Inducible nitric oxide synthase and apoptosis in murine proximal tubule epithelial cells

Abstract

Nitric oxide (NO), apoptosis, and proximal tubule dysfunction are known to be critical determinants of lipopolysaccharide (LPS)-induced renal failure in the mouse. The role of NO in apoptosis in murine proximal tubule cells (TKPTS cell line) was examined. An 18h treatment with a combination of LPS (5 μg/ml) and interferon-gamma (IFN, 100 units/ml) synergistically induced inducible nitric oxide synthase (iNOS) and produced a 20-fold increase in NO generation that was blocked by a specific iNOS blocker, L-N6–1-iminoethyl-lysine (L-NIL, 1mM). To assess the role of iNOS-derived NO in proximal tubule cell apoptosis, annexin/propidium iodide labeled cells were analyzed by flow cytometry. Neither the induction of iNOS nor its inhibition produced significant apoptotic cell death in the TKPTS cells. Two exogenous NO donors were also studied. Although both sodium nitroprusside (SNP), an iron-containing, nitrosonium cation donor, and S-nitroso-N-acetylpenicillamine (SNAP), a non-iron containing, NO generator, dose-dependently increased NO generation, only SNP dose-dependently increased apoptotic cell death in TKPTS cells (7.4 ± 1.3% apoptosis vs. 26.7 ± 3.1% apoptosis for control and SNP (500 μM), respectively; n = 5–6; P < 0.01). SNP-mediated tubule cell apoptosis was not dependent on the activation of caspases or p53 but was possibly related to and oxidative stress generated by SNP. Thus, in murine proximal tubular epithelial cells, induction of iNOS and generation of NO by LPS does not lead to tubular epithelial cell death. Studies were supported by a UAMS Pilot Study grant. K J Messer was supported by the ASPET SURF program.