Cellular Regulation of Prostaglandin Synthesis

肾小球系膜细胞(GMC)是肾小球损伤的重要参与细胞。GMC凋亡是实验性系膜增殖性肾炎肾小球高细胞增多的主要机制。GMC对凋亡的抵抗可能是损伤进展为终末期疾病的原因，因为细胞增殖不会被额外的细胞缺失所抵消。这一应用的长期目标是阐明GMC抵抗细胞凋亡的机制。本提案将验证环氧合酶2(COX-2)的假说，COX-2可从花生四烯酸中产生前列腺素，通过上调抑制一氧化氮合酶(NOS)活性和调节外排的基因来保护GMC免受凋亡。初步数据表明：1)COX-2抑制促凋亡的Bcl-2家族蛋白的凋亡，控制外源生物。这一假说是基于OUR在包括GMC在内的几种类型的细胞中的表达；2)COX-2上调一氧化氮合酶抑制蛋白(PIN)和多药耐药P-糖蛋白(MDR1)的表达；3)凋亡剂增加细胞NO的产生。Bim依赖的Bax活性将用免疫荧光法测定。在有无COX-2表达的GMC中，通过检测精氨酸向瓜氨酸的转化和通过Siever分析仪对NO氧化产物的定量，可以评估GMC中NOS活性和NO的生成。我们将在分离的肾小球和感染COX-2的重组腺病毒感染的GMC中，在存在和不存在选择性COX-2抑制剂的情况下，用质谱仪结合高效液相色谱(LC/MS)来鉴定环氧合酶活性的产物。为了评价环氧合酶活性在COX-2基因表达调控和抗凋亡作用中的作用，我们将检测COX-2产物(经LC/MS鉴定)模拟COX-2作用的能力，并将评估缺乏环氧合酶活性但保留过氧化物酶活性的COX-2突变体的抗凋亡特性。我们还将在体内实验模型中研究阻断COX-2活性对增生性肾小球肾炎、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，通过细胞凋亡来消除增殖的GMC往往是无效的。

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.