Time course of lipopolysaccharide-induced nitric oxide synthase mRNA expression in rat glomeruli

Abstract

The decrease in glomerular filtration rate that is characteristic of sepsis has been shown to result from the local glomerular inhibition of endothelial nitric oxide synthase (NOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). iNOS activation depends on de novo synthesis of both RNA and protein. Therefore it is assumed that several hours are required for its full activation. Yet the renal hemodynamic response in sepsis has been documented as early as 60 minutes after lipopolysaccharide (LPS) administration. Experiments were designed to determine the time course of LPS-induced glomerular iNOS mRNA expression and activity in rats. Rats were treated with LPS (2 mg/kg body weight ip. Kidneys were removed after 1, 2, 4, 6, and 16 hours. Glomeruli were isolated and incubated. Nitric oxide generation was measured with a Griess assay, and iNOS mRNA was studied by reverse transcriptase-polymerase chain reaction. Similar time course experiments were repeated in glomeruli isolated from normal rats and exposed to LPS in vitro. A significant increase in iNOS mRNA expression was evident as early as 60 minutes after both in vivo and in vitro administration of LPS. The quantity of iNOS mRNA reached its peak between 2 to 4 hours after administration and declined to baseline levels after 16 hours. Immunohistochemical studies were remarkable for a significant increase in the staining for iNOS in glomeruli 2 hours after the in vivo administration of LPS. Plasma nitric oxide concentration after the in vivo administration of LPS increased from a baseline level of 11.25 ± 0.8 μmol/L to a peak level of 62.9 ± 3.8 μmol/L (P < .05 vs baseline) at 4 hours and then decreased to 17.5 ± 1.9 μmol/L at 16 hours. Similar results were obtained when the glomerular generation of nitric oxide after in vivo administration of LPS was measured (2.6 ± 0.8 pmol/h/μg tissue, 17.2 ± 2.1 pmol/h/μg tissue (P < .05 vs baseline), and 0.4 ± 0.65 pmol/h/μg tissue, respectively). These results provide evidence of the rapid activation of glomerular iNOS after in vivo and ex vivo administration of LPS and thus support the role of nitric oxide in the early renal hemodynamic response to LPS.