release of lactate dehydrogenase and failure to exclude vital dyes within 15 minutes. Inclusion of glycine in the experimental medium prevented the enzyme leakage for at least 60 minutes without altering the ATP depletion or increases of Caf. The physiologic glycine concentration of 0.25 mmol/l gave 50% protection, and protection was complete at 1 mmol/l. Several other small neutral amino acids, L- and D-alanine, beta-alanine, 1-aminocyclopropane-1-carboxylate, alpha-aminoisobutyrate, and L-serine, had effects similar to glycine, but other amino acids and metabolic substrates did not. The endothelial cells were relatively resistant to damage from hydrogen peroxide, but sensitivity could be increased by preloading with Fe2+. In both non-loaded and Fe(2+)-loaded cells, hydrogen-peroxide-induced lactate dehydrogenase (LDH) release developing over 180 minutes was prevented by glycine in a fashion analogous to that seen with ionomycin damage. Mn2+ also partially protected against hydrogen peroxide injury but was not required for glycine's effects. These data demonstrate that striking modulatory effects of glycine and structurally similar amino acids that have previously been characterized in most detail using kidney tubule cells are strongly expressed in human umbilical vein endothelial cells and are involved in their response to Ca2+ and oxidant-mediated damage. These amino acid effects must be considered in the design of in vitro studies of endothelial cell injury and may contribute to endothelial cell pathophysiology in vivo.

Amino acid protection of cultured kidney tubule cells against calcium ionophore-induced lethal cell injury.

Treatment of two cultured renal tubule epithelial cell lines, MDCK and LLC-PK1, with ionomycin produced rapidly evolving models of lethal cell injury characterized by increases of cytosolic free calcium to the microM level within 15 minutes followed by lactate dehydrogenase release and failure to exclude vital dyes that began between 30 and 60 minutes and became extensive after 60 minutes. The pattern of injury was similar when the mitochondrial uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, was added to ionomycin. Carbonyl cyanide-m-chlorophenylhydrazone alone produced severe ATP depletion but not lactate dehydrogenase release. Inclusion of glycine in the experimental medium at concentrations ranging from 0.25 mM to 5 mM did not affect the increases of cytosolic free calcium or ATP depletion but was protective against enzyme release and failure to exclude vital dyes for 180 minutes. Maximal protection was achieved at glycine concentrations between 1 and 5 mM. Several other small neutral amino acids including alanine, beta-alanine, L-serine, 1-aminocyclopropane-1-carboxylic acid, and alpha-aminoisobutyric acid also had protective effects but, glucose, pyruvate, glutamate, glutamine, leucine, valine, and taurine did not. These data indicate that potent protective effects of glycine and other small neutral amino acids previously shown in fresh tubule preparations are fully expressed in cultured tubule cells of diverse origin when appropriate acute injury models are used and the protective effects are sustained for long durations. The suitability of cultured cell lines for prolonged exposure studies will provide a powerful way of further exploring mechanisms of these effects.

Relationships between intracellular amino acid levels and protection against injury to isolated proximal tubules.

Metabolism and cellular levels of glycine, alanine, and other relevant amino acids in proximal tubules were studied during models of acute injury and protection by glycine. Freeze-clamped, normal rabbit renal cortex was very rich in glycine (66.8 nmol/mg protein) and glutamate and also had substantial levels of taurine, alanine, glutamine, serine, and aspartate. Isolated proximal tubules were severely depleted of all these amino acids (glycine, 2.1 nmol/mg protein). During 37 degrees C incubation in presence of alanine, tubules recovered only glutamate to a level approximating that in vivo (38.8 nmol/mg protein, 15.2 mM). Glycine added to medium at levels ranging from 0.25 to 2 mM was actively concentrated four- to sixfold by tubule cells. Two millimolar glycine potently protected tubules from lethal cell injury induced by hypoxia, antimycin A, or ouabain. Glycine levels of injured tubules rapidly equilibrated with medium, irrespective of whether glycine was loaded by preincubation or was added concomitantly with the injury maneuver. Metabolism of glycine during protection, assessed by changes in total levels, gas chromatography-mass spectroscopy determination of the fate of [13C]glycine, and redistribution of label from [3H]glycine was minimal. The data suggest that glycine plays an essential, constitutive role in maintenance of tubule cell structural integrity independently of common metabolic pathways. Intracellular amino acid content is sufficiently labile for depletion of structurally essential amino acids to potentially occur in a variety of settings, but, even with severe ATP depletion or Na+ pump inhibition, supplemental glycine is readily available to intracellular sites of action.

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, beta-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.

Quantitative assessment of neuroprotection against NMDA-induced brain injury.

In immature rodent brain, unilateral intrastriatal injections of selected excitatory amino acid (EAA) receptor agonists, such as N-methyl-D-aspartate (NMDA), produce prominent ipsilateral forebrain lesions. In Postnatal Day (PND) 7 rats that receive a right intrastriatal injection of NMDA (25 nmol) and are sacrificed 5 days later, there is a considerable and consistent reduction in the weight of the injected cerebral hemisphere relative to that of the contralateral side (-28.5 +/- 1.9%, n = 6). In animals treated with specific NMDA receptor antagonists, the severity of NMDA-induced damage is markedly reduced. We have previously reported that the efficacy of potential neuroprotective drugs in limiting NMDA-induced lesions can be assessed quantitatively by comparison of hemisphere weights after a unilateral NMDA injection. In this study, we compared three quantitative methods to evaluate the severity of NMDA-induced brain injury and the degree of neuroprotection provided by NMDA receptor antagonists. We characterized the severity of brain injury resulting from intrastriatal injections of 1-50 nmol NMDA in PND 7 rats sacrificed on PND 12 by (i) comparison of cerebral hemisphere weights; (ii) assay of the activity of the cholinergic neuronal marker, choline acetyltransferase (ChAT) activity; and (iii) measurement of regional brain cross-sectional areas. The severity of the resulting brain injury as assessed by comparison of hemisphere weights increased linearly with the amount of NMDA injected into the striatum up to 25 nmol NMDA. The magnitude of injury was highly correlated with the degree of reduction in ChAT activity (r2 = 0.97).(ABSTRACT TRUNCATED AT 250 WORDS)