Cellular Regulation of Prostaglandin Synthesis

肾小球系膜细胞（GMC）是肾小球损伤的重要参与者。GMC凋亡是实验性系膜增生性肾炎肾小球细胞增生消退的主要机制。GMC对细胞凋亡的抗性可以解释损伤进展至终末期疾病，因为细胞增殖不被额外细胞缺失抵消。本申请的长期目标是阐明GMC对细胞凋亡的抗性机制。该提议将检验环加氧酶2（考克斯-2）（其从花生四烯酸产生异黄酮）通过基因的上调保护GMC免于凋亡的假设，所述基因抑制一氧化氮合酶（NOS）的活化活性并调节流出初步数据表明：1）考克斯-2抑制促凋亡Bcl-2家族蛋白的凋亡，控制外源性物质。该假说基于我们在包括GMC在内的几种类型的细胞中的研究：2）考克斯-2上调NOS蛋白抑制剂（PIN）和多药耐药P-糖蛋白（MDR 1）的表达; 3）凋亡剂增加细胞NO的产生。将通过免疫荧光测量Bim依赖性Bax活化。在存在和不存在考克斯-2表达的情况下，通过测量精氨酸向瓜氨酸的转化和通过用Sievers分析仪定量诱导细胞凋亡的GMC中的NO氧化产物来评价NOS的活性和NO的产生。我们将在存在和不存在选择性考克斯-2抑制剂的情况下，通过质谱联用液相色谱（LC/MS）鉴定分离的肾小球和用编码考克斯-2的重组腺病毒感染的GMC中的环氧合酶活性产物。为了评价环加氧酶活性在调控基因表达和考克斯-2的抗凋亡作用中的作用，我们将测试考克斯-2产物（通过LC/MS鉴定）模拟考克斯-2作用的能力，并将评价缺乏环加氧酶活性但保留过氧化物酶活性的考克斯-2突变体的抗凋亡性质。我们还将在体内实验模型中研究阻断考克斯-2活性对增殖性肾小球肾炎、高血压和糖尿病肾病进展的影响。这项研究的意义在于，它将为GMC对损伤的反应提供新的见解，并有助于理解为什么在进行性肾损伤过程中通过凋亡消除增殖的GMC通常是无效的。

Renal glomerular mesangial cells (GMC) are important participants in glomerular injury. GMC apoptosis is the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. Resistance of GMC to apoptosis may account for progression of injury to end stage disease since cell proliferation is not counteracted by extra cells deletion. The long term objectives of this application are to elucidate mechanisms of resistance of GMC to apoptosis. This proposal will examine the hypothesis that cyclooxygenase 2 (Cox-2), which generate prostaglandins from arachidonic acid, protects GMC from apoptosis via up-regulation of genes, that inhibit activation activity of nitric oxide synthases (NOS) and regulate efflux preliminary data indicating that 1) Cox-2 inhibits apoptosis of pro-apoptotic Bcl-2 family proteins, control of xenobiotics. The hypothesis is based on our in several types of cells including GMC; 2) Cox-2 up-regulates expression of Protein Inhibitor of NOS (PIN), and multidrug resistance P-glycoprotein (MDR1); 3) apoptotic agents increase cellular NO production. The Bim-dependent Bax activation will be measured by immunofluorescence. The activity of NOS and generation of NO will be evaluated by measurement of arginine to citrullin conversion and by quantification of NO oxidation products with Sievers analyzer in GMC induced to apoptosis in the presence and absence of Cox-2 expression. We will identify products of cyclooxygenase activity in isolated glomeruli and in GMC infected with recombinant adenovirus encoding Cox-2 by Mass Spectrometer coupled with Liquid Chromatography (LC/MS) in the presence and absence of selective Cox-2 inhibitors. To evaluate the role of cyclooxygenase activity in the regulation of gene expression and anti-apoptotic action of Cox-2 we will test the ability of Cox-2 products (identified by LC/MS) to mimic Cox-2 effects and will evaluate the anti-apoptotic properties of Cox-2 mutants which lack the cyclooxygenase activity, but retain peroxidase activity. We will also investigate the effects of blocking the Cox-2 activity on progression of proliferative glomerulonephritis, hypertension and diabetic nephropathy in in vivo experimental models. The significance of this study is that it will provide new insights into the response of GMC to injury and help to understand why elimination of proliferating GMC by apoptosis in the course of progressive renal injury is often inefficient.