Formation of lipid raft redox signalling platforms in glomerular endothelial cells: an early event of homocysteine-induced glomerular injury.
The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2*-) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91(phox) and p47(phox) in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91(phox) and p47(phox) are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs.
Yi
Fan
F
Department of Pharmacology & Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, VA, USA.
Jin
Si
S
Zhang
Fan
F
Xia
Min
M
Bao
Jun-Xiang
JX
Hu
Junjun
J
Poklis
Justin L
JL
Li
Pin-Lan
PL
D000818:Animals; D042783:Endothelial Cells / Q000166:cytology*; D006710:Homocysteine / Q000378:metabolism*; D007668:Kidney / Q000473:pathology*; D007678:Kidney Glomerulus / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley
D008055:Lipids; D008562:Membrane Glycoproteins; D006710:Homocysteine; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1