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cPLA2 derived Eicosanoids modulate Renal Fibrosis

cPLA2 衍生的类二十烷酸调节肾纤维化

基本信息

批准号：
8833705
负责人：
John Ross Montford
金额：
$ 6.16万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8833705
关键词：
Animal Model Animals Arachidonate 5-Lipoxygenase Arachidonic Acids Architecture Area Blood Vessels Bone Marrow Bone Marrow Transplantation Calcium Cell Culture Techniques Cell Line Cell model Cell physiology Cells Chronic Kidney Failure Clinical Data Coculture Techniques Connective Tissue Cytochrome P450 Cytosolic Phospholipase A2 Data Deposition Development Disease Progression Eicosanoid Production Eicosanoids Electrolytes End stage renal failure Endothelial Cells Epidemic Epithelial Cells Fibrosis Flow Cytometry Gene Silencing Generations Genetic Growth Factor Healthcare Systems Human Individual Inflammation Inflammatory Injury Kidney Kidney Diseases Knock-out Knockout Mice Lipids Lipoxygenase Liquid Chromatography Literature Mass Spectrum Analysis Measures Mediating Molecular Morbidity - disease rate Mus Myeloid Cells Myofibroblast Nature Organ Pathologic Pathway interactions Pericytes Pharmacotherapy Phenotype Phospholipase A2 Phospholipids Physiological Play Population Production Prostaglandins Recruitment Activity Research Role Signal Transduction Stimulus System Techniques Thromboxanes Time Tissues Tubular formation Ureteral obstruction Wild Type Mouse base cell type cytokine design functional disability immortalized cell interstitial kidney cell lipid mediator liquid chromatography mass spectrometry macrophage mortality mouse model pre-clinical public health relevance response tandem mass spectrometry

项目摘要

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.

描述（由申请人提供）：肾纤维化是慢性肾脏疾病的最后常见病理结果。最近的研究工作已经发现了一个巨大的炎症信号网络和负责产生肾肌成纤维细胞的细胞类型，这些细胞有助于产生和传播纤维化。一个新兴的文献基础支持类二十烷酸，广泛表征为酶裂解的生物活性脂质产物，可能在肾脏疾病，特别是肾纤维化中发挥重要作用。我们的项目旨在利用慢性肾脏疾病的动物模型以及细胞培养技术来表征类花生酸在传播肾纤维化中的贡献。具体来说，我们正在使用基因敲除小鼠来阐明胞质磷脂酶A2（cPLA2）衍生的类花生酸在产生纤维化中的作用。具体目标1将研究类花生酸在慢性肾脏疾病动物模型中传播纤维化中的作用。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）。使用液相色谱和质谱等技术，我们将能够分析UUO后这些小鼠群体中cPLA2衍生类花生酸产物的组织水平，观察纤维化和炎症程度，并进行骨髓移植以描述循环炎性细胞的贡献。我们的初步数据显示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。这些都是潜在的未探索的途径，以现有的药物治疗为目标。具体目标2将尝试使用共培养系统建立负责上述观察到的表型的细胞类型。具体而言，我们计划使用小鼠永生化细胞系，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，这与在动物中进行的研究有关。我们在人永生化上皮细胞中的初步数据表明，cPLA2基因沉默诱导的表型变化，可能表明改变了居民细胞对有害刺激的反应。