RENAL CYTOCHROME P450 EPOXYGENASE IN DIABETIC METABOLIC SYNDROME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 具体目标 肥胖和2型糖尿病是终末期肾病的主要原因。血管异常和肾功能不全通常与糖尿病有关。我们的目标是阐明细胞色素P450(CYP)环氧合酶在糖尿病代谢综合征相关血管和肾功能障碍发生发展中的调控机制(S)及其功能后果。 我们和其他人已经证实，CYP环氧合酶的代谢物，环氧二十碳三烯酸(EETs)，是有效的血管扩张剂，具有抗高血压特性。EETs还具有抗炎作用，可以保护肾脏免受高血压和糖尿病的损害。有证据表明，无法增加肾脏EET的产生会导致高盐或高脂饮食处理的大鼠患高血压。此外，我们发现在糖尿病前期肥胖大鼠中，肾脏环氧合酶的下调与乙酰胆碱诱导的松弛减弱有关。然而，目前尚不清楚CYP环氧合酶途径是否受糖尿病代谢综合征血管和肾功能障碍的发生和发展的影响或参与。我们的初步研究表明，Zucker糖尿病肥胖(ZDF)大鼠肾脏内皮细胞CYP2C23显著减少，这是一种主要的大鼠肾脏环氧合酶。此外，CYP2C23的这种减少与ZDF肾脏中功能性PPARa蛋白的减少有关。有趣的是，最近的报道表明，PPARa配体对CYP的调节可能在血管和肾功能的调节中发挥重要作用。鉴于这些结果，我们的实验室进行了初步研究，证明PPARa激动剂非诺贝特诱导的CYP酶与降低ZDF大鼠的血压、减轻肾小球肥大和肾脏纤维化有关。基于这些结果，我们假设，不适当的CYP环氧合酶调节可能阻碍血管和肾功能障碍的发生和发展，EET的过度产生将保护糖尿病代谢综合征中的血管和肾脏损伤(图)。具体来说，我们将致力于实现以下三个目标： 目的1.验证糖尿病代谢综合征时肾脏细胞色素PYP酶表达异常的假说，这可能与PPARa受体有关。 1.检测糖尿病大鼠CYP酶的表达水平和活性及PPARa的调节作用。 2.用PPARa基因敲除小鼠评价PPARa在非诺贝特对CYP调节中的作用。 3.描述负责CYP调控的转录和/或转录后机制。 4.通过检测核因子-kB、AP-1、SP1和c-JunDNA结合活性，确定参与CYP调控的其他转录因子。 目的2.验证糖尿病代谢综合征患者过量分泌EET可降低血压和保护肾脏损伤的假说。 1.观察非诺贝特和环氧合酶抑制剂PPOH对糖尿病大鼠血压的影响。 2.观察非诺贝特和PPOH对糖尿病大鼠肾脏结构和肾脏损伤分子-1的影响，探讨非诺贝特和PPOH对糖尿病大鼠肾脏损伤的影响。 3.研究EET对肾小球细胞增殖和细胞周期调节蛋白的作用 目的2.验证糖尿病代谢综合征患者过量分泌EET可降低血压和保护肾脏损伤的假说。 1.观察非诺贝特和环氧合酶抑制剂PPOH对糖尿病大鼠血压的影响。 2.观察非诺贝特和PPOH对糖尿病大鼠肾脏结构和肾脏损伤分子-1的影响，探讨非诺贝特和PPOH对糖尿病大鼠肾脏损伤的影响。 3.研究EET对肾小球细胞增殖和细胞周期调节蛋白的作用 目的3验证糖尿病代谢综合征患者EET生成下调可能参与肾血管功能障碍的发生发展，以及EET过度生成可改善肾脏微血管结构和功能的假说。 1.探讨CYP代谢产物在糖尿病代谢综合征发生发展过程中对肾脏血管结构和功能改变的作用。 2.检测基因转移对糖尿病代谢综合征患者肾血管功能的影响。 3.利用PPARa基因敲除小鼠研究PPARa在血管功能调节中的作用。 该项目产生的结果将确定预防和治疗人类肥胖和糖尿病相关肾脏疾病的潜在治疗目标和基因座。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aims Obesity and type 2 diabetes are the leading causes of end-stage renal disease. Vascular abnormality and renal dysfunction are commonly associated with diabetes mellitus. Our goal is to elucidate the regulatory mechanism(s) and functional consequences of cytochrome P450 (CYP) epoxygenases in the development and progression of vascular and renal dysfunction related to diabetic metabolic syndrome. We and others have established that CYP epoxygenase metabolites, epoxyeicosatrienoic acids (EETs), are potent vasodilators and have anti-hypertensive properties. EETs also possess anti-inflammatory actions that could protect the kidney from injury in hypertension and diabetes mellitus. There is evidence that an inability to increase renal EET production contributes to the development of hypertension in high salt or high fat diet-treated rats. .Moreover, we found that a down regulation of renal epoxygenases was associated with attenuated acetylcholine-induced relaxation in prediabetic obese rats. However, it is unclear whether the CYP epoxygenase pathway is affected by or contributes to the development and progression of vascular and renal dysfunction in diabetic metabolic syndrome. Our preliminary studies have shown a significant decrease in renal endothelial CYP2C23, a predominant rat kidney epoxygenase, in Zucker diabetic fatty (ZDF) rats. Furthermore, this decrease in CYP2C23 was correlated with a reduction of functional PPARa protein in the ZDF kidneys. Interestingly, recent reports have suggested that CYP modulation by PPARa ligands may play an important role in the regulation of vascular and renal function. In view of these results, pilot studies were conducted in our laboratory demonstrating that an induction of CYP enzymes by fenofibrate, a PPARa agonist, was associated with a reduction of blood pressure and an attenuation of glomerular hypertrophy and renal fibrosis in ZDF rats. Based on these results, we hypothesize that an inappropriate regulation of CYP epoxygenases may contrubite to the development and progression of vascular and renal dysfunction, and that overproduction of EET will protect against vascular and renal injury in diabetic metabolic syndrome (Diagram). Specifically, we will address the following three aims: Aim 1. To test the hypothesis that renal CYP enzymes are inappropriately regulated in diabetic metabolic syndrome, which may involve PPARa receptor. 1. Determine the expression level and activity of CYP enzymes and PPARa regulation in diabetic rats. 2. Evaluate the role of PPARa in the CYP regulation by fenofibrate using PPARa knockout mice. 3. Delineate the transcriptional and/or post-transcriptional mechanisms responsible for CYP regulation. 4. Identify the additional transcription factors involved in the CYP regulation by assessing the DNA binding activities of NF-kB, AP-1, SP1 and c-Jun. Aim 2. To test the hypothesis that overproduction of EET will lower blood pressure and protect against renal injury in diabetic metabolic syndrome. 1. Assess the effect of fenofibrate and PPOH (an epoxygenase inhibitor) on blood pressure in diabetic rats. 2. Determine the effects of fenofibrate and PPOH on kidney damage by evaluating the changes in kidney structure and molecular markers including nephrin, podocin and kidney injury molecule-1 in diabetic rats. 3. Characterize the action of EET on glomerular cell proliferation and cell cycle regulatory proteins Aim 2. To test the hypothesis that overproduction of EET will lower blood pressure and protect against renal injury in diabetic metabolic syndrome. 1. Assess the effect of fenofibrate and PPOH (an epoxygenase inhibitor) on blood pressure in diabetic rats. 2. Determine the effects of fenofibrate and PPOH on kidney damage by evaluating the changes in kidney structure and molecular markers including nephrin, podocin and kidney injury molecule-1 in diabetic rats. 3. Characterize the action of EET on glomerular cell proliferation and cell cycle regulatory proteins Aim 3 To test the hypothesis that a down-regulati,on of EET formation may contribute to the development and progression of renal vascular dysfunction and that overproduction of EET will improve renal microvascular structure and function in diabetic metabolic syndrome. 1. Evaluate the contribution of CYP metabolites to the altered renal vascular structure and function during the development of diabetic metabolic syndrome. 2. Determine the effect of CYP2C gene transfer on renal vascular function in diabetic metabolic syndrome. 3. Characterize the role of PPARa in the regulation of vascular function using PPARa knock out mice. The results generated from the project will identify potential therapeutic targets and loci for the prevention and treatment of obesity and diabetes-related kidney disease in humans