Date of Award

2002

Degree Name

Doctor of Philosophy

College

Graduate School of Education and Professional Development

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Gary O. Rankin

Second Advisor

Beverly C. Delidow

Third Advisor

Carl A. Gruetter

Fourth Advisor

Elsa I. Mangiarua

Abstract

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.

Subject(s)

Myoglobin -- Toxicology.

Nephrotoxicology.

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