A Mechanistic Study of Myoglobin Nephrotoxicity

Myoglobin is an endogenous protein that can become nephrotoxic under certain conditions such as crush injuries, drug overdose, and seizures where prolonged contraction of muscle leads to cell death and leakage of myoglobin. The mechanism of myoglobin-induced nephrotoxicity is not fully understood. The purpose of this study was to characterize the sequence and mechanistic events associated with the in vitro toxicity of myoglobin in renal cortical slices. Renal tissue was isolated from Fischer 344 rats. Slices of renal cortex were prepared by freehand. These slices were then incubated for 60-180 minutes with myoglobin (0-12 mg/mL) pretreated with 4 mM ascorbic acid. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, pyruvate- stimulated gluconeogenesis, and lipid peroxidation. In addition, glutathione (total and oxidized) and ATP levels were measured. Toxicity was evident at one hour by changes in gluconeogenesis, lipid peroxidation, and glutathione levels. LDH release and a decline in ATP levels were not observed until two hours of incubation with myoglobin. In short, oxidative events (namely lipid peroxidation and changes in glutathione levels) and deficit of cell function (decreased gluconeogenesis) preceded loss of viability by one hour. Pretreatment of the slices with deferoxamine (DFX) afforded protection against oxidative events and loss of membrane integrity, but not of the decrease in cell function. This finding suggested an early bifurcation in the toxicity pathway with loss of cell function residing in one path and iron-dependent oxidative events followed by loss of membrane integrity in the other. Pretreatment of the slices with exogenous reduced glutathione provided protection of all toxic events, suggesting an underlying oxidative mechanism for the loss of gluconeogenesis that is iron-independent. Furthermore, the lack of protection against LDH release and loss of gluconeogenesis by pretreatment with dimethylthiourea indicated an absence of hydroxyl radicals in the mechanism of myoglobin toxicity in the slice model. Similar to DFX, pyruvate induced a general increase in the total glutathione levels and protection against lipid peroxidation. However, in contrast to DFX, pyruvate did not provide protection against the myoglobin-induced decline in total glutathione levels with respect to the control group. The protection provided by pyruvate appears to involve detoxification of radical pathways. A comparison of the toxicity of myoglobin with its components suggested that the iron is mostly involved with the loss of membrane integrity and slightly involved with the loss of cell function. Because DFX can detoxify ferryl myoglobin as well as chelate free iron, the protection of lipid peroxidation, changes in glutathione levels, and LDH release suggest that ferryl myoglobin also might participate in the oxidative events leading to loss of membrane integrity. In contrast, the heme portion of myoglobin might target the mitochondria, initiate the production of radicals, and lead to loss of cell function indicated by loss of gluconeogenesis. In contrast to mitochondrial-substrate stimulation of gluconeogenesis, cytosolic-substrate stimulation of gluconeogenesis was not affected by the presence of myoglobin. Taken together, the experiments presented in this study suggest that myoglobin targets mitochondria and produces toxicity predominately through oxidative damage. Moreover, this study establishes three unique events concerning myoglobin toxicity. First, early and late effects of myoglobin toxicity have been delineated. Other studies have reported LDH release, lipid peroxidation, and alterations in glutathione and ATP levels, but none has ever evaluated these parameters as a function of time. Secondly, this study establishes iron-dependent and iron-independent components of myoglobin toxicity. Lastly, because myoglobin toxicity was demonstrated in a renal model that has collapsed lumens, this suggests toxicity can occur independently of luminal events

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Retinoic acid decreases ATF-2 phosphorylation and sensitizes melanoma cells to taxol-mediated growth inhibition-0

A cells. B16 cells and Melan-a cells were harvested at 80% confluence, and cytoplasmic and nuclear protein were isolated using Pierce NE-PER™ nuclear and cytoplasmic extraction reagents described in Materials and Methods. Cytoplasmic and nuclear proteins (10 μg) from both cell lines were analyzed by western blotting using polyclonal ATF-2 antibody. The autoradiogram (top right panel) was scanned using a Molecular Dynamics densitometer, and after correcting for the amount of β-actin, the relative amount of total ATF-2 in each sample was determined (bottom). The data shown are from a representative experiment, which was replicated three additional times with similar results. The top left panel illustrates the morphology of the two different cell lines prior to harvest (phase contrast, 20X). B. Phosphatase digestion of ATF-2. Nuclear extracts prepared from B16 cells were treated with 2 units of PP1 at 37°C. The reaction was stopped by the addition of SDS-sample buffer at the indicated incubation times. The samples were boiled, then blotted and detected using phospho-ATF-2 antibody. GAPDH was used as an internal control. The data are representative of three individual experiments with similar results. C. Relative amount of phospho- ATF-2 in B16 cells vs. melan-a cells. Cellular extracts (20 μg) from B16 cells and cellular extracts (40 μg) from melan-a cells (minimal amount of protein that allowed signal detection of phosphorylated ATF-2) were analyzed by western blotting using anti-ATF-2 polyclonal antibody and anti-phospho-ATF-2 polyclonal antibody, as described in Materials and Methods. The autoradiogram (top) was scanned using a Molecular Dynamics densitometer, and after correcting for the amount of total ATF-2 protein, the relative amount of phospho-ATF-2 protein in both cell lines was determined (bottom). The data shown are from a representative experiment, which was replicated three additional times with similar results.<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid decreases ATF-2 phosphorylation and sensitizes melanoma cells to taxol-mediated growth inhibition"</p><p>http://www.jmolecularsignaling.com/content/3/1/3</p><p>Journal of Molecular Signaling 2008;3():3-3.</p><p>Published online 12 Feb 2008</p><p>PMCID:PMC2265711.</p><p></p