Department of Pharmacology, Marshall University School of Medicine, 1542 Spring Valley Drive, Huntington, West Virginia 25704-9388
Received February 3, 2003; accepted April 24, 2003
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ABSTRACT |
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Key Words: myoglobinuria; substance abuse; rhabdomyolysis; renal dysfunction; pyruvate; lactate dehydrogenase; glutathione levels.
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INTRODUCTION |
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Renal vasoconstriction and tubular obstruction are functional changes that occur with myoglobin-induced renal failure (Heyman et al., 1996; Vanholder et al., 2000
Zager, 1996
; ). Cellular alterations characterized in the in vivo glycerol rat model of myoglobinuria include increased free-radical generation and ensuing lipid peroxidation (Shah and Walker, 1988
; Zager, 1992
). The lipid peroxidation appears to be iron-dependent, since pretreatment with the iron chelator deferoxamine reduced glycerol-induced renal failure in Sprague-Dawley rats (Shah and Walker, 1988
). The generation of the highly toxic substance, hydrogen peroxide, is enhanced within 18 h in the glycerol rat model (Guidet and Shah, 1989
). Hydrogen peroxide is very toxic, due to rapid entry into cells and to its ability to generate toxic reactive oxygen metabolites in the presence of iron.
Pyruvate pretreatment protected glycerol-treated Sprague-Dawley rats from renal toxicity (Salahudeen et al., 1991). Glomerular filtration rate was maintained in glycerol-treated rats at levels comparable to control values by three treatments with pyruvate. Pyruvate afforded complete protection from the myoglobin-associated rise in lipid peroxide generation. These findings suggested that the mechanism for pyruvate protection could be attributed to diminished radical formation and not increased energy substrate. Pyruvate is a 3-carbon,
-keto acid that could act, via gluconeogenesis, as an energy substrate. However, the protective effect of pyruvate for myoglobin in vitro toxicity has been less convincing. Pyruvate was not able to modify lactate dehydrogenase release induced by myoglobin following a 24-h in vitro exposure of HK-2 cells (Zager and Burkhart, 1998
). Pyruvate has been previously reported to afford protection from ischemia/reperfusion injury to the small intestine (Cicalese et al., 1996
) and cardiac tissue (Bunger et al., 1986
). Deboer and colleagues (1993) showed a greater recovery to ischemia/reperfusion injury in rat hearts when tissues were re-oxygenated with a reperfusion solution containing 2 mM pyruvate and 11 mM glucose relative to a reperfusion medium containing only 11 mM glucose. The mechanism for pyruvate protection appeared to involve a reduction in free radical injury as the presence of pyruvate decreased the level of adducts detected in the perfusate when compared to perfusion media containing only glucose (Deboer et al., 1993
).
The purpose of the present study was to first examine the ability of pyruvate to modify myoglobin cytotoxicity in an in vitro model of myoglobin toxicity. The second objective was to examine the mechanism for pyruvate-induced protection of renal cortical slices from myoglobin. These studies determined that pyruvate reduced myoglobin toxicity through a reduction in radical generation and via supply of an energy substrate.
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MATERIALS AND METHODS |
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Animals.
Male Fischer 344 rats (200250 g) were obtained from Hilltop Lab Animals, Inc. (Scottsdale, PA). All rats were given a minimum 5-day acclimation period prior to initiation of any experiments. The University Committee on Animal Care and Use reviewed and approved the protocol for animal use. Animals were maintained under a controlled ambient temperature (2123°C), humidity (4055%) and 12-h light cycle (lights on 06001800 h). Animals were provided free access to tap water and Purina Rat Chow (chunks).
Preparation of myoglobin.
Horse skeletal muscle myoglobin (Sigma, M-0630) was used for all studies. Myoglobin (0, 60, 150, or 180 mg) was dissolved in 5 ml Krebs-Ringer buffer. To each beaker, 100 ml of ascorbic acid (35.2 mg/ml water) was added to convert metmyoglobin to myoglobin, and the solutions were sonicated (30 min) until a color change from brown to red indicated successful reduction of the iron in the myoglobin. A total amount of 1 ml of myoglobin was then added to the renal slices to yield a final concentration of 0, 4, 10, or 12 mg/ml of myoglobin in a total of 3 ml.
Incubation of renal slices.
Animals were anesthetized with diethyl ether and the abdominal aorta was cut to exsanguinate the animals. The kidneys were decapsulated, excised, quartered, and immediately placed in 5 ml of ice-cold Krebs Ringer buffer and kept on ice. Renal cortical slices were prepared freehand as described previously (Valentovic et al., 1992) and placed in 10 ml ice-cold Krebs buffer. The slices from one animal were transferred to 5 ml oxygenated Krebs buffer in a 30-ml beaker. The slices were rinsed 2 times in 5 ml oxygenated Krebs buffer, each for 3 min, at 25°C in an oxygen environment with constant shaking (100 cycles/min) in a Dubnoff metabolic shaker.
The tissue (50100 mg) was transferred to 2 ml oxygenated Krebs in designated Erlenmeyer flasks and equilibrated for 10 min at 37°C under oxygen and constant shaking (100 cycles/min, Dubnoff Incubator). Renal tissue was incubated for 120 min with a final myoglobin concentration of 0, 4, 10, or 12 mg/ml, which was added in 1 ml to yield a total incubation volume of 3 ml. Pilot studies had established that 2 mg/ml myoglobin was the threshold concentration needed to induce elevated lactate dehydrogenase (LDH) leakage relative to controls. The concentration range of 412 mg/ml was based on published reports in humans (Hamilton et al., 1989; Shigemoto et al., 1997
) and corresponds with moderate to severe levels of myoglobinuria. Values above 12 mg/ml were not assessed, due to problems with maintaining solubility of myoglobin at levels of 15 mg/ml or higher.
In an initial study, tissue was exposed to myoglobin for 120 min but gluconeogenesis was stimulated between 90 and 120 min by the addition of pyruvate (100 ml, final bath concentration 10 mM). Media and tissue were collected to measure LDH leakage and glucose generation. Tissues were blotted, weighed, and added to 10% Triton X-100 to release tissue LDH. LDH release into the media was expressed as percent of total LDH. Experiments in which control tissue LDH leakage exceeded 15% were discarded and not used in the final data analyses. Glucose was expressed as mg glucose/g tissue.
In further experiments, renal slices were incubated with 012 mg/ml myoglobin in the presence of 0 or 10 mM pyruvate. Tissues were incubated for 120 min at 37°C under oxygen and constant shaking (100 cycles/min) in a Dubnoff metabolic incubator. At the end of the incubation period, cytotoxicity was evaluated using various parameters.
LDH and glucose assays.
LDH release was expressed as a percent of total. Tissues were weighed, remaining tissue LDH measured following addition of 10% Triton X-100, and LDH determined (Sigma, Kit #228). Glucose was measured using a hexokinase enzymatic assay (Sigma, Kit #18) and expressed as mg glucose/g tissue.
Adenine nucleotides.
Renal tissue was blotted, weighed, and homogenized in 1 ml of Krebs Ringer buffer, pH 7.4. The homogenate was vortexed and 250 ml was combined with 125 ml of 3 N perchloric acid and equilibrated for 5 min. The samples were centrifuged for 10 min at 2000 x g in a Sorvall MC12V microcentrifuge. A 300-ml aliquot of the supernatant was adjusted to pH 7 and centrifuged for 10 min in a microcentrifuge. The supernatant was filtered through a 0.45-mm syringe filter (Millex-HV; Millipore Cat #SLVHR04NL). The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were determined using an HPLC method adapted from Lash and Jones, (1996) as described previously (Minigh and Valentovic, 2003
). A 100-ml aliquot of sample was injected into a Beckman Model 126 HPLC equipped with a Beckman 166 variable wavelength detector. The mobile phase was a gradient of two solutions: Solvent A is 100 mM potassium phosphate buffer, pH 6.0, and Solvent B is methanol. The gradient was 7.5 min at 100% A/0% B; 7.5 minute linear gradient to 90% A/10% B; 15 min linear gradient to 75% A/25% B; 0.5 min linear gradient to 100% A/0% B, followed by 10 min at 100% A/0% B. The flow rate was 1.3 ml/min of the mobile phase. The column was an 8 x 10 mm Radial Pak C18 cartridge (Waters, Inc., Milford, MA). The wavelength for detection was 254 nm. ATP levels were calculated using a standard curve of 0.949.4 nmol ATP.
Glutathione determination.
Tissues were homogenized in 0.5% sulfosalicylic acid and adjusted to a 1-ml volume. Total glutathione was determined using a glutathione reductase and ß-nicotinamide adenine dinucleotide phosphate, reduced (NADPH) coupled reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (Andersen, 1985). Glutathione disulfide (GSSG) was measured following 2-vinylpyridine derivatization (Griffith, 1980
) and expressed as nmol/g tissue. The criterion for viability in renal slices for glutathione studies was that control tissue must have a minimum total glutathione level of 200-nmol/g tissue after 120 min incubation.
Lipid peroxidation.
Lipid peroxide generation was measured in renal slices exposed to 0, 4, 10, or 12 mg/ml myoglobin for 120 min, as described previously (Valentovic et al., 2002). The amount of malondialdehyde (MDA) was calculated based on a standard curve (range 140 nmol) using MDA (Aldrich, St. Louis, Mo) and expressed as nmol MDA/g tissue.
Statistical analysis.
Values represent mean ± SEM with n = 45 animals/group. Differences between groups were analyzed using an analysis of variance (ANOVA) followed by a Newman Keuls test at a 95% confidence interval (Sigma Stat, SPSS, Inc., Chicago, IL). Differences within treatment groups were determined using a repeated measures-ANOVA followed by a Newman Keuls test.
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RESULTS |
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DISCUSSION |
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The present study investigated whether pyruvate would reduce the in vitro toxicity of myoglobin in renal cortical slices. The current study showed that simultaneous incubation of pyruvate with myoglobin reduced myoglobin cytotoxicity. The mechanism for pyruvate attenuation of myoglobin toxicity could be mediated by two mechanisms: (1) reducing oxygen-derived free radical damage and/or (2) metabolically, by providing additional energy within the cell.
We determined that myoglobin diminished ATP levels in a concentration-dependent manner. The addition of pyruvate increased baseline ATP levels in control tissue. The presence of pyruvate was associated with higher renal ATP levels despite exposure to myoglobin. Pyruvate may reduce toxicity by maintaining higher cellular ATP levels by decarboxylation to oxaloacetate, which is a substrate for the citric acid cycle and oxidative phosphorylation. Elevation of ATP levels by the addition of pyruvate were reported by other investigators to occur in cardiac tissue perfused with a solution containing 2 mM pyruvate and glucose (Deboer et al., 1993). ATP levels were increased approximately threefold in rat hearts following the addition of pyruvate to the perfusion media. Deboer and colleagues (1993) concluded that attenuation of toxicity by pyruvate was mediated by providing metabolic substrates and by reducing free radical damage.
Pyruvate is a substrate for gluconeogenesis. In a previous study, myoglobin-reduced pyruvate stimulated gluconeogenesis (Minigh and Valentovic, 2003) prior to inhibition of ATP levels. The inhibition of gluconeogenesis by myoglobin was substrate-specific for pyruvate. Compared to control, myoglobin exposure did not inhibit gluconeogenesis stimulated by two other rate-limiting substrates, glucose-6-phosphate and fructose-1,6-diphosphate. Thus myoglobin only inhibits the gluconeogenic pathway when stimulated by pyruvate. These results suggest that the reduction in myoglobin toxicity by pyruvate may involve more than providing a source of glucose.
Effects of Glucose on Myoglobin in Vitro Toxicity
Previous studies by other investigators have indicated that glucose can act either as a free radical scavenger (Sagone et al., 1983) or as an inducer of lipid peroxidation (Catherwood et al., 2002
; Kashiwagi et al., 1996
; Yoon and Kim, 1994
). Glucose was reported to inhibit hydroxyl radical formation when added in vitro with an enzyme preparation of xanthine oxidase (Sagone et al., 1983
). Oxidative stress was increased following addition of elevated glucose concentrations to cultured cell systems using endothelial cells (Kashiwagi et al., 1996
) and mesangial cells (Catherwood et al., 2002
). Based, on these studies, we explored the possible role of glucose to modify myoglobin toxicity. Glucose could elicit intracellular effects following entry into the proximal tubular cells via a sodium-independent transport system within the basolateral membrane (Ullrich and Papavassiliou, 1985
). It is unlikely that the mechanism for partial protection of myoglobin toxicity by pyruvate is due to a direct effect of glucose, since high levels (10 mM) of glucose provided protection only with the lowest myoglobin concentration.
Pyruvate Action to Reduce Oxidative Stress
We have provided evidence that pyruvate protected renal slices from myoglobin-induced oxidative stress. Exposure of renal slices to 4, 10, or 12 mg/ml myoglobin increased lipid peroxidation. These results are different from an earlier study by Zager and Burkhart (1998) in which pyruvate did not reduce myoglobin toxicity in HK-2 cells. It is possible that pyruvate was depleted during the 24-h experimental period using the HK-2 cells. Alternatively, the difference in results may be due to the nature of HK-2 cells relative to freshly prepared renal slices.
In the present manuscript, pyruvate prevented the induction of lipid peroxidation by myoglobin in renal slices. Pyruvate also prevented an accumulation of glutathione disulfide levels by myoglobin. Although total glutathione levels were diminished by myoglobin in groups treated with or without pyruvate, the total glutathione levels were higher in all groups treated with pyruvate. Pyruvate may increase glutathione levels by 2 mechanisms. First, pyruvate and other -keto acids can undergo nonenzymatic decarboxylation in the presence of a peroxide-generating CO2 and a byproduct (Holleman, 1904
). Through this direct mechanism, pyruvate can prevent the consumption of glutathione (GSH), since pyruvate will directly detoxify peroxides. For example, Salahudeen and coworkers (1991) reported that pyruvate directly scavenged hydrogen peroxide and diminished toxicity in LLCPK1 cells. A second mechanism for increased GSH is that pyruvate can be metabolized to acetyl CoA and citrate, which can generate NADPH through the citric acid cycle. NADPH, along with glutathione reductase, converts glutathione disulfide GSSG to GSH and would maintain an increased balance of GSH/GSSG.
Finally, our increase in GSH is consistent with other laboratories that examined another model of toxicity involving reactive-oxygen-radical damage. Mallet and coworkers (2002) observed an increase in the GSH/GSSG ratio when pyruvate was present in a cardiac-ischemia /reperfusion-injury guinea pig model. These results indicated that pyruvate reduced cardiac injury by maintaining sufficient levels of GSH in the reduced state.
It is apparent that, at least in vitro, pyruvate will reduce myoglobin toxicity. The findings from our study would support the importance of reducing oxidative stress in attenuation of myoglobin in vitro toxicity. There is insufficient data to suggest whether pyruvate may be part of a treatment regimen for crush or muscle injury since in vivo, the paradigm of myoglobinuria contains many factors including dehydration, renal vasoconstriction, oxidative stress cellular injury, and tubular obstruction.
Conclusions
Myoglobin was toxic to renal cortical slices obtained from Fischer-344 rats. Addition of pyruvate for the entire 120-min exposure time reduced myoglobin toxicity. Pyruvate addition during the final 30 min of exposure did not modify toxicity. Pyruvate attenuated toxicity by two mechanisms. First, pyruvate provided energy substrates to maintain cellular ATP levels. Second, pyruvate diminished the extent of oxidative stress induced by myoglobin. These results suggest that maintaining glutathione status will reduce myoglobin-mediated radical damage.
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NOTES |
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