Metabolism Core

项目摘要/摘要：新陈代谢核心 代谢核心(Meta-Core)是华盛顿大学慢性医学中心的生物医学资源核心 国家肾脏疾病资源中心。慢性损伤的肾脏已经改变了新陈代谢，最近 数据表明，这些代谢变化在肾功能进行性下降中起到了致病作用。 询问新陈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，但是 肾脏研究人员要做到这一点有很多障碍。首先，许多研究人员对慢性病感兴趣 肾脏疾病在新陈代谢和新陈代谢检测方面没有专业知识。其次，这些化验中的许多 需要昂贵的设备，但许多实验室并不容易获得。为了解决这些问题，Meta-Core 教职员工包括Leslie Gewin，M.D.，核心总监Brian Finck，Ph.D.，副核心总监Gary Patti， 核心合作者博士和核心合作者Leah Shriver博士将与Meta- 指导核心用户设计最好的代谢测定法，回答研究问题。几种化验方法将 Meta-Core用户可用于询问细胞或整个肾脏水平的新陈代谢。这些 分析包括使用原代细胞或新分离的体外小管进行海马生物通量分析，两者都是 更能代表氧化程度较高的近端小管，而不是商业上可买到的原生人体小管 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。 肾组织体外应用放射性底物氧化法测定。~3H-棕榈酸测定，并不常见 在肾组织上进行，比更常用的14C-棕榈酸酯检测方法有优势，因为它检测到 长链脂肪酸通过电子传递链完全氧化，更容易进行 (收集3H2O，而不是14CO2)。我们也可以向用户推荐营养肥胖研究中心的高- 分辨率呼吸仪(Oroboros Oxygraph 2k)检测分离线粒体的呼吸。此外, 非靶向代谢组学和稳定同位素示踪研究将在体外和年内对原代细胞进行 活着。Meta-Core教职员工都致力于与O‘Brien共享经过验证的协议和方法 财团以及更大的科学界。此外，我们还将帮助培训肾脏科学界 如何生成原代近端小管细胞和小管，并以一种 产生一致的、可重现的结果。特别是，验证要使用的单元号的重要性，后 将强调对细胞数量的分析校正和适当的试剂浓度。Meta- CORE将利用华盛顿大学其他岩芯的专业知识和设备来生产 为降低费率提供补充服务。

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.