Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.
Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-β1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-β1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.
Sedeek
M
M
Kidney Research Centre, Ottawa Hospital Research Institute and Deptartment of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Callera
G
G
Montezano
A
A
Gutsol
A
A
Heitz
F
F
Szyndralewiez
C
C
Page
P
P
Kennedy
C R J
CR
Burns
K D
KD
Touyz
R M
RM
Hébert
R L
RL
D000818:Animals; D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000096:biosynthesis; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism* / Q000473:pathology; D003928:Diabetic Nephropathies / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis; D005947:Glucose / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D008297:Male; D051379:Mice; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D011728:Pyridones / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects
C554850:2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo(4,3-c)pyridine-3,6(2H,5H)-dione; D003573:Cytochrome b Group; D011720:Pyrazoles; D011728:Pyridones; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; C515614:Cyba protein, mouse; C060505:neutrophil cytosolic factor 1; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose