Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice

Abstract

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-β/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-β1-induced renal tubular epithelial–mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase–EET system in the pathogenesis of diabetic nephropathy.

Introduction

Approximately 20–30% of all diabetic patients develop evidence of diabetic nephropathy (DN), which represents a continuum from microalbuminuria, to overt nephropathy or macroalbuminuria, and finally end stage renal disease (ESRD) [1]. Indeed, DN is the main cause of ESRD requiring dialysis in developed countries [2]. DN is initiated by hyperglycemia and is characterized by mesangial expansion and thickening of the tubular and glomerular basement membranes due to excessive matrix accumulation, associated with progressive renal deterioration [3]. However, a recent study has indicated that tubular injury and progressive interstitial fibrosis are more closely related to DN prognosis [4]. TGF-β and its downstream signal transduction pathways play a major role in DN and have therefore been thoroughly studied [5]. Among the features of the diabetic milieu, hyperglycemia, increased non-enzymatic glycation of proteins, de novo synthesis of diacylglycerol and subsequent activation of protein kinase C, increased intracellular glucosamine production, and enhanced renal production of vasoactive agents (angiotensin II, endothelins, thromboxane) have all been shown to increase the expression of TGF-β in cultured renal cells and in animal models of DN [6]. With the emerging evidence that TGF-β is involved in the pathogenesis and progression of DN, potential treatments with specific factors that interrupt the activity of TGF-β have become promising and attractive.

Epoxyeicosatrienoic acids (EETs), cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acids (AAs), have diverse biological effects within the cardiovascular system and have been identified as endothelium-derived hyperpolarizing factors, which activate calcium-sensitive potassium channels resulting in hyperpolarization of the resting membrane potential and the relaxation of vascular smooth muscle cells [7]. CYP epoxygenases are highly expressed in cardiomyocytes and vascular endothelial cells [8]. CYP epoxygenase overexpression, and the corresponding increase in EET levels, promotes an angiogenic phenotype, including endothelial cell proliferation, migration, and capillary tubule formation, as well as significantly protects endothelial cells from TNF-α induced apoptosis [9], [10]. EETs upregulate eNOS in bovine aortic endothelial cells via activation of MAPK, protein kinase C, PI3-kinase/AKT, and MAPK signaling pathways [11], [12]. 14,15-EET mediated mitogenic signaling pathway in a kidney epithelial cell line, which involved EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF [13]. EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF [14]. Blocking PPARγ by GW9662 abolished the EET-mediated anti-inflammatory effect, which indicates that PPARγ is an effector of EETs [15]. In the pancreas, CYP epoxygenase expression and EET production suggest that EETs also may be involved in pancreatic hormone secretion [16]. Indeed, 5,6-EET directly stimulates insulin release from isolated rat pancreatic islets in vitro [17].

CYP, family 2, subfamily J, polypeptide 5 epoxygenase (CYP2J5) produces EETs and is highly expressed in the proximal tubule and collecting duct of mice [18]. The inhibition of soluble epoxide hydrolase 2 (EPHX2), the primary EET metabolizing enzyme, results in increased EET levels, which has been shown to attenuate the progression of renal damage associated with hypertension and type II diabetes [19]. The human ortholog of mouse CYP2J5 is CYP2J2 [20]. Our previous CYP2J3 gene therapy studies have demonstrated alleviation of insulin resistance and diabetic symptoms in db/db mice and fructose-induced insulin resistant rats [21]. We hypothesize that endothelial CYP2J2 overexpression and the resultant increase in EET production will attenuate DN in streptozotocin (STZ)-treated mice. Furthermore, we postulate that TGF-β1-induced changes in cultured kidney cells will be attenuated by the administration of exogenous EETs.