Obesity-induced vascular inflammation is an early pathological change for the development of diabetic nephropathy. Studies have previously demonstrated that high fat diet (HFD) treatment decreased cytochrome P450 epoxygenase-mediated epoxyeicosatrienoic acids (EETs) production which in turn triggers vascular dysfunction and renal inflammation. We hypothesize that HFD up-regulates let-7b miRNA to decrease epoxgenase-mediated EETs production triggering vascular dysfunction and renal injury. Injection of let-7b mimetic has let-7b (1 nmol/day i.v) into rats decreased renal and hepatic cyp2c23 and cyp2j epoxygenases expression (P< 0.05). To determine whether high fat diet up-regulates let-7b to decrease EETs production, we use sEH gene (Ephx2) knock-out mice (KO) as a model for high EETs availability as EETs are rapidly hydrolyzed by the soluble epoxide hydrolase (sEH) to less active metabolites. WT and Ephx2 (-/-) mice were fed normal (ND, 14% fat) or HFD (60 % fat) for 3 months. HFD treatment was associated with 2 fold up-regulation of renal let-7b miRNA and this effect was coincided with down-regulation of cyp2c44 and cyp2j epoxygenases and decreased EETs levels. Decreased EETs levels in HFD fed rats was also associated with significant podocyte loss and elevation in podocalyxin excretion (56±4 ng/day) when compared to rats fed ND (33±7 ng/day) and these changes were significantly reduced in Ephx2 (-/-) mice. Furthermore, HFD treatment markedly elevated T helper cells expressing inflammatory IL-17 (CD3+CD4+TH17+) and decreased anti-inflammatory regulatory T cells (Tregs, CD3+CD4+FOXP3+) when compared to ND and these changes were also significantly attenuated in Ephx2 (-/-) mice. In-vitro, treatment of cultured glomerular endothelial cells with palmitate (200 μM) for 48 hours increased let-7b miRNA expression and decreased cyp2J mRNA and the tight junction protein ZO-1 expression levels and these changes were significantly reduced by transfecting cells with let-7b inhibitor (50 nM). Our data suggest that up-regulation of let-7b miRNA decreases epoxygenase-mediated EETs production to trigger vascular dysfunction and inflammation in the kidney of obese mice via shifting T cells polarization to favor inflammatory Th17 cells activation.
Cytochrome P450 system components and nitric oxide synthase activity in mouse liver under conditions of retinoid stores absence