Isoflurane alters proximal tubular cell susceptibility to toxic and hypoxic forms of attack.

BACKGROUND: Fluorinated anesthetics can profoundly alter plasma membrane structure and function, potentially impacting cell injury responses. Because major surgery often precipitates acute renal failure, this study assessed whether the most commonly used fluorinated anesthetic, isoflurane, alters tubular cell responses to toxic and hypoxic attack. METHODS: Mouse proximal tubule segments were incubated under control conditions or with a clinically relevant isoflurane dose. Cell viability (lactate dehydrogenase release), deacylation (fatty acid, such as C20:4 levels), and adenosine triphosphate (ATP) concentrations were assessed under one or more of the following conditions: (a) exogenous phospholipase A2 (PLA2) or C20:4 addition, (b) Ca2+ overload (A23187 ionophore), (c) increased metabolic work (Na ionophore), and (d) hypoxia- or antimycin A-induced attack. Isoflurane's effect on NBD phosphatidylserine uptake (an index of plasma membrane aminophospholipid translocase activity) was also assessed. RESULTS: Isoflurane alone caused trivial deacylation and no lactate dehydrogenase release. However, it strikingly sensitized to both PLA2- and A23187-induced deacylation and cell death. Isoflurane also exacerbated C20:4's direct membrane lytic effect. Under conditions of mild ATP depletion (Na ionophore-induced increased ATP consumption; PLA2-induced mitochondrial suppression), isoflurane provoked moderate/severe ATP reductions and cell death. Conversely, under conditions of maximal ATP depletion (hypoxia, antimycin), isoflurane conferred a modest cytoprotective effect. Isoflurane blocked aminophospholipid translocase activity, which normally maintains plasma membrane lipid asymmetry (that is, preventing its "flip flop"). CONCLUSIONS: Isoflurane profoundly and differentially affects tubular cell responses to toxic and hypoxic attack. Direct drug-induced alterations in lipid trafficking/plasma membrane orientation and in cell energy production are likely involved. Although the in vivo relevance of these findings remains unknown, they have potential implications for intraoperative renal tubular cell structure/function and how cells may respond to superimposed attack.

Ceramide accumulation during oxidant renal tubular injury: mechanisms and potential consequences.

UNLABELLED: Ceramide is an important signaling molecule that is typically generated via sphingomyelinase (SMase)-mediated sphingomyelin (SM) hydrolysis. Although diverse forms of renal injury elicit ceramide accumulation, the molecular determinants of this change and its contribution to tissue damage are poorly defined. The present study uses iron (Fe/hydroxyquinoline)-mediated injury of cultured human proximal tubular (HK-2) cells to gain additional insights into these issues. A 4-h Fe exposure doubled ceramide levels in the absence of cell death. This was independent of de novo synthesis, since ceramide synthase inhibition (with fumonisin B1) had no effect. Oxidant stress directly suppressed, rather than stimulated, SMase activity by: (1) decreasing SMase levels; (2) depleting SMase-stimulating glutathione; and (3) increasing SM resistance to SMase attack. Fe suppressed cell sphingosine levels (3 to 4 times ceramide/sphingosine ratio increments), suggesting a possible ceramidase block. Fe did not directly affect HK-2 ceramidase levels. However, arachidonic acid (C20:4) accumulation, a consequence of oxidant-induced phospholipase A2 (PLA2) activation, markedly suppressed ceramidase and stimulated SMase activity. Exogenous C20:4, as well as PLA2 (in doses simulating Fe-induced deacylation) recapitulated Fe's ceramide-generating effect. Because C20:4 is directly cytotoxic, it was hypothesized that ceramide might offset some of C20:4's adverse effects. Supporting this possibility were the following: (1) C20:4 exacerbated Fe toxicity; (2) this was abrogated by ceramide treatment; and (3) ceramide blunted Fe-mediated cell death. CONCLUSIONS: (1) ceramide accumulation during acute cell injury can be an adaptive response to PLA2 activation/C20:4 generation; (2) C20:4-induced ceramidase inhibition, coupled with SMase stimulation, may trigger this result; and (3) these ceramide increments may exert a "biostat" function, helping to offset C20:4/PLA2- and "catalytic" iron-mediated tubular cell death.

Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure.

UNLABELLED: Recent evidence indicates that a "sphingomyelin signaling pathway" exists: in response to heterogeneous influences, sphingomyelin is hydrolyzed, liberating ceramide, and subsequently its sphingoid base, sphingosine. Ceramide and sphingosine can influence diverse cellular processes, including cell differentiation, proliferation, protein trafficking, and apoptosis. Each of these processes have important implications for post-ischemic acute renal failure (ARF). However, sphingosine and ceramide expression during the induction of ischemic/reperfusion injury have not been previously assessed. To this end, CD-1 mice were subjected to 45 minutes of unilateral renal ischemia +/- reperfusion, followed by cortical sphingosine, ceramide, and sphingomyelin assessments. Contralateral kidneys served as controls. Ischemia caused approximately 50% sphingosine and ceramide decrements. During reperfusion, sphingosine rebounded to normal values. Conversely, ceramide rose to, and was maintained at, supranormal levels (approximately 175% of controls). Subsequent studies performed with hypoxic or oxygenated isolated proximal tubules suggested that these changes: (1) had a multifactorial basis; (2) were partially simulated by enhanced PLA2 activity; (3) and were dissociated from alterations in net sphingomyelin content. To assess the potential pathogenic relevance of the documented ceramide increments, cultured human proximal tubule (HK-2) cells were subjected to ATP depletion/Ca2+ ionophore- or PLA2-induced attack with or without exogenous C2 ceramide loading. Ceramide worsened both forms of injury without exerting an independent lethal effect. Conversely, ceramide markedly attenuated arachidonic acid cytotoxicity. This occurred without any decrease in arachidonate uptake, suggesting a direct cytoprotective effect. IN CONCLUSION: (1) sphingosine and ceramide fluxes are hallmarks of early ischemic/reperfusion injury; (2) these changes occur via divergent metabolic pathways; and (3) that ceramide increments can affect divergent injury pathways, and that sphingosine and ceramide have potent cell signaling effects, suggest that the currently documented sphingosine/ ceramide fluxes could have important implications for the induction phase and evolution of post-ischemic ARF.

Phospholipase A2: a potentially important determinant of adenosine triphosphate levels during hypoxic-reoxygenation tubular injury.

During the course of O2 deprivation-induced proximal tubular injury, profound alterations in ATP homeostasis exist. This study sought to characterize direct cellular determinants of these abnormalities further. Mouse proximal tubular segments (PTS) were isolated and their adenine nucleotide profiles were determined during hypoxic-reoxygenation injury. The extent of oxidant stress, Ca2+ overload, cytoskeletal disruption, and phospholipase activity were experimentally manipulated by H2O2, Ca2+ ionophore, cytochalasin D, or PLA2 addition, respectively. Hypoxia induced the expected deterioration in adenylate profiles, and a persistent defect in ATP homeostasis was observed during reoxygenation (decreased ATP/ADP ratios and absolute ATP content). H2O2, Ca2+ ionophore, and cytochalasin D had no significant impact on adenylate profiles. However, doses of PLA2 that had no overt effect on normal tubules caused 50 to 75% reductions in both hypoxic and reoxygenation ATP/ADP ratios and absolute ATP content. This effect was completely reproduced by the addition of arachidonic acid (C20:4). No other test fatty acid (C16:0, C18:1, C18:3) reproduced this result. Despite its profound negative impact on hypoxic/reoxygenation ATP concentrations, PLA2 and C20:4 each decreased lethal cell injury (lactate dehydrogenase release), as previously reported. The reductions in ATP and lethal cell injury were not mechanistically linked, because C18:1 and C18:3 reproduced the protective action of C20:4 without altering adenine nucleotide profiles. Ouabain, mannitol, or plasma membrane fatty acid "scavenger" therapy (albumin) did not improve the posthypoxic/PLA2-induced depressions in ATP. The addition of C20:4 caused a modest decrease in posthypoxic tubule oxygen consumption, compared to controls. It was concluded that: (1) PLA2 can be a major determinant of ATP concentrations during both hypoxic and reoxygenation tubular injury; (2) this action is mediated via C20:4 release; (3) a primary defect in mitochondrial ATP production, rather than increased ATP consumption, is likely to be responsible for this action.

Phospholipase A2-induced cytoprotection of proximal tubules: potential determinants and specificity for ATP depletion-mediated injury.

Addition of phospholipase A2 (PLA2) to isolated proximal tubular segments (PTS) has previously been shown to decrease hypoxic cell death without altering ATP concentrations. The study presented here was undertaken to identify determinant(s) of this protection, and to define the spectrum of injuries against which it can operate. PTS were extracted from mouse kidneys and subjected to diverse forms of injury (hypoxia/reoxygenation, antimycin A, Ca2+ ionophore, amphotericin B, FeSO4, and myohemoglobin). In subtoxic doses, addition of PLA2 significantly reduced hypoxic- and antimycin A-induced injury (percentage of lactate dehydrogenase release); however, a dose-dependent exacerbation of all other forms of injury resulted. The ability of PLA2 to mitigate hypoxic injury remained intact despite the inhibition of Na,K-ATPase (ouabain) or the inducement of cytoskeletal disruption (cytochalasin D). However, it was negated by minimally toxic amphotericin B or Ca2+ ionophore doses, indicating its dependence on preserved ionic gradients. Nevertheless, neither lowering/removing buffer Ca2+ or NaCl concentrations, nor hypertonic mannitol addition reproduced the cytoprotective effect of PLA2. PLA2 induced synergistic deacylation in hypoxic tubules, suggesting that unsaturated fatty-acid accumulation might mediate its cytoprotective effect. The fact that the addition of exogenous arachidonate, but not palmitate, to tubules protected against hypoxia, but worsened nonhypoxic forms of injury, supported this hypothesis. Since arachidonate might induce "feedback" inhibition of intracellular PLA2, the ability of an intracellular phospholipase inhibitor (ONO-RS-082; Biomol, Plymouth, PA) to blunt hypoxic damage was tested. This agent fully reproduced the cytoprotective effect of PLA2. It was concluded that: (1) PLA2-induced cytoprotection is relatively specific for ATP depletion injury; (2) it is dependent on, but not explained by, maintenance of NaCl and Ca2+ gradients; (3) it does not require Na,K-ATPase activity or cytoskeletal integrity for its expression; and (4) extracellular PLA2, via arachidonate release, may cause feedback inhibition of intracellular PLA2, thereby protecting critical intracellular targets from attack.

Iron, heme oxygenase, and glutathione: effects on myohemoglobinuric proximal tubular injury.

UNLABELLED: This study assessed the impacts of iron, heme oxygenase (HO), hydroxyl radical (.OH), and glutathione (GSH) on the initiation phase of myohemoglobinuric proximal tubular injury using a novel model system. Rhabdomyolysis was induced in rats by glycerol injection and four hours later proximal tubular segments (PTS) were isolated. They were incubated for 0 to 90 minutes either in the presence or absence of an iron chelator (deferoxamine; DFO), .OH scavengers, an .OH trapping agent (salicylate; to gauge .OH production), GSH, or catalase. In selected experiments, an HO inhibitor (Sn protoporphyrin) was given at the time of glycerol injection to assess HO's acute effects on the evolving injury. Cell death and lipid peroxidation were quantified by % LDH release and malondialdehyde (MDA) generation, respectively. PTS from normal rats served as controls. Post-glycerol PTS manifested progressive LDH release (47 +/- 2%) and 20-fold MDA increments during the incubations, whereas only 11 +/- 1% LDH release and no MDA generation was observed in the normal PTS. DFO completely prevented both parameters of glycerol-induced injury. HO inhibition exerted an acute protective effect, despite previous in vivo data suggesting that HO is a cytoprotectant. Neither .OH scavengers nor catalase mitigated post-glycerol injury, the latter correlating with reduced, not increased, .OH production. GSH slightly decreased LDH release while causing a paradoxical threefold MDA increment. The latter was iron dependent (blocked by DFO), was expressed in normal PTS, and it could be reproduced by equimolar cysteine. That GSH increased iron-dependent lipid peroxidation in a cell free system (exogenous phosphatidylcholine) indicated that GSH metabolism to cysteine was not a requirement for this reaction. IN CONCLUSION: (1) chelatable iron can fully account for heme protein-triggered proximal tubular injury; (2) HO contributes to this injury, presumably by causing iron release; (3) the heme-induced injury appears to be mediated by non-.OH oxidizing intermediates; (4) GSH can exert both anti- and pro-oxidant effects; and (5) i.m. glycerol injection, followed by proximal tubular isolation, represents a new and highly useful model for studying direct determinants of heme protein cytotoxicity.