cPLA2 derived Eicosanoids modulate Renal Fibrosis

描述(申请人提供)：肾脏纤维化是慢性肾脏疾病的最终常见病理表现。最近的研究发现了一个由炎症信号和细胞类型组成的巨大网络，这些细胞负责产生肾脏肌成纤维细胞，这些细胞在创造和传播纤维化方面发挥了重要作用。一个新的文献基础支持二十烷类化合物可能在肾脏疾病，特别是肾脏纤维化中发挥重要作用，它广泛地被描述为酶切割的生物活性脂肪产物。我们的项目试图利用慢性肾脏疾病的动物模型和细胞培养技术来表征二十烷类化合物在促进肾脏纤维化中的作用。具体地说，我们正在使用基因敲除小鼠来阐明胞浆磷脂酶A2(CPLA2)衍生的二十烷基类化合物在造成纤维化中的作用。具体目标1将研究二十烷类化合物在慢性肾脏疾病动物模型中在传播纤维化中所起的作用。我们将在野生型(WT)和cPLA2基因敲除(KO)小鼠中使用单侧输尿管梗阻(UUO)。通过使用高效液相色谱和质谱仪等技术，我们将能够分析UUO后这些小鼠群体中cPLA2衍生二十烷类化合物的组织水平，观察纤维化和炎症的程度，并进行骨髓移植以描绘循环炎症细胞的贡献。我们的初步数据显示，在UUO后，前列腺素、脂氧合酶和细胞色素P450产物的组织表达上调。这些都是现有药物疗法靶向的潜在未知途径。具体目标2将尝试使用共培养系统来确定对上述观察到的表型负责的细胞类型。具体地说，我们计划使用一种小鼠永生化细胞系，MCT细胞与从我们的实验小鼠中分离的巨噬细胞进行共培养。通过细胞特异性基因敲除，我们可以观察这些细胞系之间的分子串扰，以确定导致损伤的主要细胞类型，并将其与动物研究相联系。我们在人类永生化上皮细胞中的初步数据表明，cPLA2基因沉默诱导了表型变化，这可能表明常驻细胞对伤害性刺激的反应发生了变化。

DESCRIPTION (provided by applicant): Renal fibrosis is the final common pathologic finding in chronic kidney disease. Recent research efforts have uncovered a vast network of inflammatory signals and cell types responsible for generating renal myofibroblasts that are instrumental in creating and propagating fibrosis. An emerging literature base supports that eicosanoids, broadly characterized as biologically active lipid products of enzymatic cleavage, may play a major role in renal disease, in particular renal fibrosis. Our project seeks to characterize the contribution of eicosanoids in propagating renal fibrosis using an animal model of chronic kidney disease as well cell culture techniques. Specifically, we are using genetic knockout mice to elucidate the role that cytosolic phospholipase A2 (cPLA2) derived eicosanoids have in creating fibrosis. Specific aim 1 will investigate the role that eicosanoids play in propagating fibrosis in an animal model of chronic kidney disease. We will be using unilateral ureteral obstruction (UUO) in wild type (WT) and cPLA2 genetic knockout (KO) mice. Using techniques such as liquid chromatography and mass spectrometry we will be able to profile tissue levels of cPLA2 derived eicosanoid products in these mouse populations after UUO, look at degree of fibrosis and inflammation, and perform bone marrow transplants to delineate the contribution from circulating inflammatory cells. Our preliminary data shows upregulated tissue expression of prostanoid, lipoxygenase, and cytochrome P450 products after UUO. These are potential unexplored pathways to target with existing drug therapies. Specific aim 2 will attempt to establish the cell type responsible for the phenotypes observed above using a co-culture system. Specifically, we plan to use a mouse immortalized cell line, MCT cells in co-culture with macrophages isolated from our experimental mice. By cell specific genetic knockout we can observe the molecular cross-talk between these cell lines to determine the major cell type contributing to injury relating this to the studies done in animals. Our preliminary data in human immortalized epithelial cells demonstrates that cPLA2 gene silencing induces phenotypic changes that may indicate an altered resident cell response to injurious stimuli.