Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells

Kidney Int. 2004 Nov;66(5):1977-87. doi: 10.1111/j.1523-1755.2004.00968.x.


Background: We recently demonstrated that homocysteine (Hcys) increases superoxide (O2-) production via NADH/NADPH oxidase in renal mesangial cells. This O2- production contributes to increased expression of tissue inhibitor of metalloproteinase (TIMP-1) and consequent deposition of collagen in response to Hcys. However, the mechanism by which Hcys activates NADH/NADPH oxidase remains unknown. Given that ceramide is an intracellular activator of this oxidase in several cell types, the present study tests the hypothesis that Hcys activates NADH/NADPH oxidase through a ceramide-mediated signaling pathway in rat mesangial (MG) cells, resulting in O2- production.

Methods: Rat MG cells were incubated with L-homocysteine (L-Hcys) to determine the mechanism by which Hcys activates NADH/NADPH oxidase. Thin layer chromatography (TLC), Western blot analysis, Rac GTPase activity pull down assay, and NADH/NADPH oxidase activity measurements were performed.

Results: TLC analysis demonstrated that L-Hcys increased de novo production of ceramide in MG cells. L-Hcys and increased ceramide did not alter the amount of NADH/NADPH oxidase subunit p47phox and p67phox in both membrane and cytosolic fractions from MG cells. However, L-Hcys or ceramide markedly increased the level of GTP-bound Rac, which was accompanied by enhanced activity of NADH/NADPH oxidase. These Hcys or ceramide-induced actions were substantially blocked by a Rac GTPase inhibitor, GDPbetaS, and a de novo ceramide synthesis inhibitor, fumonisin B1 (FB1).

Conclusion: These results indicate that Hcys activates NADH/NADPH oxidase by stimulating de novo ceramide synthesis, and subsequently enhancing Rac GTPase activity in rat MG cells. This ceramide-Rac GTPase signaling pathway may mediate Hcys-induced oxidative stress in these glomerular cells.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • Cells, Cultured
  • Ceramides / biosynthesis
  • Ceramides / physiology*
  • Chromatography, Thin Layer
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Fumonisins / pharmacology
  • Glomerular Mesangium / cytology
  • Glomerular Mesangium / enzymology*
  • Guanosine Diphosphate / analogs & derivatives*
  • Guanosine Diphosphate / pharmacology
  • Homocysteine / pharmacology*
  • Isoenzymes / metabolism
  • Multienzyme Complexes / metabolism*
  • NADH, NADPH Oxidoreductases / metabolism*
  • NADPH Oxidases / metabolism*
  • Rats
  • Sphingomyelin Phosphodiesterase / metabolism
  • Thionucleotides / pharmacology
  • rac GTP-Binding Proteins / antagonists & inhibitors
  • rac GTP-Binding Proteins / metabolism*


  • Ceramides
  • Enzyme Inhibitors
  • Fumonisins
  • Isoenzymes
  • Multienzyme Complexes
  • Thionucleotides
  • Homocysteine
  • Guanosine Diphosphate
  • fumonisin B1
  • guanosine 5'-O-(2-thiodiphosphate)
  • NADH oxidase
  • NADH, NADPH Oxidoreductases
  • NADPH Oxidases
  • Sphingomyelin Phosphodiesterase
  • rac GTP-Binding Proteins