Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes.
Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91(phox)) and cytosolic subunit (p47(phox)). Then, confocal microscopy showed that gp91(phox) and p47(phox) could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-beta-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91(phox) or LR-p47(phox) transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91(phox) and p47(phox) were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.
Zhang
Chun
C
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Hu
Jun-Jun
JJ
Xia
Min
M
Boini
Krishna M
KM
Brimson
Christopher
C
Li
Pin-Lan
PL
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D005372:Filipin / Q000494:pharmacology; D005434:Flow Cytometry; D006710:Homocysteine / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D050199:Podocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D012333:RNA, Messenger; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C113267:nephrin; D006710:Homocysteine; D005372:Filipin; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1