MECHANISMS OF LIPID A TOXICITY IN RENAL PROXIMAL TUBULES

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DESCRIPTION: (Adapted from the Investigator's Abstract) Acute renal failure is a frequent and often fatal consequence of severe bacterial infection in humans. The investigators's previous studies indicate that activation of the enzyme nitric oxide synthase (NOS) is responsible for the cytotoxic effects of bacterial endotoxin (lipopolysaccharide) in the kidney proximal tubule. Lipid A, the biologically active moiety of lipopolysaccharide, acts by releasing intracellular Ca2+ which in turn activates NOS resulting in NO generation. Although NO generation is detected within minutes, cell death occurs much 2+ later. Nevertheless, cytotoxicity can be prevented by (a) inhibiting Ca2+ release, (b) inhibitors of NOS, or (c) antioxidants. The proposed studies will address the hypothesis that early activation of NOS and subsequent oxidant stress are the major determinants of lipid A toxicity. In specific aims 1 and 2, the investigators will examine the biochemical events triggered by lipid A and NO that ultimately lead to cytotoxicity. In specific aim 3 they will examine the role of NOS activation and oxidant generation in a model of endotoxin-induced acute renal failure. To accomplish all of the above, the following specific aims are proposed; 1.) they will examine the role of Ca2+ in the activation of proximal tubule NOS using two complimentary approaches. The first is enzymatic characterization using a partially purified preparation. The second approach utilizes intact isolated tubules stimulated with lipid A. 2.) the investigators will examine the role of NO generation in the cytotoxicity of lipid A. The PI will evaluate the effectiveness of NOS inhibitors, NO scavengers and oxidant scavengers, and Ca 2+ release inhibitors on the development of sublethal to lethal injury. 3.) The PI will examine the role of NO in endotoxin-induced renal failure in vivo. Using a model of endotoxin-induced renal failure, they will examine the role of NO and oxidants in lipid A and lipopolysaccharide-induced acute renal failure that develop over a similar time course as in their in vitro studies. An understanding of the regulation of NOS activity and the consequence of NO generation is crucial to the understanding of the role of NO in proximal tubule pathophysiology and the development of new therapies to treat the human disease.