Ceramide is known to play a role in the cell signaling pathway involved in apoptosis. Most studies suggest that enhanced
ceramide generation is the result of hydrolysis of
sphingomyelin by sphingomyelinases. However, the role of
ceramide synthase in enhanced
ceramide generation has not been previously examined in
hypoxia-reoxygenation injury. In the present study, we demonstrated that 60-min
hypoxia of rat renal tubular epithelial NRK-52E cells in a gas chamber with 95% N2-5% CO2 with
glucose deprivation resulted in a significant increase in
ceramide generation. The
ceramide level further increased after reoxygenation for 60 min. Exposure of cells to
hypoxia-reoxygenation resulted in a significant increase in
ceramide synthase activity without any significant change in
acid or neutral
sphingomyelinase. The
hypoxia-reoxygenation of NRK-52E cells was also associated with the release of
endonuclease G (EndoG) from mitochondria to cytoplasm measured by Western blot analysis and
endonuclease activity assay. It further led to the fragmentation of
DNA and cell death. A specific inhibitor of
ceramide synthase,
fumonisin B1 (50 microM), suppressed
hypoxia-reoxygenation-induced
ceramide generation and provided protection against
hypoxia-reoxygenation-induced EndoG release, DNA fragmentation, and cell death. Taken together, our data suggest that
hypoxia-reoxygenation results in an activation of
ceramide synthase rather than
sphingomyelinase and that
ceramide synthase-dependent
ceramide generation is a key modulator of EndoG-mediated cytotoxicity in
hypoxia-reoxygenation injury to renal tubular epithelial cells.