A Causative Role for Redox Cycling of Myoglobin and Its Inhibition by Alkalinization in the Pathogenesis and Treatment of Rhabdomyolysis-induced Renal Failure*

Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by ∼80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it’s concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.

In 1941 Bywaters and Beall (1) described four patients who developed acute renal failure, associated with dark brown urinary granular casts following crush injury. This syndrome has subsequently been termed rhabdomyolysis, since it occurs as a consequence of massive breakdown of muscle. It is usually associated with trauma, but may occur in a variety of clinical settings such as hyperthermia, seizures, muscle ischemia, or exposure to toxins such as alcohol or drug overdose. The associated muscle breakdown releases myoglobin (Mb) 1 into the circulation, which then becomes deposited in the kidney causing renal failure in 30% of cases. Rhabdomyolysis accounts for 7% of cases of acute renal failure in the United States (2,3).
The mechanism of the renal failure in rhabdomyolysis has been attributed to both intense renal vasoconstriction and renal tubular necrosis (4 -6). Recent studies have suggested a potential role for free radicals in the pathogenesis of myoglobinuria-induced renal failure based on the findings that in rats with rhabdomyolysis, levels of malondialdehyde in the kidney are increased (2-3-fold). There is an induction of antioxidant enzymes and consumption of antioxidants, and administration of antioxidants partially protects against the renal failure (7)(8)(9)(10)(11). The modest increases of malondialdehyde levels are difficult to interpret, since it is well recognized that measurements of aldehyde lack specificity as a marker of lipid peroxidation, and others have failed to replicate these findings (12). To explain how myoglobinuria could induce an oxidant injury to the kidney, it has been suggested that Mb may release free ferrous (Fe 2ϩ ) iron, which could then induce lipid peroxidation as a result of the generation of hydroxyl radicals via the Fenton reaction (13,14). However, it has been difficult to demonstrate hydroxyl radical production in the kidneys of animals with myoglobinuria and administration of desferrioxamine at doses that effectively chelate all free iron in the urine is only partially effective in preventing renal failure (15).
An alternative mechanism could involve the heme group in Mb itself, which can redox cycle between different oxidation states and promote lipid peroxidation reactions as a consequence of it's ability to decompose lipid hydroperoxides to peroxyl and alkoxyl radicals (16). This is achieved catalytically by a redox cycle between the ferric (Fe 3ϩ ) and ferryl ([FeϭO] 2ϩ ) Mb oxidation states, the latter of which can directly initiate lipid oxidation (17,18) (Fig. 1). In muscle cells, the iron in Mb is maintained predominantly in the reduced ferrous (Fe 2ϩ ) state, which is unable to participate in such reactions. However, the redox state of Mb in renal tubules has not been determined. We hypothesized that Mb in the extracellular milieu, i.e. in renal tubules, would undergo autooxidation to the ferric form (MetMb), which is then catalytically competent to promote lipid peroxidation reactions. Importantly, unlike the Haber-Weiss/Fenton reactions involving free iron, this reaction is not dependent on the generation of superoxide or hydrogen peroxide; it can be driven solely by endogenous lipid hydroperoxides (17)(18)(19)(20). This hypothesis may also explain the partial efficacy of desferrioxamine to protect against renal failure in the rat model of rhabdomyolysis, since desferrioxamine is capable of reducing ferryl-Mb and its associated globin radical to the ferric form (21,22). Interestingly, nitric oxide also partially protects against the renal failure (23). In accord with our hypothesis, this might be explained by an effect of nitric oxide to inhibit heme-dependent lipid peroxidation (24).
Mb-induced lipid peroxidation could explain the occurrence of renal tubular necrosis in myoglobinuria, but how this could be causally linked to the reduction in renal blood flow that also characterizes the nephropathy of rhabdomyolysis is not immediately obvious. One hypothesis that could be advanced is that vasoactive products of lipid peroxidation are generated that cause renal vasoconstriction. In this regard, one of the most attractive candidates would be the isoprostanes (IsoPs), which we previously identified as a group of prostaglandin (PG)-like compounds that are formed nonenzymatically in vivo as products of free radical-induced peroxidation of arachidonic acid (25). PGF 2 -, E 2 -, and D 2 -like compounds are formed in vivo by this process (25,26). Importantly, two IsoPs formed in abundance, namely 15-F 2t -IsoP (27) (8-iso-PGF 2␣ ) and 15-E 2t -IsoP (8-iso-PGE 2 ), are very potent renal vasoconstrictors that appear to interact with a unique receptor (25,28,29).
An intriguing question then arises as to whether an effect on oxidant injury could be mechanistically linked with the observation that alkalinization of the urine significantly attenuates the renal dysfunction (2,12). A plausible and accepted explanation is that alkalinization enhances the solubility of Mb and thus its elimination from the kidney (12). However, we considered an alternative explanation based on the recent demonstration that Mb-induced lipid peroxidation reactions are accelerated at acidic pH (30).
In this study, therefore, we examined the role of oxidative injury to the kidney in the pathogenesis of the renal failure associated with rhabdomyolysis in a rat model by measuring the specific marker of lipid peroxidation, F 2 -IsoPs, which has emerged as one of the most reliable approaches to assess oxidative stress status in vivo (31). For this purpose, F 2 -IsoPs were measured both in urine and in renal lipids. A novel aspect of the formation of IsoPs is that they are initially formed in situ esterified to tissue lipids and subsequently released (32). Since IsoPs generated in renal lipids would, in part, be directly released into the urine, measurement of urinary F 2 -IsoPs can provide an index of lipid peroxidation that occurs in the kidney (31). More direct evidence localizing oxidant injury to the kidney can also be obtained by measurement of levels of F 2 -IsoPs esterified in renal lipids. Furthermore, we explored the hypothesis that the protective effect of alkalinization operates by stabilizing ferryl-Mb, thus reducing its potential to induce lipid peroxidation.

MATERIALS AND METHODS
Preparation and Oxidation of Human LDL-Human LDL was isolated by differential centrifugation (33). Horse heart Mb (determined to be MetMb) was purchased from Sigma, Poole, Dorset, United Kingdom. MetMb (0 -10 M final concentration) was added to LDL (50 or 100 g/ml) in Hanks' buffered salts solution in which the pH was adjusted as indicated and incubated at 37°C for the time specified. Ferryl-Mb was prepared immediately before use by mixing 15 M hydrogen peroxide with 10 M MetMb. Control experiments involved incubation in phosphate-buffered saline alone or together with hydrogen peroxide (15 M). The reaction was terminated by addition of butylated hydroxytoluene (100 M). All experiments were performed in the presence of the iron chelator, DTPA (diethylenetriaminepentaacetic acid) (10 M), to exclude free iron-mediated oxidation. Electrophoretic mobility of LDL was determined using a lipoprotein electrophoresis system (Beckman Co). Gels were fixed, stained with Sudan black B, and results related to mobility of unoxidized LDL (33).
Animal Experiments-The rat model of glycerol-induced rhabdomyolysis used has been described previously (12). Animals were divided into three groups designated as controls, rhabdo, or rhabdo-alk. Control animals (n ϭ 8) were untreated. Animals in the rhabdo group (n ϭ 6) were injected with glycerol (10 ml/kg of a 50% solution in saline), which produces a nonlethal form of renal failure associated with heavy myoglobinuria. The third group (rhabdo-alk, n ϭ 6) was retreated for 24 h with water containing potassium bicarbonate (150 mM) and injected with potassium bicarbonate (2.5 ml/kg of 150 mM potassium bicarbonate) 1 h prior to injection with glycerol. Urine was collected from 0 to 24 h after injection of glycerol, after which the animals were sacrificed and kidneys snap-frozen in liquid N 2 for measurement of esterified F 2 -IsoPs, or frozen in isopentane (at Ϫ56°C), and stored at Ϫ80°C. For the purpose of measuring renal esterfied F 2 -IsoPs a further three animals in the rhabdo group and a further four animals in the rhabdo-alk group were also studied. Kidney frozen in isopentane was embedded in OCT medium (Tissue-Tek, Miles Inc., IN) and 10-m frozen sections cut onto glass slides for spectrophotometric analysis.
Extraction and Measurement of F 2 -IsoPs-Esterified F 2 -IsoPs in LDL and kidney and free F 2 -IsoPs in urine were extracted and quantified by stable isotope dilution mass spectrometric assay as described, except [ 2 H 4 ]8-iso-prostaglandin F 2␣ (Cayman Chemical Co., Ann Arbor, MI) was used as the internal standard (34). Urinary excretion of F 2 -IsoPs was normalized to creatinine clearance to correct for the widely differing degrees of renal function, i.e. excretion rate/creatinine clearance. This is calculated as shown below and results expressed as pg/ml Cr.Cl. Optical Spectroscopy Analysis of Mb in the Kidney-Examination, under a microscope of thin (10 m) sections of rhabdomyolytic kidney showed, in contrast to normal kidney sections, a red/brown pigment to be located within the tubules. This pigment could easily be obtained from the mounted section by washing the 10-m sections with 200 l of sodium phosphate buffer, pH 7.4, containing 10 M DTPA. This extract was diluted to 1 ml with the same buffer. The spectrum of the extract from the kidney was compared with purified MetMb. Sodium dithionite (1 mM) was added to produce the deoxy form for comparison with purified deoxy-Mb. Optical spectroscopy was performed using a Varian Cary 5E UV-visible spectrophotometer.
Electron Paramagnetic Resonance (EPR) Spectroscopy Analysis of Mb in the Kidney-Rat kidney was cut into pieces of similar size and frozen in liquid nitrogen. One EPR sample, always made of one kidney, consisted of 20 -30 such pieces (ϳ0.3 g). When placed directly into a Bruker finger Dewar (inner diameter ϭ 4.5 mm) without using an EPR tube, such a sample occupied the volume of the Dewar, the length of which was always greater than the working zone of the cavity, 22 mm. This ensured that an EPR spectrum was always taken of the same volume (the cavity working zone) fully filled with the sample, the packing of this volume with tissue pieces being the only variable factor. This packing factor, however, was not a problem, since an EPR spectrum taken of the same sample after reloading into the Dewar (thus providing a different packing) differed by less than 5% in signal intensity. Furthermore EPR spectra of samples made from different parts of the same kidney differed by less than 10% in signal intensity. The tissue pieces were retained in the finger of the Dewar with a quartz rod to avoid movement of the pieces caused by liquid nitrogen boiling. Since sufficient kidney tissue was used to extend the sample beyond the working zone, the rod did not cause a problem with either frequency tuning or induce any background signals. All EPR measurements were performed at 77 K. The EPR spectra were measured on a Bruker EMX spectrometer with an ER 041XG microwave bridge (X-band). A 4103TM cavity was used. The experimental conditions comprised: microwave frequency, 9.2689 GHz; microwave power, 3.16 milliwatts; modulation frequency, 100 kHz; modulation amplitude, 7 G; time constant, 0.041 s; sweep time, 84 s; receiver gain, 104; number of scans, 4; data points, 2048/scan.
Where integration of the EPR signals was possible, the concentrations are quoted in M by comparing the double integrals to that of known standards separately measured in an EPR tube (35). Due to uncertainties in comparing signal intensities from different types of EPR sample (tissue slice placed directly in the Dewar versus frozen standard in EPR tube), we estimate that the systematic error of these absolute measurements (in terms of M spins) could be as high as 50%, but the relative intensities of the signals in controls versus rhabdomyolytic kidneys are accurate to within 5%. When integration of the EPR signal was not straightforward due to overlapping signals, the signal intensity was normalized to the average intensity seen in the control rats. It is important to note that it is not the peak intensity, but pure line shape second integral, that is proportional to concentration. The low spin ferric signals are much wider than the rhombic signal at g ϭ 4.3; therefore, at equal concentrations of the two centers, the peak intensity of the low spin signal will be smaller. In addition the EPR absorbance "efficiency" of the paramagnetic centers (EPR signal second integral/concentration) depends on the respective g factors.
Assessment of the pH Dependence of Lipid Hydroperoxide Consumption by Mb and Decay of Ferryl-Mb-A solution of MetMb (10 M) was prepared and the reaction initiated by addition of 40 M 13(S)-hydroperoxy-9(E),11(Z)-octadecadienoic acid (13-HPODE) (Cascade Biochem, Reading, United Kingdom). Consumption of 13-HPODE was followed by monitoring the loss of the conjugated diene chromophore at 234 nm, which occurs as 13-HPODE is converted to an epoxy peroxyl radical (Fig. 1). The single exponential progress curves were fitted by a regression analysis. The pH dependence of the decay of ferryl-Mb was determined by mixing 10 M MetMb with 20 M hydrogen peroxide to form ferryl-Mb, and the decay of ferryl-Mb was followed by monitoring the increase in absorbance at 408 nm due to reformation of MetMb.
Statistics-Urinary F 2 -IsoP excretion in animals and esterified F 2 -IsoPs in LDL were compared between groups using the nonparametric Mann Whitney U test and were considered significant when p Ͻ 0.05. Where mean values are reported, the S.E. is also indicated. For changes in creatinine clearance, the unpaired t test was used.

Formation of F 2 -IsoPs during Oxidation of LDL by
MetMb-We initially examined the capacity of Mb to induce the formation of F 2 -IsoPs using LDL as a model lipid containing system. MetMb (10 M) caused marked lipid peroxidation of LDL resulting in a ϳ50-fold increase over base line in esterified levels of F 2 -IsoPs (p Ͻ 0.01). This was accompanied by a corresponding increase in the relative electrophoretic mobility of the LDL, indicative of significant oxidative modification of ApoB-100 (Table I) (Table II). However, ferryl-Mb treatment resulted in the formation of approximately 3-fold higher concentrations of F 2 -isoprostanes than MetMb. Table II also shows that ferryl-Mb treatment resulted in a greater degree of ApoB-100 modification confirming pervious literature reports that ferryl-Mb is a more potent inducer of lipid peroxidation than MetMb (17). For comparison with Table I it should be noted that the greater amounts of F 2 -isoprostanes generated in the first experiment are due to a prolonged incubation period (24 h) compared with the data in Table II (8 h).
Studies in the Rat Model of Rhabdomyolysis-Following induction of rhabdomyolysis in the rat, there was a profound reduction (mean 76.4%) in creatinine clearance in the rhabdo group compared with the control group (p Ͻ 0.02, Fig. 2). Alkalinization of the urine significantly attenuated the fall in creatinine clearance (p Ͻ 0.02, rhabdo versus rhabdo-alk, Fig.  2A). The urinary excretion of F 2 -IsoPs was also markedly increased (mean 7.3-fold) in the rhabdo group compared with the control group (p Ͻ 0.01, Fig. 2B). Of considerable interest was the finding that the enhanced urinary excretion of F 2 -IsoPs in the rhabdo group was markedly suppressed (mean 78%) by alkalinization (p Ͻ 0.01, rhabdo versus rhabdo-alk) to an extent that the difference between urinary levels of F 2 -IsoPs in the control and rhabdo-alk groups were not significant (p ϭ 0.17, Fig. 3B). Data for urinary F 2 -isoprostanes were corrected for creatinine clearance, since we have found that urinary excretion rate decreases in parallel to creatinine clearance. 2 By comparison, if corrected for urinary creatinine alone, the mean urinary excretion was 33 Ϯ 6 pg/mg creatinine in the controls and 203 Ϯ 42 and 26 Ϯ 12 pg/mg creatinine in the rhabdo and rhabdo-alk groups, respectively (p Ͻ 0.02 for rhabdo versus other groups). Urinary excretion rates of F 2 -IsoPs were 19 Ϯ 4, 19 Ϯ 6, and 14 Ϯ 2 ng/day in the control, rhabdo, and rhabdoalk groups, respectively (no significant difference between groups). However, the data when presented as urinary excretion rate alone does not reflect the obvious increase in lipid peroxidation observed in the kidney. Thus, levels of F 2 -IsoPs esterified in renal lipids were also found to be strikingly elevated (mean 7.5-fold) in the rhabdo group compared with the control group (p Ͻ 0.01, Fig. 3) and was significantly attenuated by alkalinization (p Ͻ 0.05). These findings unequivocally    Table I it should be noted that the greater amounts of F 2 -isoprostanes generated in Table I are due to a prolonged incubation period (24 h) compared with the data in Table II  established the occurrence of oxidant injury in the kidney in this animal model of rhabdomyolysis. Following induction of rhabdomyolysis in the rat, deposition of Mb in the medulla and inner cortex of the kidney is apparent by visual inspection. The spectrum of an extract washed from a rhabdomyolytic kidney section is shown in Fig. 4A. The major feature is the Soret peak at 406 nm, typical of MetMb (36,42). The visible region (inset) shows peaks at approximately 502 and 630 nm, also characteristic of MetMb. The peaks at 540 and 580 nm may be assigned to a small contribution from oxy-Mb (ϳ10% of the total Mb) or to sulf-Mb, both of which have absorption bands at these wavelengths. Analysis following reduction and deoxygenation of the Mb extract with sodium dithionite is shown in Fig. 5B. Both a red shift of the Soret peak from 406 to 430 nm and the appearance of the peak at 550 nm in the visible region indicate the conversion of Mb to the deoxy ferrous form. However, the absorbance peak at 620 nm remains after the addition of dithionite. This absorbance band is consistent with the interaction of sulfide or sulfur-containing compounds, e.g. glutathione, with the porphyrin ring in ferryl-Mb forming sulf-Mb (37). An alternative explanation is that the band at 620 nm in the deoxy form is due, as reported by Catalano et al. (38), to cross-linking of the heme to the protein mediated by ferryl-Mb. Whether the presence of this band at 620 nm is due to sulf-Mb or to hemeprotein cross-linking, it is highly likely that the Mb has passed through the ferryl state, since this intermediate is required for the formation of both species.
The ferric oxidation state of Mb (Fe 3ϩ ), but not the ferrous or ferryl forms, is detectable by EPR spectroscopy. Thus quantitative changes in the MetMb concentration can be measured using this technique. Significant changes were seen in EPR spectra from the kidneys of rhabdomyolytic rats (Fig. 5B) compared with controls (Fig. 5A). These data have been quantified and the results reported in Table III. Specifically, there was a large increase in the g ϭ 6 high spin ferric heme signal, corresponding to metmyoglobin, and a low spin heme signal at g x ϭ 2.58; g y ϭ 2.28 (Table III). At the same time a new rhombic iron signal with the same g ϭ 4.3 but unknown ligands appears in the spectrum from the rhabdomyolysic kidneys, suggestive of an increase in low molecular weight ferric pools in the rhabdomyolytic kidneys.
Decreases are seen in the other cellular EPR signals, e.g. cytochrome P450 and iron-sulfur enzymes, which may be explained as a result of general cellular damage and associated edema in the kidney. The low spin heme signal evident in the spectra B and C has different characteristics to cytochrome P450 with a different shape and slightly different g2. Since the high spin form of MetMb is very much enhanced in spectra B and C, it is likely to be the source of this new signal. Alkalinization had no significant effect on any of the components of the EPR spectra in the rhabdomyolytic kidneys, including the size of the new iron signal at g ϭ 4.3 (Fig. 5, C and B, Table II). In particular there was no change in the concentration of MetMb following alkalinization (Table III).
Effect of Alkalinization on LDL Oxidation-The hypothesis that the oxidative potential of Mb to induce lipid peroxidation is pH-dependent was explored using MetMb-induced oxidation of LDL in vitro. LDL (three preparations) were incubated with 5 M MetMb at a pH range of 5.5-8.0. The results obtained indicate that Mb-induced formation of F 2 -IsoPs is highly pHdependent, being markedly suppressed at alkaline pH (Fig. 6).

Effect of Alkalinization on the Stability of Ferryl-Mb and Consumption of Lipid Hydroperoxides-Oxidation of lipids by
Mb is a lipid hydroperoxide-dependent process (17). To further probe the mechanism for the pH dependence of Mb-induced lipid oxidation, consumption of the lipid hydroperoxide, 13-HPODE, by Mb was assessed as a function of pH. Calculated rate constants for consumption of 13-HPODE by MetMb at different pH values is shown in Table IV. The pH profile shows that consumption of 13-HPODE is low at pH values above 7 but increased 35-fold with a reduction in pH from 8.0 to 5.0. This profile is consistent with the pH dependence for the formation of F 2 -IsoPs in LDL (Fig. 6). To determine whether alkalinization stablilized the decay of ferryl-Mb, ferryl-Mb was incubated as above and the decay rate of ferryl-Mb followed spectrophotometrically as a function of pH. The decay rate of ferryl- Mb   FIG. 2. Effect of alkalinization on renal function and urinary F 2 -IsoP excretion in rats with rhabdomyolysis. A, creatinine clearance in normal rats and in rats with glycerol-induced rhabdomyolysis (Rhabdo) and the effect of alkalinization (Rhabdo-Alk). ** denotes p Ͻ 0.02 for rhabdo versus normal (Norm); * denotes p Ͻ 0.02 for rhabdo-alk versus rhabdo; # denotes p ϭ 0.048 for rhabdoalk versus normal (n ϭ 5 or 6 in each group). B, urinary excretion of F 2 -IsoPs in normal rats and in rats with rhabdomyolysis and the effect of alkalinization. The ** denotes p Ͻ 0.01 for rhabdo versus normal; * denotes p Ͻ 0.01 for rhabdo-alk versus rhabdo; # denotes p ϭ 0.17 for rhabdo-alk versus normal. was markedly reduced by 450-fold at pH 8.0 compared with that at pH 5.0 (Table IV). DISCUSSION The mechanistic basis for the pathogenesis of the renal failure of rhabdomyolysis is poorly understood. The results obtained in this study demonstrating striking increases in the urinary excretion of F 2 -IsoPs in rats with rhabdomyolysis provide compelling evidence that lipid peroxidation is a feature of rhabdomyolysis. A finding of elevated levels of F 2 -IsoPs in the urine is highly suggestive, albeit not definitive, evidence that this resulted from oxidant injury to the kidney. However, we found that levels of F 2 -IsoPs esterified in renal lipids of rats with rhabdomyolysis were also markedly increased, which unequivocally localizes the occurrence of oxidant injury in the kidney. Importantly, we also found that alkalinization of the urine, which protects against renal failure, significantly suppresses the urinary excretion of F 2 -IsoPs, supporting a causative link between oxidant injury and the renal dysfunction.
The molecular mechanisms by which Mb deposited in the kidney induces oxidant injury could occur as a result of the FIG. 5. The EPR spectra of the kidney from rats with rhabdomyolysis and controls. The spectra represent the averaged spectra obtained from four to six samples (i.e. four to six animals) shown as a function of the magnetic field strength in Gauss (G). Spectrum A, normal rat kidneys (n ϭ 4); spectrum B, kidneys from rats with rhabdomyolysis (n ϭ 6); spectrum C, kidneys from rats with rhabdomyolysis and alkalinization (n ϭ 6). The EPR spectrum of normal rat kidney (A) comprises a number of signals: the high spin ferric heme signal at g ϭ 6, the signal at g ϭ 4.3 from the non-heme ferric iron with rhombic symmetry (serum transferrin and low molecular weight non-protein-bound iron coordinated with HCO 3 Ϫ ), the signal arising from the low spin ferric heme in cytochrome P450 (g x ϭ 2.42 and g y ϭ 2.25), the Mn 2ϩ signal (a six-line signal centered at g ϭ 2 of which only the first three components are visible in this spectrum), the free radical (FR) signal (g ϭ 2.003), the Mo 5ϩ signal (g ϭ 1.97), and the signals from the mitochondrial iron-sulfur (IS) enzymes NADH dehydrogenase and succinate dehydrogenase (g ϭ 1.94 -1.86). Two new signals appear In the rhabdomyolysis kidneys (spectra B and C), which are not apparent in the control kidneys (spectrum A). First there is a low spin ferric heme iron with g x ϭ 2.58; g y ϭ 2.28. This is most likely to be a low spin form of metmyoglobin and has similar g values to the MetMb-sulfide complex described in Ref. 37. The second new signal becomes apparent following subtraction of the control signal from that in the rhabdomyolytic kidneys in the g ϭ 4.3 region (spectrum B-A) and is due to a new high spin rhombic ferric iron component. The g values for the EPR signals are indicated near corresponding vertical bars.

FIG. 4. Optical spectra of an extract of Mb from rhabdomyolytic kidney before (A) and after reduction and deoxygenation with sodium dithionite (B).
The inset shows the expanded region from 450 to 700 nm. Spectrum A contains peaks characteristic of MetMb, i.e. 406, 502, and 630 nm. Spectrum B is typical of deoxy ferrous Mb exhibiting peaks at 430 and 550 nm. A peak at 620 nm, indicative of sulf-Mb, is present in both spectrum A and spectrum B, but partially masked by the MetMb peak in spectrum A.
release of free iron from Mb or, as suggested in the current study, from a direct pro-oxidant effect of the heme protein itself. In order for Mb to catalyze lipid peroxidation, ferrous Mb must be oxidized to the ferric form, which is competent to induce lipid peroxidation by redox cycling with ferryl-Mb. This is a highly reactive form of Mb, which can potently induce lipid peroxidation (Tables I and II). In support of the latter hypothesis, we found that the redox form of Mb deposited in the kidney is MetMb.
Importantly, in addition to explaining the occurrence of renal tubular necrosis, oxidant injury may also provide a plausible explanation for the occurrence of renal vasoconstriction in this disorder, which contributes to the renal dysfunction. The finding that IsoPs are markedly overproduced in myoglobinuria is potential highly relevant in that both 15-F 2t -IsoP and 15-E 2t -IsoP are very potent renal vaoconstrictors (25,26,28). Furthermore, 15-F 2t -IsoP has been shown to induce secretion of endothelin-1 (39), which is also a potent renal vasoconstrictor. Perhaps relevant to the latter finding is the recent report that bosentan, a combined Endothelin A and B receptor antagonist, partially protects against renal failure in rhabdomyolysis (40). Thus, although it remains to be proven, it is attractive to speculate that overproduction of IsoPs may be responsible for the intense renal vasoconstriction that occurs in myoglobinuria.
Our findings also provide novel insights into mechanism by which alkalinization of the urine protects against the renal failure of rhabdomyolysis. The accepted explanation is that alkalinization enhances the solubility of Mb, and thus its excretion from the kidney (12). Clearly, this could explain the finding that alkalinization reduced urinary excretion of F 2 -IsoPs. However, EPR analysis failed to detect a reduction in the amount of high spin heme (MetMb) deposited in the kidney following treatment with alkali. Thus, an alternative explanation was considered based on the hypothesis for an effect of alkaline pH to reduce the capacity of Mb to catalyze lipid peroxidation. This hypothesis was supported by the finding that Mb-induced formation of F 2 -IsoPs in LDL was increasingly suppressed with increasing pH over the range of 5.5-8.0. Further studies undertaken in an attempt to elucidate the mechanistic basis for the pH dependence of the capacity of Mb to induce lipid peroxidation revealed that the rate of the ferryl-Mb to ferric Mb transition is strongly pH-dependent, being very low at pH Ͼ7.0, indicating stabilization, i.e. reduced reactivity, of ferryl-Mb at alkaline pH. The reason for this pH dependence may reside in the fact that the protonated form of ferryl heme (Fe(IV)OH Ϫ ) is the reactive species, which is formally equivalent to a hydroxyl radical coordinated to ferric heme. It is important to note that renal tubular fluid is relatively acidic and the pH of the surrounding interstitium decreases further during low blood flow states (41). Thus, MetMb deposited in the renal tubules is in a milieu that greatly promotes the ability of Mb to induce lipid peroxidation.
In summary, a number of important new insights regarding the mechanism of the pathogenesis of the renal failure of rhabdomyolysis have emerged from these studies. First, compelling evidence was obtained for the occurrence of oxidant injury to the kidney in rhabomyolysis. Second, the observation that IsoP production is increased may explain the occurrence of intense renal vasoconstriction in this disorder. Third, the identification of MetMb as the principal redox form of Mb deposited in the kidney provides a mechanistic basis to explain how myoglobinuria can cause lipid peroxidation independent of free iron and conventional Fenton reactions. Fourth, data obtained suggest that the mechanism by which alkalinization protects against renal failure is not by increasing solubility of Mb as previously proposed, but is consistent with stabilization of the highly reactive ferryl-Mb. These findings may have wider implications to the general functioning of heme proteins during  MetMb and decay of ferryl-Mb Consumption of 13-HPODE was followed by monitoring the loss of the conjugated diene chromophore at 234 nm, which occurs as 13-HPODE is converted to an epoxy peroxyl radical. The pH dependence of the decay of ferryl-Mb was determined by mixing 10 M MetMb with 20 M hydrogen peroxide to form ferryl-Mb and the decay of ferryl-Mb was followed by monitoring the increase in absorbance at 408 nm due to reformation of MetMb.  and kidneys from rats with rhabdomyolysis with alkalinization No significant difference was seen in any EPR signal between the rhabdomyolysis kidney and the rhabdomyolysis ϩ alkalinization kidney. The maximum concentration limit of signals that were undetectable in the control kidney was determined by comparing the estimated signal size at different concentrations with the noise in the EPR spectrum. Concentrations were established from the second integrals of pure line shapes of the signals obtained under non-saturating conditions. These second integrals were compared with those of standards at known concentrations.  changes of intracellular pH and provide a rationale to further explore whether the administration of antioxidants in conjunction with alkaline therapy might have an additive effect in preventing renal injury as suggested by others (8). This would impact significantly on our ability to preserve renal function in patients with rhabdomyolysis.