INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MITOCHONDRIAL DYSFUNCTION has long been considered to play a central role in the development of cell injury during ischemia-reperfusion and hypoxia-reoxygenation (12). However, the contributions of various biochemical alterations seen in this setting have not been fully defined, and it has been difficult to distinguish primary events from secondary processes associated with generalized cellular damage caused by ATP depletion. Several developments have changed this situation. A major advance has been recognition of the mitochondrial permeability transition (MPT), a porous defect of the inner mitochondrial membrane. Developing initially as a potential sensitive megachannel regulated by a mitochondrial matrix cyclophilin, the MPT evolves to become a proteinaceous membrane pore with a size exclusion limit of ~1,500 Da, and thereby compromises mitochondrial integrity following diverse stimuli (2, 12, 24, 32). Independently or in association with the MPT, mitochondrial outer membranes may also become permeable under specific injurious circumstances to proteins residing in the intermembrane space (17, 30, 49, 69). Mitochondrial release of one such protein, cytochrome c, has twofold effects. Because of its role as an electron shuttle, dislocation of cytochrome c compromises respiration (49, 58). As a cytosolic cofactor required to activate caspase 9, it can trigger apoptosis (49, 58, 69).

Both the MPT and mitochondrial release of cytochrome c have been invoked as factors involved in the death of cells during or following hypoxia and ischemia (12, 29, 32, 49). Cyclosporine A has been shown to bind mitochondrial cyclophilin and suppress development of the MPT (2, 68). Coordinate suppression of both the MPT and cell killing by cyclosporine has suggested that the MPT is a determinant of lethal outcome during hypoxia (34, 40) and post-hypoxic or post-ischemic reoxygenation (14, 25, 44, 57). Bax-mediated cytochrome c release in the absence of the MPT contributes to both necrosis and apoptosis of cultured kidney tubule cells during prolonged hypoxia and hypoxia-reoxygenation (48, 49).

Proximal tubules have relatively little or no glycolytic capacity, making them dependent on aerobic mitochondrial metabolism for ATP synthesis (1, 47, 67). Accordingly, ATP concentrations in freshly isolated proximal tubules decline steeply during hypoxia, in spite of the presence of glucose (62). We have found that the tubule cells develop a severe mitochondrial functional deficit that is expressed during reoxygenation following >30-min hypoxia, despite availability of substrates optimized for aerobic proximal tubule metabolism, and glycine to maintain plasma membrane integrity (62, 64). The abnormality is characterized by incomplete recovery of mitochondrial membrane potential (_m) and cellular ATP, impaired respiration utilizing substrates that donate electrons to respiratory complex I, and persistence of hypoxia-induced mitochondrial matrix condensation (64). Respiratory functions of complexes II, III, and IV remain largely intact (64). The lesion is partially ameliorated by chemical inhibitors of the MPT, including cyclosporine (62). In recent studies (64), we have shown that a metabolic strategy, that promotes anaerobic mitochondrial metabolism to generate ATP and maintain _m during hypoxia, can prevent development of the mitochondrial lesion and, importantly, can also reverse the mitochondrial defects and enable cellular recovery, even if it is introduced only during reoxygenation, after completion of the hypoxic period (64).

Anaerobic mitochondrial metabolism can generate ATP and maintain mitochondrial energization via two pathways (Fig. 1): A) substrate-level phosphorylation during the conversion of -ketoglutarate to succinate by -ketoglutarate dehydrogenase (23, 27, 28, 42); and B) electron transport in complexes I and II driven by reduction of fumarate to succinate coupled to the oxidation of reduced ubiquinone that is generated via NADH from citric acid cycle (CAC) reducing equivalents (23, 27, 42, 52). These reducing equivalents are shown in Fig. 1 as being provided by -ketoglutarate dehydrogenase because that reaction will be favored with concomitant -ketoglutarate supplementation, but any source of NADH can serve this purpose. In our recent studies, stimulation of these metabolic pathways by supplementation of the tubules with -ketoglutarate + aspartate (-KG/ASP) strikingly ameliorated the energetic deficit that developed during hypoxia-reoxygenation (64). The pathways shown in Fig. 1 indicate that other CAC intermediates and related compounds should be able to substitute for -KG and aspartate. These include glutamate, which is relatively abundant in the kidney (61), and could, therefore, be a large source of -KG, as well as malate or fumarate, which require less metabolism than aspartate to promote pathway B (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Metabolic pathways for protective effects of citric acid cycle (CAC) metabolites. A: substrate level phosphorylation. During hypoxia or anaerobiosis, -ketoglutarate (-KG) is metabolized to succinate (SUC) with production of GTP that transphosphorylates ADP to ATP. B: anaerobic respiration in electron transport (ET) complexes I and II. Reduction of fumarate (FUM) to succinate coupled by the FAD-containing flavoprotein of succinate dehydrogenase (complex II) to oxidation of reduced ubiquinone (CoQRED) drives proton extrusion by complex I, which increases membrane potential (m) and can generate an additional molecule of ATP using the mitochondrial F1F0-ATPase. In the process, NADH is anaerobically oxidized to NAD+, which can then promote further metabolism of -KG. Transamination of aspartate (ASP) to oxalacetate (OAA) serves as a source of malate (MAL) and fumarate. Not shown is the conversion of OAA to MAL that also oxidizes NADH formed during the decarboxylation of -KG and, thus, favors that reaction independently of anaerobic electron transport. C: aerobic bypass of complex I. When electron transport resumes during reoxygenation, succinate provided directly or produced via pathways A and B bypasses complex I of the electron transport chain, allowing ATP production in complexes III and IV.

These considerations and our recently reported findings (64) raise important questions. The strong protection against mitochondrial hypoxic injury afforded by provision of -KG and aspartate during hypoxia is readily explained by postulating a single process. By anaerobically generating ATP and/or maintaining _m, they preempt the development of damage to respiratory complex I and other inner mitochondrial membrane components. However, there are at least two explanations for the beneficial effects afforded by provision of the substrates only during reoxygenation, and they are not mutually exclusive. One is that the same mechanisms of substrate-level phosphorylation and anaerobic respiration that prevent the lesion from developing during hypoxia are responsible. The other explanation is that, during reoxygenation, succinate, which is a product of both pathways A and B (Fig. 1), donates electrons to complex III via complex II (succinate dehydrogenase), bypassing the limitation of metabolism of complex I substrates (pathway C in Fig. 1). In this fashion, succinate-dependent aerobic respiration via normal electron transport can support _m and generate ATP, even in mitochondria with impaired function of complex I, and thereby repair and rescue cells. The absence of the terminal electron acceptor, oxygen, would preclude benefit from succinate by this mechanism during hypoxia.

In the present studies, we have systematically investigated whether one or more of a range of CAC intermediates and related compounds other than -KG/ASP can modify the hypoxia-reoxygenation mitochondrial insult and the resulting cellular energetic deficit of proximal tubule cells. The ability of the metabolites to prevent or reverse injury was assessed by measuring the recovery of cell ATP concentration and _m during reoxygenation. These studies were designed not only to further delineate the mechanisms of mitochondrial damage and protection, but also to provide information bearing on the likelihood of their expression during proximal tubule injury in vivo, where both tissue and circulating levels of the full spectrum of available metabolites must be considered. Our results indicate that the protective effects of -KG/ASP on both mitochondrial energization and recovery of cell ATP can be duplicated to various degrees by other CAC intermediates, either alone, or in combination. However, the protective effects are specific for subsets of metabolites depending on whether protection occurs during hypoxia or reoxygenation. Moreover, the data indicate that the aerobic pathway of protection provided by succinate is important and that the recovery process, once initiated by protective substrates, is maintained even if they are withdrawn. These observations provide new insights into a fundamental mode of mitochondrial dysfunction during a common form of cell injury in the kidney and other tissues, and suggest potentially powerful approaches for modifying the lesion and subsequent damaging processes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolation of tubules. Proximal tubules were prepared from kidney cortex of female New Zealand White rabbits (1.5-2.0 kg; Oakwood Farms, Oakwood, MI) by digestion with combinations of Worthington Type I (Worthington, Freehold, NJ) and Sigma Blend Type H or F collagenase and centrifugation on self-forming Percoll gradients as described (61, 62, 64).

Experimental procedure. Incubation conditions generally followed our published protocols (62, 64). Tubules were suspended at 3.0-5.0-mg tubule protein/ml in a 95% O₂/5% CO₂-gassed medium containing (in mM) 110 NaCl, 2.6 KCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.25 CaCl₂, 1.2 MgCl₂, 1.2 MgSO₄, 5 glucose, 4 sodium lactate, 0.3 alanine, 5.0 sodium butyrate, 3% dialyzed dextran (Pharmacia, T-40), and 2 mM glycine. The medium was also supplemented with 0.5 mg/ml bovine gelatin (75 bloom) to suppress aggregation of the isolated tubules during the prolonged experimental incubation periods. After 15-min preincubation at 37°C, tubules were resuspended in fresh medium with experimental agents and regassed with either 95% O₂-5% CO₂ (controls) or 95% N₂-5% CO₂ (hypoxia). Hypoxic tubules were kept at pH 6.9 to simulate tissue acidosis during ischemia in vivo (62). After 60 min, samples were taken for analysis. The remaining tubules were washed twice to remove any experimental substrates being tested for their efficacy only during hypoxia and were then resuspended in fresh 95% O₂-5% CO₂-gassed, pH 7.4 medium, with experimental agents as needed. In the reoxygenation medium, 2.0-mM sodium heptanoate replaced sodium butyrate, and, to insure availability of purine precursors for ATP resynthesis, 250 µM AMP or ATP was added (62) in most experiments. The supplemental medium, purine, eliminates any effect of hypoxia-induced decreases of the intracellular purine pool (59) to limit recovery of ATP and, thus, allows the cell ATP levels to be a better index of the functional state of the mitochondria. After 60 or 120 min of reoxygenation, samples were taken again for analysis. Cell ATP and lactate dehydrogenase (LDH) release were measured as previously described (62). Other parameters were assayed as in the following sections.

Staining with _m-sensitive dyes. For staining with tetramethylrhodamine methyl ester [(TMRM), Molecular Probes, Eugene, OR)] (36), at the end of the desired experimental period, a 0.5 ml aliquot of the tubule suspension was mixed with an equal volume of room temperature phosphate-buffered saline containing 1.0 µM TMRM. After 1 min, the tubules were pelleted, washed twice in an ice-cold solution containing (in mM) 110 NaCl, 25 Na-HEPES, pH 7.2, 1.25 CaCl₂, 1.0 MgCl₂, 1.0 KH₂PO₄, 3.5 KCl, 5.0 glycine, and 5% polyethylene glycol (average MW 8000), and then held in this solution in the dark at 4°C until they were examined by confocal microscopy. For staining with the carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1, Molecular Probes) (45, 54), an aliquot from a 1,000× stock solution in dimethyl sulfoxide was mixed with an equal volume of calf serum, dispersed as an intermediate 100× stock solution in phosphate-buffered saline, and then added at the end of the desired experimental period to a final concentration of 5 µg/ml in the tubule suspension. The suspension was regassed with O₂/CO₂ and incubated in the dark for an additional 15 min at 37°C, then tubules were pelleted, washed three times in the same solution as used for the TMRM studies, and held in that solution in the dark at 4°C until either viewing of individual tubules by confocal microscopy or measurements of fluorescence on samples of the whole suspension. In some studies, vital dye exclusion was concomitantly assessed by inclusion of 2 µg/ml propidium iodide along with the TMRM or for the last 1-2 min of the period of JC-1 exposure.

Laser-scanning confocal microscopy of TMRM and JC-1 stained tubules. Samples of the washed tubules were loaded into a Dvorak-Stotler chamber (Lucas-Highland, Chantilly, VA) and allowed to settle for 10-15 min in the cold, then rapidly viewed with a 100 × Plan Apochromat lens (NA 1.4) using a Nikon Diaphot microscope attached to a Bio-Rad MRC 600-laser scanning confocal system equipped with a krypton/argon mixed-gas laser at the wavelength settings described with the results and the Figs. Illustrations shown with the results are representative of changes that were uniformly seen in tubules from 3-5 separate experiments.

Measurement of JC-1 fluorescence in suspension (54). Immediately after sampling and washing, a 300-µl aliquot of the tubules containing 1.2-1.5 mg protein was brought up to 2.5 ml with additional ice-cold wash solution and then scanned during continuous gentle stirring using a Photon Technology International (Monmouth Junction, New Jersey) Alphascan fluorometer at 488-nm excitation/500-625-nm emission collected in right angle mode of the fluorometer. Under these conditions, the peak of the green fluorescence of the monomeric form of the dye was at 530 nm and the red fluorescence of the J-aggregates peaked at 590 nm. This procedure allowed for collection of data for both forms of the dye from a single rapid scan so that there was no deterioration of the signal from photobleaching or continued mixing and warming in the chamber.

Measurement of cytochrome c release. At the end of the desired experimental period, tubules were pelleted and resuspended in a solution containing (in mM) 250 sucrose, 10 KCl, 1.5 MgCl_2, 1 EDTA, 1 EGTA, 10 K-HEPES, pH 7.1, 10 phenylmethylsulfonyl fluoride 0.25 mg/ml digitonin, 16 µg/ml benzamidine, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A at room temperature. After 5 min incubation at room temperature, which allowed release of all lactate dehydrogenase, the suspensions were centrifuged at 12,000 g for 2 min Pellets and supernatants were saved at 80°C for analysis of cytochrome c distribution by immunoblotting with a monoclonal antibody to cytochrome c (clone 7H8.2C12, Pharmingen, San Diego, CA) as previously described (48). The relative distribution of cytochrome c between the supernatants and pellets was quantitated by densitometry using Kodak 1D software version 2.0.2 (Kodak, Rochester, NY).

Determination of total amino-acid levels. Amino acids were measured on neutralized trichloroacetic acid extracts of the tubule suspension by a Varian-9012 high pressure liquid chromatography system equipped with Auto Sampler-9100. Precolumn derivatization with o-phthalaldehyde and fluorescence detection were employed as previously (61).

Assay of succinate. Succinate was assayed on neutralized trichloroacetic acid extracts of the tubule suspensions exactly as in (4) except for the use of fluorometric detection to monitor NADH consumption. Succinate was converted to succinyl-CoA by reaction with coenzyme A and inosine triphosphate in the presence of succinyl-CoA synthetase (Roche Molecular Bioproducts, Indianapolis, IN). The inosine diphosphate formed was used to convert phosphoenolpyruvate to pyruvate in the presence of pyruvate kinase. The pyruvate was then reduced by NADH to lactate in the presence of lactate dehydrogenase. NADH consumption was linear for succinate concentrations up to 30 µM.

Determination of ¹⁵N-labeled metabolites. For GC-MS analysis of ¹⁵N-labeled amino acids, a 50-µl aliquot of neutralized trichloracetic-acid extract of the whole tubule suspension processed as for determination of ATP (62) was applied to an AG-50 column (100-200 mesh; 0.5 × 2.5 cm). The column was washed with 3 ml of deionized H₂O. Amino acids were eluted with 3 ml of NH₄OH. For determination of ¹⁵N isotopic enrichment, amino acids were converted to t-butyldimethylsilyl derivatives (37). ¹⁵N enrichment in glutamine and glutamate was monitored using the following ions: m/z 432, 432, for glutamine and m/z 432, 433 for glutamate. Calculation of ¹⁵N atom% excess (APE) was carried out as described (38). The production of ¹⁵N-labeled amino acid was calculated as ¹⁵N nmol/mg protein = C × APE / 100 where C is the total concentration measured by HPLC.

Reagents. Reagents were from Sigma (St. Louis, MO) unless otherwise indicated and were of the highest grade commercially available. Agents solubilized in ethanol or dimethyl sulfoxide were delivered from 1,000× stock solutions. All substrates tested were provided from 100×, pH-adjusted stocks of their Na⁺ salts, except for acetoacetate, which was the Li⁺ salt. [¹⁵N]aspartate was obtained from MSD Isotopes (Quebec, Canada). It behaved identically to unlabeled aspartate with respect to all experimental effects. Cyclosporine A was from Calbiochem (San Diego, CA).

Statistics. Paired and unpaired t-tests were used as appropriate. Where experiments consisted of multiple groups they were analyzed statistically by analysis of variance for repeated measure or independent-group designs as needed. Individual-group comparisons for the multi-group studies were then made by using the Newman-Keuls test for multiple comparisons (SigmaStat, SPSS, Chicago, IL). P < 0.05 was considered to be statistically significant. The Ns given represent the numbers of separate tubule preparations studied.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Prevention and reversal of hypoxia-reoxygenation-induced energy deficits by combinations of CAC metabolites. Tubules subjected to 60 min hypoxia and 60 min of reoxygenation with no further additions to the glucose, lactate, alanine, and fatty acid that normally serve as optimal substrates for the preparation (1, 47, 61, 62, 64) had severely impaired recovery of cell ATP levels [no extra substrate (NES) group in Fig. 2A]. This energetic deficit occurred despite the presence of glycine, which prevents plasma membrane damage, as measured by LDH release and vital dye exclusion under these conditions [(62, 64) and studies described below]. Addition to the medium of supplemental purine to provide precursors for resynthesis of ATP (62), increased ATP levels in both control and reoxygenated tubules, but did not eliminate the large difference between the two conditions (Fig. 2A). Cytochrome c was retained in the mitochondria throughout 60 min of hypoxia and was not detected in the cytosol (Fig. 2B). During reoxygenation, only negligible amounts of cytochrome c were seen in the cytosol, the vast bulk being retained intra-mitochondrially (Fig. 2B).

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Energetic deficit after hypoxia-reoxygenation and its modification by mitochondrial permeability transition (MPT) inhibitors and -KG/ASP. A: ATP levels of reoxygenated tubules. Tubules were subjected to 60 min hypoxia (2 mM glycine, pH 6.9) and 60 min reoxygenation (2 mM glycine, pH 7.4) with either no extra substrate (NES), or 4 mM -ketoglutarate + 4 mM aspartate (-KG/ASP) during reoxygenation alone, or cyclosporine A (5 µM) plus butacaine (30 µM) plus L-carnitine (2 mM) (CBC) during both hypoxia and reoxygenation. Reoxygenation was studied both without and with supplemental exogenous purine (250 µM ATP) to promote recovery of cell ATP. Control values shown are for parallel flasks from the same preparations incubated under oxygenated conditions for the same total duration as the experimental groups. The purine-supplemented control group had 250 µm ATP in its medium for the last 60 min of incubation. Cell ATP levels are means ± SE for N = 4-6, *significantly different from corresponding -KG/ASP group, #significantly different from corresponding CBC group, and significantly different from corresponding control group. B: immunoblot of cytochrome c in membrane pellets containing mitochondria and the corresponding cytosol supernatants. Tubules were incubated under oxygenated control conditions for either 75 min (75'C) or 135 min (135'C), for 60 min hypoxia (H), or for 60 min hypoxia followed by 60 min reoxygenation (H/R) with or without 4 mM -KG/ASP. Repeated conditions are from separate flasks. To provide a visible signal, the amount of protein for the cytosolic fractions was increased 3 fold relative to its actual proportion between the 2 types of samples. The percentages of total cytochrome c in the supernatants were 0.50 ± 0.12 in the H/R group without supplemental substrates and 0.21 ± 0.07 in the -KG/ASP-treated group (N = 3).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Benefit of alternative combinations of CAC metabolites. A: the basic experimental design was similar to that of the Fig. 2A studies except that extra test substrates were provided during hypoxia (H) alone or during hypoxia plus reoxygenation (H+R), and supplemental exogenous purine (250 µM AMP) was used during reoxygenation of all flasks. Glutamate plus malate (GLU/MAL) and -ketoglutarate plus malate (-KG/MAL), like -KG/ASP, were delivered at a final concentration of 4 mM of each substrate. Cell ATP levels are means ± SE for N = 3-5, *significantly different from corresponding no extra substance (NES) group and #significantly different from corresponding oxygenated control group. B: concentration dependence of protective effects of -KG/MAL. Tubules were subjected to hypoxia plus reoxygenation as in Fig. 2A with 250 µM AMP as the supplemental exogenous purine. -KG/MAL was added only during reoxygenation at the indicated concentrations followed by measurement at the end of reoxygenation of ATP and of the 590/530 nm JC-1 fluorescence ratio. Values are means ± SE for N = 4-8 at each concentration of -KG/MAL. Every -KG/MAL flask had a paired unsupplemented flask prepared from the same hypoxia sample. Values for these unsupplemented flasks did not differ among the groups and are pooled for clarity of presentation as the 0 mM point. All concentrations of -KG/MAL except 0.01 mM significantly increased ATP and the JC-1 fluorescence ratio relative to the paired unsupplemented flasks.

Increases of mitochondrial energization induced by protective substrates during reoxygenation. Mitochondrial membrane potential (_m) is both an important direct marker of integrity of the inner mitochondrial membrane and a regulator of the MPT pore (24, 32, 36, 53). It is, thus, a critical parameter in understanding the mechanism of the substrate effects, despite the limitations of available techniques for its measurement in intact cells (35). We investigated changes of _m in the tubules using two different membrane-permeant, cationic fluorophores, TMRM and JC-1 (36, 45, 54). In control tubules, TMRM stained the mitochondria brightly in their typical basolateral locations (Fig. 4a). TMRM uptake was entirely blocked by the mitochondrial uncoupler, FCCP (Fig. 4d). During reoxygenation without supplemental substrates, the majority of cells exposed to TMRM at the end of the experimental period displayed mitochondrial uptake (Fig. 4, b and e), but in most of them the signal was substantially weaker than that seen in the controls (Fig. 4a), consistent with a reduced but not absent level of energization. The majority of tubules treated with -KG/ASP (Fig. 4c and f) had bright-basal punctate staining similar to the controls. These studies, utilizing TMRM, provide high-resolution confocal images for visualization and show clearly that mitochondria were not completely deenergized. However, self-quenching by TMRM (15) complicates comparison of the signals in the control and injured tubules because it tends to exaggerate the signals from the partially energized mitochondria in the reoxygenated tubules that have lower levels of TMRM uptake. To further assess differences between the various experimental conditions we used JC-1.

View larger version (65K): [in this window] [in a new window]   Fig. 4.   Mitochondrial tetramethylrhodamine methyl ester (TMRM) uptake. Tubules were stained with TMRM and propidium iodide after: oxygenated control incubation (a); 60 min hypoxia followed by 60 min reoxygenation with no extra substrates (b,e) or with 4 mM -KG/ASP during reoxygenation (c,f); or 5 µM FCCP + 5 mM glycine for 15 min (d). Optical sections were viewed at 568 nm excitation, 585 nm emission. Panels a-d are cuts through the midplanes of the tubules showing the basally oriented mitochondria around the periphery of the tubule profile. The unstained areas in the center of each tubule are the apical portions of the cells, which are devoid of mitochondria, and the tubule lumens. Nuclei are seen as the circular areas devoid of staining. The full tubule profile in panel d occupies approximately the same area and is similarly oriented to those in the other panels, but is poorly seen because the mitochondria are unstained. The brightly stained circular structures are the rare propidium iodide-positive nuclei that were no more frequent than those seen in controls. Panels e and f are higher magnification cuts through the basal sections of the cells to show mitochondria at higher resolution. Bars in panels a and e are 10 µm. Results are representative of 8-10 tubules from 4-5 different preparations viewed under each experimental condition.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   JC-1 fluorescence in solution. A: tubules were incubated under oxygenated control conditions, or for 60 min hypoxia plus 60 min reoxygenation as in Fig. 3A with either no extra substrate (NES) or 4 mM of either -KG/ASP, -KG/MAL, or GLU/MAL during reoxygenation. These incubations were followed by staining with JC-1, washing, and scanning of suspensions of the intact tubules at 488 nm excitation and 500-625 nm emission. The tracings shown are representative of those obtained from 10-20 experiments under each of the conditions. B: JC-1 ratios (590/530 nm) for multiple individual experiments done under the same conditions as shown for Fig. 5A plotted against the ATP levels of the same samples. Each point is the result of a single experiment. Also shown are JC-1 ratios for tubules incubated with 5 µM FCCP in the presence of 5 mm glycine for 15 min.

View larger version (113K): [in this window] [in a new window]   Fig. 6.   Confocal microscopy of red JC-1 aggregates after hypoxia plus reoxygenation. Tubules were stained with JC-1 after: oxygenated control incubation (a); 5 µM FCCP + 5 mM glycine for 15 min (b); or 60 min hypoxia followed by 60 min reoxygenation with no extra substrates (c) or with 4 mM glutamate + malate during reoxygenation (d), then viewed by confocal microscopy at 568-nm excitation, 585-nm emission. Magnifications are the same for all panels. Bar: 10 µm. Unavoidable photobleaching results in dropout of the signal from the most severely affected mitochondria in the no extra substrate group.

Substrate specificity of protection. The data from the present studies presented up to this point, along with our earlier work (64), have indicated that combinations of CAC substrates that support anaerobic mitochondrial metabolism powerfully modify the defects of mitochondrial energization and recovery of cell ATP seen during hypoxia-reoxygenation. To further test the hypothesis that both anaerobic and aerobic mechanisms contribute to these effects (Fig. 1) and to determine whether the substrate combinations initially assessed were, in fact, providing the strongest protection, it was necessary to systematically evaluate the full range of CAC intermediates and related metabolites under both aerobic and anaerobic conditions. Our initial studies of this type focused on efficacy of substrates provided only during reoxygenation (Fig. 7). Based on those data, we then selectively assessed the active metabolites for their protective effects during hypoxia (Fig. 8). For clarity of presentation and analysis, the results for the large number of compounds tested during reoxygenation are grouped according to whether they are intrinsic intermediates of the CAC (Fig. 7, A and B) or require additional metabolism before entry in the cycle (Fig. 7, C and D). For the most part, this classification was also predictive of the efficacy of the metabolites.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Protection by individual CAC intermediates and related metabolites delivered only during reoxygenation. At the end of 60 min hypoxia in the presence of 2 mM glycine at pH 6.9 tubules were resuspended in oxygenated medium at pH 7.4 with 2 mM glycine and 250 µM AMP. This suspension was divided in half so that one of the aliquots received no extra substrate (NES) and the other aliquot was supplemented with the indicated substrate or substrate combination (all additions 4 mM). ATP levels (panels A and C) and 590/530 nm JC-1 fluorescence ratios (panels B and D) measured after 60 min reoxygenation are means ± SE for N = 5-12, *significantly different from the corresponding paired NES group. Values for oxygenated control tubules incubated in parallel (not shown) were ATP, 20.0 ± 0.69 nmol/mg protein; 590/530 nm JC-1 fluorescence ratio, 4.38 ± 0.10. AcAc, acetoacetate; OHB, -hydroxybutyrate.

View larger version (42K): [in this window] [in a new window]   Fig. 8.   Protection by individual CAC intermediates and related metabolites delivered only during hypoxia or during hypoxia and reoxygenation. Tubules were incubated with either no extra substrate or with 4 mM -ketoglutarate plus malate (-KG/MAL), or the indicated individual substrates (each at 4 mM) during 60 min hypoxia in the presence of 2 mM glycine at pH 6.9, then were washed and incubated either with or without the same extra substrate during reoxygenation at pH 7.4 with 2 mM glycine and 250 µM AMP. A: cell ATP values at the end of reoxygenation. Oxygenated control (not graphed) is the same as the value given with Fig. 7. B: JC-1 fluorescence ratios at the end of reoxygenation. Oxygenated control (not graphed) is the same as the value given with Fig. 7. C: cell ATP values at the end of hypoxia. Oxygenated control (not graphed) was 7.10 ± 0.33 nmol/mg protein. This value is lower than the control ATP for the reoxygenation period because it precedes the addition of AMP to the medium. D: succinate levels at the end of hypoxia. Oxygenated control and end hypoxia with no extra substrate succinate levels (not graphed) were 0.51 ± 0.05 and 0.39 ± 0.10 nmol/mg protein, respectively. All values are means ± SE for N = 4-12, *significantly different from the corresponding no extra substrate group. #Significantly different from corresponding no extra substrate and extra substrate during only hypoxia groups.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Production of 15N-labeled amino acids from [15N]aspartate with and without -KG. Tubules were supplemented with [15N]aspartate (4 mM) or [15N]aspartate+-KG (4 mM each) during either 60 min of oxygenated control incubation, 60 min of hypoxia, or 60 min of reoxygenation (REOXY) following 60 min of hypoxia. All other conditions during hypoxia and reoxygenation are the same as for Fig. 8. Results are the product of 15N isotopic enrichment (atom% excess/100) times concentration (nmol/mg protein). Values are means ± SE for 3 experiments. *Significantly different from corresponding hypoxia value; #significantly different from corresponding aspartate alone condition.

View larger version (34K): [in this window] [in a new window]   Fig. 10.   Maintenance of improved mitochondrial function after withdrawal of protective substrates. Tubules were subjected to hypoxia + reoxygenation as for the Fig. 7 studies with either no extra substrate (NES) during reoxygenation or substrate supplementation during reoxygenation as follows: 4 mM -KG/MAL for 60 min of reoxygenation (A/M 60'), 4 mM -KG/MAL for 120 min of reoxygenation (A/M 120'), 4 mM succinate for 60 min of reoxygenation (SUC 60'), 4 mM succinate for 120 min of reoxygenation (SUC 120'). Tubules were sampled for ATP levels at 60 min reoxygenation (60' REOX) and for ATP levels and JC-1 fluorescence at 120 min reoxygenation (120' REOX). Values are means ± SE from three experiments, each with duplicate samples for every condition. All 120' REOX values are significantly greater than the corresponding 60' REOX values. All substrate-supplemented values are significantly greater than the corresponding NES group values. All -KG/MAL values are significantly greater than the corresponding succinate values. Control values are for preparations incubated under oxygenated conditions for the same total durations as the experimental groups and are significantly greater than the corresponding values for the experimental groups.

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Lactate dehydrogenase (LDH) release after glycine (GLY) withdrawal. Tubules were subjected to 60 min hypoxia followed by 120 min reoxygenation in the presence of either no extra substrate (NES) or with 4 mM -KG/MAL during reoxygenation. In the indicated groups designated as -GLY for the 060 min and 60120 min reoxygenation (REOX) periods, glycine was withdrawn either immediately at the start of reoxygenation or at 60 min of reoxygenation. LDH release was measured at 60 and 120 min of reoxygenation. Tubules for all groups were similarly washed and resuspended in fresh medium at both the start of reoxygenation and at 60 min of reoxygenation, so the values for LDH release shown are the amounts released during the 0-60 and 60-120 min reoxygenation periods. Data are means ± SE from four experiments. *P < 0.05 vs. corresponding -KG/MAL group. LDH release by oxygenated control tubules that had not been subjected to hypoxia averaged 1.4 ± 0.1% during the 0 to 60-min period and 1.1 ± 0.05% during the 60- to 120-min period irrespective of the presence of glycine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The studies in this paper provide new insights into: a) the basis for mitochondrial alterations during a ubiquitous form of tissue injury highly relevant to proximal tubule pathology during ischemic acute renal failure, b) their relationships to general mechanisms of mitochondrial dysfunction of widespread recent investigative interest, and c) a powerful effect of CAC metabolites to ameliorate the biochemical lesion.

The MPT and cytochrome c release have received considerable attention during the past several years as mechanisms by which mitochondria become damaged and contribute to cell death (12, 24, 30, 32, 49, 58, 69). Despite the severity of the insult studied, neither of these processes accounted for the energetic deficit in the proximal tubules. The MPT has been implicated in several models of hypoxia-reoxygenation injury (14, 21, 22, 34, 40, 44, 57) and might reasonably have been expected to occur. Relatively long-lived, solute-selective substates of the MPT have been described in isolated mitochondria (6), and could contribute to the deenergization observed in the tubules, as well as explain the amelioration of the lesion by chemical inhibitors of the MPT (62). The main respiratory abnormality in reoxygenated tubules is in complex I (64), which is similar to the behavior of mitochondria isolated from whole tissues after ischemia and ischemia-reperfusion (12, 20, 46). This damage to complex I might conceivably be a precursor lesion that eventually leads to the MPT, since recent work suggests that, in addition to previously implicated components of the pore such as the adenine nucleotide translocase (68), complex I proteins may form part of the MPT pore (18). However, the increase of inner membrane permeability to ions resulting from the MPT (2, 24, 32, 36) would have precluded the partial retention of _m observed in the reoxygenated tubules, making it unlikely that the MPT had developed over the time frame of our studies. The microfluorometric observations (Figs. 4 and 6) are important for this conclusion because they demonstrate that the changes of _m occur in individual cells rather than as shifts in relative proportions of cells that either remain fully energized or become fully deenergized. It is theoretically possible that individual mitochondria within cells could develop the MPT and consequently become fully deenergized, while others remain fully energized, thus decreasing the overall _m-dependent fluorescence without eliminating it entirely. However, the confocal observations with TMRM, which was relatively resistant to photobleaching, suggest that mitochondria in affected cells mostly developed uniform partial losses of _m (Fig. 4). This conclusion is further supported by our ultrastructural studies, which have shown that all mitochondria in the majority of unprotected, reoxygenated tubule cells have a condensed configuration rather than the swelling expected for the MPT (64).

Leakage of cytochrome c from mitochondria into the cytosol has been invoked as a damage mechanism that contributes to cell death (16, 17, 30, 49, 69). Dislocation of the cytochrome from its normal location in the space between the inner and outer mitochondrial membranes interrupts electron transport, thereby inhibiting oxidative phosphorylation, and its presence in the cytosol can trigger apoptosis. Loss of the cytochrome can occur via either selective permeabilization of outer mitochondrial membranes or the membrane damage that accompanies the MPT. However, the defects of ATP synthesis that were seen in the reoxygenated tubules were not associated with significant losses of mitochondrial cytochrome c (Fig. 2B). These results importantly complement our prior observation that respiratory function of complex IV, which depends on electrons donated by cytochrome c, remains intact during the cellular insult caused in proximal tubules by 60 min hypoxia and 60 min reoxygenation (64). We have detected little or no cytochrome c release from the isolated proximal tubules during up to 180 min of hypoxia (not shown), a period that is sufficient to induce substantial release of the protein and subsequent apoptosis in cultured proximal tubule cells (48). It will be of interest to determine why this mechanism of injury is so suppressed in fully differentiated proximal tubules. The mitochondrial lesion can certainly be lethal, but, as shown by the Fig. 11 studies, cell death is predominantly by the glycine-sensitive plasma membrane lesion that causes necrosis and rapid LDH release.

Mitochondrial anaerobic substrate-level phosphorylation and respiration are used for energetic support by diving mammals (27) and have been demonstrated to maintain low levels of functionally significant ATP synthesis in hypoxic heart and kidney (8, 23, 28, 31, 41-43, 55, 65), as well as to be beneficial for survival during hemorrhagic shock (10), although their precise mechanism of action in the injury settings has been controversial because of the small amounts of ATP produced anaerobically (65). We have shown that maintenance of ATP by the protective substrates during hypoxia is a result of substrate-level phosphorylation, while anaerobic respiration in complexes I and II supports _m (64). These effects and the data in the present studies provide an explanation for the benefits of the substrates on organ function despite the relatively small amounts of ATP produced. This ATP, because of its continued availability in the mitochondrial matrix, in combination with the concomitant increases of _m prevents and reverses a persistent, severe state of mitochondrial dysfunction involving damage to complex I that precedes the MPT and cytochrome c release. The selective importance of the substrate effects at the mitochondrial level is emphasized by observations that the substrates do not alter the plasma membrane damage measured by LDH release or failure to exclude vital dyes during hypoxia in the absence of glycine [(60) and additional data not shown] when oxidative phosphorylation is limited by oxygen deprivation. In contrast, during reoxygenation, improvement of mitochondrial function by the substrates under aerobic conditions that permit resumption of oxidative phosphorylation generates ATP to prevent the plasma membrane damage and LDH release that occurs if glycine is withdrawn from tubules without protective substrates that have not recovered mitochondrial function (Fig. 11).

Supplementation with protective substrates produced relatively large parallel increases of both cellular ATP levels and mitochondrial energization during reoxygenation, with the JC-1 fluorescence values in the best-protected groups reaching control levels. This indicates that the protective effects of the substrates can induce recovery of essentially normal _m. Although reportedly not an issue for JC-1 (51), cellular entry of potentiometric fluorophores, like their mitochondrial uptake, can be plasma membrane potential dependent so that decreases of the plasma membrane potential as expected during ATP depletion, due to inhibition of the Na⁺ pump, could reduce mitochondrial uptake of the fluorophores independently of changes of _m (35, 54). This consideration doesn't affect our conclusion that mitochondria of the unprotected tubules are not completely deenergized because, despite any limitation of cellular uptake, mitochondrial energization is detected with both TMRM and JC-1. Decreased fluorophore uptake across the plasma membrane resulting from ATP depletion-induced plasma membrane depolarization could exaggerate the differences between the unprotected and substrate-protected tubules during reoxygenation. However, we have recently shown for oxygenated control tubules as well as for posthypoxic tubules, reoxygenated both with and without protective substrates, that JC-1 fluorescence after uptake of the fluorophore in digitonin-containing intracellular buffer is similar to that of tubules loaded with JC-1 in the usual fashion without permeabilization (63). There is evidence that the proton motive force across the inner mitochondrial membrane, most of which consists of _m, must be maintained at 80-90% of its maximal value for oxidative phosphorylation to occur (35). Thus the capacity of even moderately deenergized mitochondria for ATP synthesis by oxidative phosphorylation may be severely impaired or completely suppressed.

The substrate-induced increases of ATP and of the 590/530 nm JC-1 fluorescence ratio measured during reoxygenation (Figs. 2A, 3A and B, 5B, 7A and B, 8A and B, and 10) are much larger than the increments of ATP (Fig. 8C) and JC-1 fluorescence (64) produced by the substrates during hypoxia, and, as shown by the Fig. 10 studies, do not require continued presence of high concentrations of the supplemental protective substrates. Therefore, the improvement during reoxygenation results from a combination of primary effects of protective substrates on the 'rescue' pathways (Fig. 1) followed by global, self-perpetuating restoration of aerobic mitochondrial function. The large, progressive increases of ATP during the second hour of reoxygenation (Fig. 10) are particularly impressive in this regard.

Our measurements of ¹³C-labeled metabolites in tubules incubated with [¹³C]aspartate during the hypoxia-reoxygenation maneuvers have provided direct evidence for operation in the tubules of the anaerobic pathways of -KG/ASP metabolism shown in Fig. 1 (64). Aspartate is theoretically advantageous in combination with -KG because it provides oxalacetate that serves as a direct substrate acceptor for NADH formed during the oxidation of -KG to succinyl-CoA and, thereby, promotes continuing anaerobic substrate-level phosphorylation (23, 64). Our studies in the present paper with the additional substrate combinations, however, show that malate in combination with -KG is just as effective as aspartate, probably because pathway B in Fig. 1 can also utilize the NADH formed from oxidation of -KG and, thus, maintain continued substrate level phosphorylation from anaerobic metabolism of -KG.

The experiments testing individual substrates (Fig. 8) provide strong support for the involvement of both the anaerobic and aerobic protective mechanisms shown in Fig. 1. During hypoxia, interconversion of CAC intermediates by normal forward operation of the cycle is limited and electron transport is blocked except for the cycling between complexes I and II in pathway B. Under these conditions, only -KG, which feeds directly into pathway A to promote substrate-level phosphorylation, and malate and fumarate, which feed into pathway B to promote anaerobic respiration, were unequivocally beneficial when provided individually. In this regard, it should be noted that Fig. 1 shows the interaction between pathways A and B to illustrate the synergistic effect of stimulating both of them simultaneously, but coupling between -KG metabolism in pathway A and anaerobic respiration in pathway B is not obligate. NADH from any source will maintain anaerobic respiration in complexes I and II. The measurements of succinate production during hypoxia (Fig. 8D) indicate that anaerobic metabolism yielding succinate is required for protection. The substrates that were effective during hypoxia (-KG, malate, and fumarate) all induced accumulation of succinate. The substrates that were ineffective during hypoxia (glutamate, citrate, and aspartate) did not. The data that ATP levels during hypoxia were increased by -KG, but not by malate or fumarate, are consistent with the involvement of separate pathways in the protective effects of -KG, as opposed to those of malate and fumarate (Fig. 8C), and provide additional evidence for the conclusion from our prior work (64) that anaerobic respiration in complexes I and II accounts for increments of _m during hypoxia, but does not contribute to increases of ATP. The effects of the two pathways were at least partly additive since -KG, malate, and fumarate individually did not protect as strongly as the substrate combinations that stimulate both pathways.

During reoxygenation, all the CAC intermediates tested, and aspartate, were protective (Fig. 7). The efficacy of citrate and aspartate individually during reoxygenation suggests that under aerobic conditions there is enough forward operation of the CAC before correction of the lesion to provide sufficiently high levels of -KG from the added citrate to drive the substrate-level phosphorylation rescue pathway, and enough -KG from endogenous metabolites, to support transamination of aspartate to allow it to be utilized. The strong benefit of succinate during reoxygenation could be due to provision of -