Metabolism Core

新陈代谢核心

• 批准号: 10747722
• 负责人: Leslie S Gewin
• 金额: $ 27.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747722
• 关键词: Address; Affect; Biological Assay; Cell Count; Cells; Chronic; Chronic Kidney Failure; Communities; Consult; Consultations; Data; Disease Progression; Disease model; Doctor of Medicine; Doctor of Philosophy; Education; Electron Transport; Equipment; Faculty; Functional disorder; Genes; Genus Hippocampus; Glucose; Human; In Vitro; Injury; Injury to Kidney; Isotopes; Kidney; Kidney Diseases; Measures; Metabolic; Metabolism; Methods; Mitochondria; Obesity; Palmitates; Pathway interactions; Pharmaceutical Preparations; Play; Pre-Clinical Model; Process; Proteins; Protocols documentation; Pyruvate; Radioactive; Reagent; Recommendation; Renal function; Renal tubule structure; Reproducibility; Reproducibility of Results; Research; Research Personnel; Resolution; Resources; Respiration; Role; Services; Spirometry; Standardization; Tissues; Tracer; Training; Tubular formation; Universities; Update; Validation; Washington; Writing; biomedical resource; design; fatty acid oxidation; in vivo; instrumentation; interest; liquid chromatography mass spectrometry; long chain fatty acid; metabolomics; nutrition; oxidation; protein expression; response; stable isotope; transcriptomics

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.

项目摘要/摘要：新陈代谢核心 新陈代谢核心（Meta-Core）是华盛顿大学慢性病学中心内的生物医学资源核心 肾脏疾病国家资源中心。慢性损伤的肾脏有新陈代谢，最近。 数据表明，这些代谢变化在肾功能逐渐下降中发挥着致病作用。 探究代谢的能力对于理解慢性肾脏病的病理生理学至关重要，但是 肾脏研究人员这样做存在许多障碍，首先，许多研究人员对慢性病感兴趣。 其次，肾脏疾病没有代谢和代谢测定方面的专业知识。 需要许多实验室不容易获得的昂贵设备，为了解决这些问题，Meta-Core 诞生了。 教职人员包括核心主任 Leslie Gewin 医学博士、Brian Finck 博士、副核心主任 Gary Patti、 核心合作者博士和核心合作者 Leah Shriver 博士将与 Meta- 核心用户将指导和设计最佳代谢测定来回答研究问题。 Meta-Core 用户可以在细胞或整个肾脏水平上询问新陈代谢。 测定包括使用原代细胞或离体新鲜分离的小管进行海马生物通量分析，两者 比市售的人原代小管更能代表高度氧化的近端小管 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，3H-棕榈酸酯测定，不常见。 在肾组织上进行，比更常用的 14C-棕榈酸酯检测具有优势，因为它可以检测 通过电子传递链完全氧化长链脂肪酸并且更容易进行 （收集 3H2O 而不是 14CO2）我们还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）用于检测分离线粒体的呼吸。 将在体外和体内对原代细胞进行非靶向代谢组学和稳定同位素示踪剂研究 Vivo 的 Meta-Core 教师都致力于与 O’Brien 分享经过验证的协议和方法。 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，并以以下方式使用 Seahorse 生物通量分析： 产生一致、可重复的结果，特别是验证使用后的细胞数量的重要性。 将强调细胞数量的分析校正以及试剂的适当浓度。 核心将利用华盛顿大学其他核心的专业知识和设备来生产 附加服务可降低费率。