Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases.
OBJECTIVE
We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.


METHODS
Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.


RESULTS
Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.


CONCLUSIONS
Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.
Eid
Assaad A
AA
University of Texas Health Science Center, Department of Medicine, San Antonio, Texas, USA.
Gorin
Yves
Y
Fagg
Bridget M
BM
Maalouf
Rita
R
Barnes
Jeffrey L
JL
Block
Karen
K
Abboud
Hanna E
HE
D000818:Animals; D042926:Cytochrome P-450 CYP4A / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000503:physiopathology*; D004195:Disease Models, Animal; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D050199:Podocytes / Q000473:pathology / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004791:Enzyme Inhibitors; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D042926:Cytochrome P-450 CYP4A; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C422943:Nox4 protein, mouse; D005947:Glucose