Superoxide Generation by Renal Proximal Tubule Nitric Oxide Synthase

Abstract

Lipid A, the biologically active component of lipopolysaccharide, initiates a specific cytotoxic signaling cascade in the renal proximal tubule that involves a rapid release of intracellular calcium, the activation of nitric oxide synthase (NOS) and NO production. Superoxide (O2−) generation is also a component of this cascade and both NO and O2−are required for the development of oxidant stress and cytotoxicity. Here we examined whether NOS activity was responsible for O2−generation. In renal proximal tubules isolated from the rat, the NOS inhibitorNG-monomethyl-l-arginine (l-NMMA) but notd-NMMA blocked lipid A (50 μg/ml)-stimulated O2−generation as measured by the reduction of cytochrome c during a 30-min incubation period. Whenl-arginine (2 mM) was added to the tubule suspensions, O2−generation was significantly inhibited, while NO2−(a marker of NO generation) was significantly increased. The addition ofl-arginine also reduced lipid A-stimulated malondialdehyde formation at 30 min (a marker of lipid peroxidation) and lactate dehydrogenase release at 90 min (a marker of cell death). Thus, lipid A-stimulated the generation of both NO and O2−via NOS activation. Furthermore, increasingl-arginine availability shifted NOS activity toward NO generation and reduced oxidant injury. These results offer an explanation of why scavengers of NO or oxygen radicals ameliorate endotoxin-induced acute renal failurein vivo.