Homocysteine enhances TIMP-1 expression and cell proliferation associated with NADH oxidase in rat mesangial cells.
BACKGROUND
Recent studies in our laboratory demonstrated that chronic hyperhomocysteinemia (hHcys) induced glomerular sclerosis. The mechanism mediating hHcys-induced glomerular damage remains unknown. The present study was designed to test a hypothesis that homocysteine (Hcys) increases the production by nicotinamide adenine dinucleotide (NADH) oxidase and thereby stimulates the formation of tissue inhibitor of metalloproteinase (TIMP-1) in rat mesangial cells, consequently leads to glomerulosclerosis.


METHODS
Rat mesangial cells were incubated with L-homocysteine (L-Hcys) to determine the effects of Hcys on cell proliferation and metabolism of extracellular matrix (ECM). Northern blot, Western blot, oligonucleotide transfection, measurements of NADH oxidase activity and levels, and cell proliferation assay were performed.


RESULTS
In cultured rat mesangial cells, treatment with L-Hcys (40 to 160 micromol/L) markedly increased the mRNA levels of TIMP-1 and Gp91 and led to accumulation of collagen I, which were accompanied by enhanced cell proliferation and NADH oxidase activity in mesangial cells. These Hcys-induced biochemical and functional changes were substantially blocked by a NADH oxidase inhibitor, diphenylene iodonium chloride (DPI) or a superoxide dismutase (SOD) mimetic, hydroxyl-tetramethylpiperidin-oxyl (TEMPOL). Moreover, blockade of NADH oxidase subunit, phox22, by its antisense oligodeoxynucleotide also eliminated the increase in NADH oxidase activity induced by L-Hcys.


CONCLUSIONS
These results indicate that Hcys-induced alterations of ECM metabolism in mesangial cells are associated with enhanced NADH oxidase activity and that oxidative stress-stimulated up-regulation of TIMP-1 may play an important role in the deposition of collagen or ECM elements in the glomeruli during hHcys.
Yang
Zhi-Zhang
ZZ
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Zou
Ai-Ping
AP
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006710:Homocysteine / Q000494:pharmacology*; D020138:Hyperhomocysteinemia / Q000378:metabolism / Q000503:physiopathology; D008562:Membrane Glycoproteins / Q000235:genetics; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009861:Onium Compounds / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*
D000975:Antioxidants; D024042:Collagen Type I; D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D009861:Onium Compounds; D010750:Phosphoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013113:Spin Labels; D019715:Tissue Inhibitor of Metalloproteinase-1; D006710:Homocysteine; C007517:diphenyleneiodonium; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase; C001803:tempol