Pyruvate Attenuates Myoglobin in Vitro Toxicity

Monica A. Valentovic1 and Jennifer Minigh

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myoglobinuria is a complication of crush injury as well as substance abuse. This study examined whether pyruvate modified myoglobin in vitro renal toxicity. Renal slices from Fischer-344 rats were incubated for 120 min with 0–12 mg/ml myoglobn. In an initial study, gluconeogenesis was stimulated by the addition of 10 mM pyruvate during the final 30 min. In all other studies, renal slices were incubated with myoglobin in the presence of 0 or 10 mM pyruvate for 120 min. Myoglobin increased lactate dehydrogenase (LDH) release and this was not modified by the presence of pyruvate for the last 30 min of the incubation. Myoglobin toxicity was reduced by coincubation of myoglobin with pyruvate for 120 min. LDH leakage was increased 1.2-, 1.7-, and 1.8-fold above control by 4, 10, and 12 mg/ml myoglobin, compared to 1.2, 1.3, and 1.3 fold in slices coincubated with 10 mM pyruvate, respectively. Myoglobin diminished adenosine triphophate (ATP) levels but pyruvate maintained a 5x higher level of ATP within the slices. Glucose (10 mM) provided protection only for the low concentration (4 mg/ml) of myoglobin. Myoglobin induced oxidative stress while pyruvate prevented the rise in lipid peroxidation and glutathione disulfides by myoglobin. Myoglobin diminished total glutathione levels in pyruvate-treated tissue, but glutathione levels remained higher than tissues incubated in the absence of pyruvate. These results indicate that pyruvate reduced toxicity by preventing oxidative stress and via a supply of an energy substrate.

Key Words: myoglobinuria; substance abuse; rhabdomyolysis; renal dysfunction; pyruvate; lactate dehydrogenase; glutathione levels.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Crush injury with subsequent release of myoglobin induces acute renal failure. Myoglobinuria symptoms include muscle pain, brown discoloration of urine, and tubular casts (David, 2000Go). Historically, myoglobinuria and its effects on the kidney were first described during World War II (Bywaters and Beall, 1941Go). More recently, rhabdomyolysis and renal dysfunction have become a complication in clinical cases of drug overdose and substance abuse (Hampel et al., 1983Go; Loper, 1989Go). In one study, 24% of cocaine users that were treated in the emergency room had rhabdomyolysis (Welch et al., 1991Go). Individuals who abuse solvents by inhalation (huffing) and glue sniffers may also develop rhabdomyolysis (Kao et al., 2000Go).

Renal vasoconstriction and tubular obstruction are functional changes that occur with myoglobin-induced renal failure (Heyman et al., 1996Go; Vanholder et al., 2000Go Zager, 1996Go; ). Cellular alterations characterized in the in vivo glycerol rat model of myoglobinuria include increased free-radical generation and ensuing lipid peroxidation (Shah and Walker, 1988Go; Zager, 1992Go). 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, 1988Go). The generation of the highly toxic substance, hydrogen peroxide, is enhanced within 18 h in the glycerol rat model (Guidet and Shah, 1989Go). 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., 1991Go). 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, {alpha}-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, 1998Go). Pyruvate has been previously reported to afford protection from ischemia/reperfusion injury to the small intestine (Cicalese et al., 1996Go) and cardiac tissue (Bunger et al., 1986Go). 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., 1993Go).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Horse skeletal muscle myoglobin (Sigma, M-0630), deferoxamine mesylate, 2-thiobarbituric acid, reduced glutathione, and glutathione disulfide were obtained from Sigma Chemical Company (St. Louis, MO). The lactate dehydrogenase kit (Sigma, 228) and the Infinity glucose reagent kit (Sigma Kit #18) were purchased from Sigma Chemical Company. The solvents were high-pressure liquid chromatography (HPLC) grade. All other chemicals were purchased from various commercial sources.

Animals.
Male Fischer 344 rats (200–250 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 (21–23°C), humidity (40–55%) and 12-h light cycle (lights on 0600–1800 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., 1992Go) 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 (50–100 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 4–12 mg/ml was based on published reports in humans (Hamilton et al., 1989Go; Shigemoto et al., 1997Go) 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 0–12 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)Go as described previously (Minigh and Valentovic, 2003Go). 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 0.94–9.4 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, 1985Go). Glutathione disulfide (GSSG) was measured following 2-vinylpyridine derivatization (Griffith, 1980Go) 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., 2002Go). The amount of malondialdehyde (MDA) was calculated based on a standard curve (range 1–40 nmol) using MDA (Aldrich, St. Louis, Mo) and expressed as nmol MDA/g tissue.

Statistical analysis.
Values represent mean ± SEM with n = 4–5 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxic Effect of Myoglobin on Renal Slices
LDH leakage in vehicle-treated tissue was below 15% throughout all studies, indicating that the slices were viable. A 120-min exposure to 4–12 mg/ml myoglobin induced cytotoxicity within renal cortical slices. LDH leakage (Table 1Go) was increased relative to vehicle control values, in a concentration-dependent manner, by 4–12 mg/ml myoglobin. Pyruvate-stimulated gluconeogenesis was diminished by myoglobin when compared to control values. Renal slices were exposed for 90 min to myoglobin, followed by the addition of pyruvate and an additional 30 min incubation to stimulate gluconeogenesis. Pyruvate-stimulated gluconeogenesis was diminished by myoglobin at all concentrations and minimal levels of glucose formation were monitored following exposure to 10 and 12 mg/ml myoglobin.


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TABLE 1 The Effect of Myoglobin on Renal Slice LDH Leakage and Pyruvate-Stimulated Gluconeogenesis
 
Reduction in Myoglobin Toxicity by Pyruvate
Myoglobin cytotoxicity was reduced by concurrent incubation of renal slices for 120 min in the presence of 10 mM pyruvate (Fig. 1Go). Although LDH leakage was increased (p < 0.05) above vehicle-treated values by 4–12 mg/ml myoglobin; the extent of LDH leakage was diminished by pyruvate. Exposure to 12 mg/ml of myoglobin produced a 70% increase in LDH leakage relative to control, in the absence of pyruvate, compared to a 30% increase in LDH leakage when pyruvate was present. In addition, the concentration-dependent rise in LDH leakage was not observed when pyruvate and myoglobin were incubated concurrently for 120 min. Partial protection by pyruvate of myoglobin-induced increased LDH leakage was not observed in the initial studies in which pyruvate was added during the final 30 min of myoglobin exposure (Table 1Go), suggesting that either the duration or the late addition of pyruvate was not sufficient to modify LDH leakage.



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FIG. 1. Pyruvate reduces LDH leakage induced by myoglobin. Renal slices were exposed to the designated levels of myoglobin for 120 min as described in Materials and Methods. Slices were coincubated for the 120-min period in the presence of 0 (–PYR) or 10 mM pyruvate (+PYR). Values represent the mean ± SEM with n = 5/group. LDH leakage was expressed as percent of total release by 10% Triton X-100. Values with dissimilar superscripts have statistical differences (p < 0.05) within respective pyruvate treatments.

 
ATP levels were diminished (p < 0.05) in the absence of pyruvate, by exposure to 10 and 12 mg/ml myoglobin (Fig. 2Go). ATP levels in the 4, 10, and 12 mg/ml myoglobin-treated tissue were diminished to 90, 75, and 63%, respectively, of vehicle treated values. A 120-min incubation of renal slices with pyruvate maintained higher renal ATP levels when compared to slices incubated in the absence of pyruvate (Fig. 2Go). ATP depletion by myoglobin was partially reversed by the presence of pyruvate. ATP levels in tissue coincubated with pyruvate were diminished (p < 0.05) below control values only when slices were exposed to the highest myoglobin concentration of 12 mg/ml. ATP levels in the 4, 10, and 12 mg/ml myoglobin- and pyruvate-treated tissue were 97, 106, and 67%, respectively, of vehicle-treated values.



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FIG. 2. Myoglobin-induced changes in ATP levels. Renal slices were exposed to the designated levels of myoglobin for 120 min as described in Materials and Methods. Slices were coincubated for the 120-min period in the presence of 0 (–PYR) or 10 mM pyruvate (+PYR). Values represent the mean ± SEM with n = 5/group. Values with dissimilar superscripts have statistical differences (p < 0.05) within respective pyruvate treatments.

 
Effect of Glucose on LDH Leakage
Pyruvate is a substrate for gluconeogenesis in renal cortical slices. Further studies explored whether glucose generated from renal slices by gluconeogenesis reduced myoglobin toxicity. Renal slices were coincubated with myoglobin and 1.67 mM glucose. The level of glucose tested was selected based on previous studies in our lab, in which the level of glucose in the medium was monitored over time following the addition of pyruvate to untreated renal slices. The average glucose levels in 4 different control tissue experiments were 0.8 mM, 1.1 mM, and 1.36 mM at 60, 90, and 120 min after the addition of pyruvate. Although the average after a 120 min exposure to pyruvate was 1.36 mM, we selected the highest value (1.67 mM) in the range of data points. Based on this time study, experiments conducted with 1.67 mM glucose provide slices with exposure to glucose for a longer duration than that which occurs with pyruvate. Myoglobin induced a concentration-dependent rise in LDH leakage in the presence of 1.67 mM glucose (Fig. 3Go). These experiments indicated that glucose does not provide the same protection from LDH leakage as does pyruvate for myoglobin toxicity. Additional studies were conducted to examine myoglobin toxicity in the presence of 10 mM glucose (Fig. 3Go). Myoglobin did induce a concentration-dependent rise in LDH leakage in the presence of 10 mM glucose (Fig. 3Go). The addition of 10 mM glucose did prevent a rise in LDH release in slices exposed to 4 mg/ml of myoglobin, while myoglobin induced increased LDH release in the presence of 10 and 12 mg/ml myoglobin. These results suggest that glucose does not provide the same protection from myoglobin-induced LDH leakage as pyruvate.



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FIG. 3. The effect of glucose on myoglobin induced LDH leakage. Renal slices were incubated for 120 min with 1.67 mM or 10 mM glucose and the designated levels of myoglobin. Values represent the mean ± SEM with n = 4/group. LDH leakage was expressed as a percentage of total release by 10% Triton X-100. Groups with dissimilar superscripts have statistical differences (p < 0.05) within a specific glucose treatment.

 
Effect of Pyruvate on Myoglobin-Induced Lipid Peroxidation
Pyruvate did not alter baseline lipid peroxidation levels in control tissue. Lipid peroxidation was elevated (p < 0.05) in renal cortical slices exposed exclusively to myoglobin at concentrations of 4, 10, and 12 mg/ml relative to the control group (Fig. 4Go). The presence of pyruvate protected renal slices from myoglobin-induced lipid peroxidation. Lipid peroxidation was not elevated by any concentration of myoglobin tested in the presence of pyruvate. Additionally, pyruvate produced lower lipid peroxidation levels at all concentrations of myoglobin when compared to tissues exposed to myoglobin in the absence of pyruvate. These findings support a hypothesis that pyruvate reduced oxidative stress induced by myoglobin exposure.



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FIG. 4. Pyruvate protects against myoglobin induced lipid peroxidation. Lipid peroxidation was measured following 120-min exposure to 0–12 mg/ml myoglobin in the presence of 0 (–PYR) or 10 mM pyruvate (+PYR). Lipid peroxidation was expressed as nmol malondialdehyde (MDA)/g tissue. Values represent the mean ± SEM with n = 4/group. An asterisk (*) indicated a difference (p < 0.05) from +PYR group at the respective myoglobin concentration. Dissimilar superscript letters denote different (p < 0.05) groups within the respective pyruvate (PYR) treatment groups.

 
Pyruvate Protection against Myoglobin-Induced Glutathione Depletion
Exposure for 120 min to 4 and 12 mg/ml myoglobin decreased (p < 0.05) total glutathione levels below control values in tissues coincubated with or without pyruvate (Fig. 5Go). The presence of pyruvate increased baseline total renal glutathione levels relative to untreated tissue incubated in the absence of pyruvate. Despite a decrease in total glutathione levels by myoglobin in the pyruvate treated group, glutathione levels were maintained at a level higher to control tissue levels, in the absence of pyruvate, at 120 min. These results suggest that although myoglobin reduced total glutathione levels, the presence of pyruvate preserved glutathione status at a higher level.



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FIG. 5. Total glutathione (GSH) levels following 120 min exposure to myoglobin incubated with 0 or 10 mM pyruvate. Renal slices were exposed to 0, 4, or 12 mg/ml of myoglobin for 120 min, as described in Materials and Methods. Slices were coincubated for the 120-min period in the presence of 0 (–PYR) or 10 mM pyruvate (+PYR). Total glutathione (GSH) levels were expressed as nmol/g tissue. Values represent the mean ± SEM with n = 5/group. An asterisk (*) indicates a difference (p < 0.05) from respective 0 mg/ml control within PYR pretreatments. A (filled diamond) indicates a difference (p < 0.05) from tissue incubated without pyruvate (–PYR) within the designated myoglobin concentration.

 
Glutathione disulfide levels were elevated above control values in renal slices incubated for 120 min with myoglobin in the absence of pyruvate, suggesting induction of oxidative stress (Fig. 6Go). Glutathione disulfide levels remained comparable to control values following a 120-min exposure to myoglobin in the presence of pyruvate. These results suggest that pyruvate reduced myoglobin toxicity by preventing induction of oxidative stress by myoglobin.



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FIG. 6. Percent glutathione disulfide (GSSG) following 120 min exposure to myoglobin incubated with 0 or 10 mM pyruvate. Renal slices were exposed to 0, 4, or 12 mg/ml of myoglobin for 120 min as described in Materials and Methods. Slices were coincubated for the 120-min period in the presence of 0 (-PYR) or 10 mM pyruvate (+PYR). Glutathione disulfide (GSSG) levels were expressed as a percentage of total glutathione. Values represent the mean ± SEM with n = 5/group. An asterisk (*) indicates a difference (p < 0.05) from respective 0 mg/ml control within respective PYR pretreatments. A diamond (filled diamond) indicates a difference (p < 0.05) from tissue incubated without pyruvate (–PYR) within the designated myoglobin concentration.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Free radical generation and ensuing lipid peroxidation (Shah and Walker, 1988Go; Zager, 1992Go; Zager and Burkhart, 1997Go) are perceived as a vital component for manifestation of myoglobin-induced renal failure. The radicals involved in toxicity appear to be iron-dependent, since pretreatment with the iron chelator deferoxamine reduced glycerol-induced renal failure in Sprague-Dawley rats (Shah and Walker, 1988Go). Increased levels of the highly toxic substance, hydrogen peroxide, have been documented in the glycerol model of myoglobinuria (Guidet and Shah, 1989Go). Hydrogen peroxide is highly toxic, due to its physicochemical characteristics of easy diffusibility into cells and generation of toxic reactive oxygen metabolites, including superoxide anion and hydroxyl radicals.

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., 1993Go). 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, 2003Go) 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., 1983Go) or as an inducer of lipid peroxidation (Catherwood et al., 2002Go; Kashiwagi et al., 1996Go; Yoon and Kim, 1994Go). Glucose was reported to inhibit hydroxyl radical formation when added in vitro with an enzyme preparation of xanthine oxidase (Sagone et al., 1983Go). Oxidative stress was increased following addition of elevated glucose concentrations to cultured cell systems using endothelial cells (Kashiwagi et al., 1996Go) and mesangial cells (Catherwood et al., 2002Go). 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, 1985Go). 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)Go 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 {alpha}-keto acids can undergo nonenzymatic decarboxylation in the presence of a peroxide-generating CO2 and a byproduct (Holleman, 1904Go). 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.


    NOTES
 
1 To whom correspondence should be addressed. Fax (304) 696-7391. E-mail: alentov{at}marshall.edu. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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