Integrated multi-tissue 13C flux analysis platform to assess renal metabolism in vivo

用于评估体内肾脏代谢的集成多组织 13C 通量分析平台

• 批准号: 10727785
• 负责人: Jamey D. Young
• 金额: $ 23.78万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727785
• 关键词: Address; Animal Model; Animals; Biochemical Pathway; Biological Assay; Cardiovascular Diseases; Catheterization; Catheters; Cell Separation; Chronic; Chronic Kidney Failure; Clinic; Closure by clamp; Collaborations; Computer software; Conscious; Databases; Development; Diabetic Nephropathy; Diagnosis; Disease; Dyslipidemias; Educational workshop; Endocrine; Endothelium; Fasting; Future; Genetic; Gluconeogenesis; Glucose; Glucose Clamp; Goals; Health; Heart; Hepatic; Hyperinsulinism; Hypertension; Hypoglycemia; Individual; Infusion procedures; Insulin; Insulin Resistance; Intervention; Isotope Labeling; Isotopes; Kidney; Kidney Diseases; Knock-out; Knowledge; Label; Liver; Location; Measurement; Measures; Metabolic; Metabolic syndrome; Metabolism; Methods; Mission; Modification; Mus; Nitric Oxide; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Outcome; Pathogenesis; Pathologic; Pathway interactions; Pattern; Pharmacologic Substance; Phenotype; Physiological; Procedures; Public Health; Publications; Publishing; Regulation; Research; Research Personnel; Resolution; Risk; Sampling; Services; Slice; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Technology; Technology Assessment; Testing; Time; Tissue Extracts; Tissues; Tracer; Training; United States National Institutes of Health; Work; db/db mouse; dietary; disorder prevention; euglycemia; heart metabolism; in vivo; innovation; kidney cortex; kidney medulla; kidney metabolism; liver metabolism; mass spectrometric imaging; mathematical model; metabolic phenotype; metabolomics; mouse model; new technology; novel; novel strategies; nutrient metabolism; regional difference; response; software development; stable isotope; technology development

PROJECT SUMMARY/ABSTRACT Prior studies have assessed disease-associated changes in metabolic fluxes within a single organ but have not attempted to simultaneously examine flux alterations within multiple organs that together control whole-body nutrient metabolism. Furthermore, current isotope tracer technologies do not have the ability to dissect metabolic flux differences between spatial locations within a single organ. The overall objective of this proposal is to develop a multi-tissue 13C metabolic flux analysis (MFA) platform to assess kidney metabolism and its interrelationship with liver and heart metabolism. The rationale for this technology is that it will enable investigators to address important questions about in vivo regulation of renal metabolism that cannot be answered through studies of single organs or isolated cells/tissues. The research builds from our recently published study wherein renal and hepatic contributions to gluconeogenesis (GNG) were simultaneously assessed in fasted mice. To our knowledge, this was the first time 13C MFA had been applied to assess interactions between liver and kidney fluxes in a live animal. There is now a critical need to, first, expand this flux measurement technology to include glycolytic tissues and, second, test whether regional differences in metabolic fluxes can be distinguished within the kidney. The first aim will establish a multi-organ 13C MFA platform to simultaneously quantify in vivo metabolism of gluconeogenic and glycolytic tissues of the kidneys, liver, and heart. Mice with veinous and arterial catheters will receive 13C-glucose infusions during hyperinsulinemic-euglycemic or hyperinsulinemic-hypoglycemic clamps. In each condition, the isotopes, measurements, and mathematical modeling procedures will be optimized to precisely determine metabolic fluxes in each tissue. We will then apply this novel platform to assess metabolic flux alterations in a relevant animal model of diabetic kidney disease: BKS db/db mice with endothelial nitric oxide knockout (eNOS−/−). The second aim will expand 13C-labeled metabolite measurements to assess spatially resolved metabolism in the kidney. Untargeted high-resolution LC-MS/MS profiling will be applied to tissue extracts, and metabolites enriched by 13C-glucose will be identified using novel software that screens for hits against the KEGG Compound Database. Then, MALDI-based imaging mass spectrometry (IMS) of kidney tissue slices will be applied to locate these labeled metabolite features, determine their spatial patterns of 13C enrichment, and perform 13C MFA to assess metabolic fluxes within different kidney regions. The proposed research is innovative because it will establish new technologies for quantifying flux phenotypes of integrated multi-tissue metabolic networks in live animals. The analysis platform will be implemented as a core service of the Vanderbilt Mouse Metabolic Phenotyping Center and Vanderbilt O’Brien Kidney Center. The research is significant because it will enable future studies to assess how metabolism is dysregulated during progression of kidney disease and how treatments that target renal pathways impact whole-body metabolic health.

项目总结/摘要 先前的研究已经评估了单个器官内代谢通量的疾病相关变化，但没有 试图同时检查共同控制全身的多个器官内的通量变化 营养代谢此外，目前的同位素示踪剂技术不具有解剖 单个器官内的空间位置之间的代谢通量差异。本提案的总体目标是 是开发一个多组织13 C代谢通量分析（MFA）平台，以评估肾脏代谢及其 与肝脏和心脏代谢的相互关系。这项技术的基本原理是， 研究者解决关于肾代谢的体内调节的重要问题， 通过对单个器官或分离的细胞/组织的研究来回答。这项研究建立在我们最近 已发表的一项研究，其中肾脏和肝脏对胚胎发生（GNG）的贡献同时存在， 在禁食小鼠中评估。据我们所知，这是第一次将13 C MFA应用于评估 在活体动物中肝脏和肾脏流量之间的相互作用。现在迫切需要首先扩大 这种通量测量技术包括糖酵解组织，其次，测试糖酵解组织的区域差异是否存在 可以在肾内区分代谢通量。第一个目标是建立一个多器官13 C MFA 同时定量肾脏的生血管和糖酵解组织的体内代谢的平台， 肝脏和心脏具有静脉和动脉导管的小鼠将接受13 C-葡萄糖输注， 高胰岛素-正常血糖或高胰岛素-低血糖钳夹。在每种情况下，同位素， 测量和数学建模程序将被优化，以精确地确定代谢 每个组织中的流量。然后，我们将应用这个新的平台，以评估代谢通量的改变，在相关的 糖尿病肾病动物模型：内皮型一氧化氮敲除（eNOS-/-）的BKS db/db小鼠。的 第二个目标是扩展13 C标记的代谢物测量，以评估空间分辨代谢， 肾将对组织提取物和代谢物进行非靶向高分辨率LC-MS/MS分析 富含13 C-葡萄糖的细胞将使用新的软件进行鉴定，该软件筛选针对KEGG的命中物 复合数据库。然后，将对肾组织切片进行基于MALDI的成像质谱（IMS）。 应用于定位这些标记的代谢物特征，确定其13 C富集的空间模式，以及 进行13 C MFA以评估不同肾脏区域内的代谢通量。拟议的研究是 创新，因为它将建立新的技术，用于量化整合的多组织的通量表型 活体动物的代谢网络。分析平台将作为 范德比尔特小鼠代谢表型中心和范德比尔特奥布莱恩肾脏中心。这项研究是 重要的是，它将使未来的研究能够评估代谢在进展过程中如何失调 以及针对肾脏通路的治疗如何影响全身代谢健康。