Metabolism Core

项目概要/摘要：代谢核心 代谢核心（元核心）是华盛顿大学慢性 肾脏疾病国家资源中心。慢性损伤的肾脏会改变新陈代谢， 数据表明，这些代谢变化在肾功能的进行性下降中起着致病作用。 询问代谢的能力对于理解慢性肾脏疾病的病理生理学至关重要， 肾脏研究人员这样做有许多障碍。首先，许多对慢性病感兴趣的研究者 肾病患者不具备代谢和代谢测定方面的专业知识。第二，这些检测中的许多 需要昂贵的设备，许多实验室不容易获得。为了解决这些问题，Meta-Core 由Leslie Gewin，M.D.，核心总监，Brian Finck博士，副核心总监，加里帕蒂， 博士，核心合作者，Leah Shriver博士，核心合作者，将与Meta进行协商， 核心用户指导和设计最佳的代谢分析来回答研究问题。几种测定将 Meta-Core用户可在细胞或整个肾脏水平上查询代谢。这些 测定包括使用原代细胞或离体新鲜分离的小管的海马生物通量分析， 比市售的人原代小管更能代表高度氧化的近端小管 细胞或条件永生化的小管细胞。我们可以测量脂肪酸或葡萄糖/丙酮酸的氧化， 肾组织离体使用放射性底物氧化测定。3 H-棕榈酸酯测定，不经常 在肾组织上进行，比更常用的14 C-棕榈酸酯测定法更有优势，因为它检测 长链脂肪酸通过电子传递链的完全氧化， （收集3 H2O而不是14 CO2）。我们也可以向用户推荐营养肥胖研究中心的高- 分辨率呼吸计（Oroboros Oxygraph 2k）来检测分离的线粒体中的呼吸。此外，本发明还提供了一种方法， 非靶向代谢组学和稳定同位素示踪剂研究将在体外和体内对原代细胞进行。 vivo.元核心教师都致力于与奥布莱恩分享验证的协议和方法 联盟以及更大的科学界。此外，我们将帮助培训肾脏科学界 如何产生初级近端小管细胞和小管，并以某种方式使用海马生物通量分析， 产生一致的、可重复的结果。特别是，验证细胞数量的重要性， 将强调对细胞数目的分析校正和试剂的适当浓度。Meta- 核心将利用华盛顿大学其他核心的专业知识和设备， 以较低的价格提供补充服务。

Project Summary/Abstract: Metabolism Core The Metabolism Core (Meta-Core) is a Biomedical Resource Core within the Washington University Chronic Kidney Disease National Resource Center. Chronically injured kidneys have altered metabolism, and recent data suggests that these metabolic changes play a causative role in progressive decline in kidney function. The ability to interrogate metabolism is crucial to understanding chronic kidney disease pathophysiology, but there are a number of barriers for kidney researchers to do so. First, many investigators interested in chronic kidney disease do not have expertise in metabolism and metabolic assays. Second, many of these assays require expensive equipment not readily available to many labs. To address these issues, the Meta-Core faculty consisting of Leslie Gewin, M.D., Core Director, Brian Finck, Ph.D., Associate Core Director, Gary Patti, Ph.D., Core Collaborator, and Leah Shriver, Ph.D., Core Collaborator, will perform consultations with Meta- Core users to guide and design the best metabolic assay to answer the research question. Several assays will be available to the Meta-Core users to interrogate metabolism at the cellular or whole kidney level. These assays include Seahorse bioflux analyses with the use of primary cells or freshly isolated tubules ex vivo, both more representative of the highly oxidative proximal tubules than commercially available primary human tubule cells or conditionally immortalized tubule cells. We can measure oxidation of fatty acids or glucose/pyruvate in kidney tissue ex vivo using radioactive substrate oxidation assays. The 3H-palmitate assay, not often performed on kidney tissue, has advantages over the more commonly used 14C-palmitate assay as it detects complete oxidation of long chain fatty acids through the electron transport chain and is easier to perform (collect 3H2O rather than 14CO2). We can also refer users to the Nutrition Obesity Research Center’s high- resolution respirometer (Oroboros Oxygraph 2k) to detect respiration in isolated mitochondria. In addition, untargeted metabolomics and stable isotope tracer studies will be performed on primary cells in vitro and in vivo. The Meta-Core faculty are all committed to sharing validated protocols and methods with the O’Brien Consortium as well as larger scientific community. In addition, we will help train the kidney scientific community how to generate primary proximal tubule cells and tubules and use the Seahorse bioflux analysis in a way that yields consistent, reproducible results. In particular, the importance of validating cell number to use, post- analysis correction for number of cells, and proper concentrations of reagents will be emphasized. The Meta- Core will leverage the expertise and equipment of other Cores at Washington University to produce complementary services for a reduced rate.