1 Division of Nephrology, Department of Internal Medicine, University of Michigan and Veteran's Administration Medical Center, Ann Arbor, Michigan 48109; 2 Departments of Pathology and Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78284; and 3 Division of Child Development, Children's Hospital of Philadelphia and Dept. of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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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 -ketoglutarate (
-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic
metabolism of
-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
-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
-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was withdrawn.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.
rabbit; kidney; -ketoglutarate; glycine; succinate; adenosine-5'-triphosphate
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INTRODUCTION |
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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).
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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.
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METHODS |
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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 MgCl2, 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.
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RESULTS |
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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).
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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.
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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.
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DISCUSSION |
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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
-KG from forward operation of the CAC, but much more
likely derives from the ability of succinate to bypass the respiratory
block for complex I-dependent substrates that fundamentally
characterizes the lesion (64). This would generate ATP and
increase
m, with resulting correction of the complex I dysfunction and other elements of the underlying lesion. The experiments in Fig. 10 clearly show that supplemental succinate, like
-KG/MAL, can be removed from the medium at 60 min reoxygenation without impairing further recovery, indicating sustained improvement of
the underlying lesion.
It may be questioned how substrates, such as malate and fumarate, that
act primarily through pathway B, can protect when provided individually if complex I, which is an essential component for pathway B, is damaged. During hypoxia, we presume that the
presence of malate or fumarate, from the start of hypoxia, prevents the damage to complex I by maintaining m and, thereby,
allows continuing activity of pathway B. During
reoxygenation, the provision of succinate by these substrates would
bypass complex I, allowing recovery of ATP and
m to
effect repair of the respiratory chain defect. This consideration lends
further support to a major role for the aerobic-bypass pathway of
succinate metabolism in the protective effects during reoxygenation.
Neither acetoacetate, which induces a highly oxidized state of
mitochondrial pyridine nucleotides, nor -hydroxybutyrate, which
promotes their reduction (11), had substantial effects on
the mitochondrial dysfunction of the tubules. Oxidant injury has been
implicated in complex I dysfunction (20, 33), but we have
not found strong or consistent effects of antioxidants on the tubule
lesion (data not shown). Pyruvate is protective for ischemic myocardium
(29) and anoxic hepatocytes (5), has
important antioxidant effects during tissue injury (50), and could also feed into the CAC, but had no activity in our model by itself.
Glutamate alone had a surprisingly weak protective effect during
reoxygenation and was ineffective during hypoxia. We also tested
glutamine, which had no protective effect at all (data not shown).
Rabbit proximal tubules, in contrast to those from the rat and the dog,
produce glutamine from glutamate (9), which can shunt
glutamate away from -KG. Under oxygenated conditions, however,
substantial conversion of glutamate to
-KG by both transamination and glutamate dehydrogenase occurs simultaneously with glutamine synthesis in rabbit tubules (9). During hypoxia, glutamate dehydrogenase activity will be limited by the high
NADH/NAD+ ratio (39). We have been able to
enhance the protection provided by glutamate during both hypoxia and
reoxygenation by combining it with pyruvate, which will favor its
conversion to
-KG by transamination (data not shown).
The behavior of glutamate is of relevance to the question of whether
these substrate effects are already fully expressed during ischemia-reperfusion of the intact kidney due to the normal tissue content of metabolites. Glutamate is present at mM levels in renal cortex (61), but was relatively ineffective. In contrast,
levels of -KG in the circulation (10-30 µM), and renal cortex
(100-300 µM) (7, 26), are below or at the low end
of the effective concentration range shown in Fig. 3B. The
already low cortical levels of
-KG drop sharply during ischemia of
the rat kidney in vivo (26), and we have similar data for
the rabbit kidney (not shown), suggesting that, as in the isolated
tubules, glutamate and other endogenous substrates do not maintain
sufficient
-KG availability for protection of mitochondrial function
during injury conditions in vivo. Among the other protective
metabolites, aspartate, malate, fumarate, and citrate all approximate
0.5 to 1.0 mM in vivo (66). Further dose-dependence
studies will be necessary to assess whether these metabolites alone
or in combination are of benefit at these concentrations. However, the
persistent mitochondrial condensation during reoxygenation that
characterizes the lesion in the isolated tubules (64) is
seen in at least a subpopulation of proximal tubules during reperfusion
after clamp ischemia in vivo, including tubules in the cortex where
reperfusion is relatively complete (19). Cortical ATP
levels are also slow to recover during reperfusion (56).
This suggests that the benefits of CAC metabolites on mitochondrial
function are not fully provided by the available endogenous
substrates and that effective delivery of appropriate substrates could
improve recovery of mitochondrial function during ischemia-reperfusion
in vivo.
In summary, the present studies extend and complement our earlier work
(62, 64) to provide evidence for a pivotal mitochondrial defect during hypoxia-reoxygenation injury to proximal tubules involving impaired function of complex I and mitochondrial
deenergization. The mitochondrial dysfunction produces a severe
cellular energy deficit that is highly subject to modulation as a
function of the availability of specific CAC metabolites. Metabolism of
-KG to succinate by
-KG dehydrogenase, and cycling of electrons
in complexes I and II driven by conversion of fumarate to succinate, generate ATP and support
m anaerobically during
hypoxia to prevent the lesion. The same processes plus bypass of the
complex I block by succinate can reverse the lesion during
reoxygenation. The small direct effects on ATP production and
m from metabolism via the anaerobic pathways under
the injury conditions are then amplified with large increases of ATP
recovery and mitochondrial energization during continued reoxygenation,
reflecting a sustained global improvement of mitochondrial function
under aerobic conditions. These observations provide insight into a
critical factor that likely regulates the resistance of tubules to
ischemia-reperfusion injury and that may be subject to enhancement in
vivo. They are also highly pertinent to the use of isolated tubules for
studies of the mechanisms of sublethal structural changes during ATP
depletion states insofar as they allow assessment in a controlled and
highly manipulable fashion of more severe hypoxic insults followed by metabolic recovery, than would otherwise be possible.
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ACKNOWLEDGEMENTS |
---|
We appreciate the technical assistance of Magaly Abarzua, Rebecca Anderer, Julie Davis, Yuan Hua Wen, and Ilana Nissim.
![]() |
FOOTNOTES |
---|
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34275 and DK-39255, and Office of Naval Research Grant N00014-95-1-584 to J. M. Weinberg; National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37139 to M. A.Venkatachalam; and National Institute of Diabetes and Digestive and Kidney Diseases DK-53761 to I. Nissim.
Preliminary reports of some of the data appeared in abstract form as: J Am Soc Neprol 9: 591A, 1998.
Address for reprint requests and other correspondence: J. M. Weinberg, Nephrology Research, Rm. 1560, MSRB II, Univ. of Michigan Medical Center, Ann Arbor, MI 48109-0676 (Email: wnberg{at}umich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 February 2000; accepted in final form 19 July 2000.
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