Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice
Highlights
► This study is the first report about the protection role of CYP2J2 in diabetic nephropathy in vivo. ► Endothelial overexpression of CYP2J2 attenuates activation of the TGF-β/Smad signaling pathway in diabetic kidney mice. ► EETs attenuate TGF-β1-induced tubular epithelial–mesenchymal transdifferentiation on HK-2 cells. ► EETs attenuate the effects of TGF-β1 through a mechanism that involves activation of EGFR and PPARγ.
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.
Section snippets
Materials
Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories (Soham, UK). Enhanced chemiluminescence reagent was purchased from Pierce Chemical (Rockford, IL), pre-stained molecular weight standards were from Bio-Rad (Hercules, CA), and polyvinylidene difluoride membranes were from Schleicher and Schuell (Dassel, Germany). Antibodies against p-samd3, smad3, smad4, smad7 and α-SMA were purchased from Sigma–Aldrich (St. Louis, MO).
Metabolic characteristics and renal function in streptozotocin-induced diabetic mice
Table 1 lists the biochemical and physiological parameters of WT and Tie2-CYP2J2 transgenic mice in both control and STZ-treated groups. Administration of STZ to mice significantly increased the ratio of kidney weight to body weight, blood glucose, HbA1c (A1C), blood urea nitrogen, plasma creatinine, urinary microalbumin levels and decreased urinary osmolarity compared with control mice group(P < 0.05), indicating the characteristics of diabetic renal dysfunction and establishment of the early
Discussion
In this study, we injected one-low dose STZ (50 mg/kg body weight) daily by I.P. for 5 consecutive days to induce a diabetic state characterized by hyperglycemia and hypoinsulinemia in mice. We examined the effects of endothelial CYP2J2 overexpression on diabetic nephropathy, including renal function and morphology. We found that endothelial CYP2J2 overexpression attenuated the detrimental effects associated with DN. Specifically, CYP2J2 overexpression decreased hyperglycemia, improved renal
Acknowledgments
This work was supported by funds from the National Education Ministration project, Nature Science Foundation Committee projects (Nos. 30930039, 30700377) and Wuhan City grant.
References (55)
- et al.
Diabetic nephropathy: important pathophysiologic mechanisms
Diabetes Res Clin Pract
(2008) - et al.
TGF-beta and fibrosis in different organs—molecular pathway imprints
Biochim Biophys Acta
(2009) - et al.
Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets
Biochem Biophys Res Commun
(1983) - et al.
Molecular cloning, enzymatic characterization, developmental expression, and cellular localization of a mouse cytochrome P450 highly expressed in kidney
J Biol Chem
(1999) - et al.
Utility of endogenous creatinine clearance as a measure of renal function in mice
Kidney Int
(2004) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
Mesangial immune injury, hypertension, and progressive glomerular damage in Dahl rats
Kidney Int
(1984) - et al.
Mice lacking Smad3 are protected against cutaneous injury induced by ionizing radiation
Am J Pathol
(2002) - et al.
Transforming growth factor-beta regulates tubular epithelial–myofibroblast transdifferentiation in vitro
Kidney Int
(1999) - et al.
Advanced glycation end products induce tubular epithelial–myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway
Am J Pathol
(2004)