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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要先前的研究已经评估了与疾病相关的单个器官中代谢通量的变化，但没有试图简单地检查多个器官中的通量变化，这些器官共同控制全身营养代谢。此外，当前的同位素示踪技术没有能力剖析单个器官内空间位置之间的代谢通量差异。该提议的总体目标是开发一个多组织13C代谢通量分析（MFA）平台，以评估肾脏代谢及与肝脏和心脏代谢的相互关系。该技术的理由是它将启用调查人员解决有关肾脏代谢体内调节的重要问题通过研究单器官或孤立的细胞/组织的回答。这项研究是根据我们最近的发表的研究，其中肾脏和肝脏对糖异生（GNG）的贡献只是在禁食小鼠中进行评估。据我们所知，这是第一次应用13C MFA评估现场动物中的现场和肾脏磁通之间的相互作用。现在，关键需要首先扩展这种通量测量技术包括糖酵解组织，其次，测试区域差异是否存在代谢通量可以在肾脏中区分。第一个目标将建立多器官13C MFA 简单地量化儿童糖化和糖酵解组织的体内代谢的平台，肝脏和心脏。具有钢干和人工制品的小鼠在期间将获得13c-葡萄糖输注高胰岛素血糖或高胰岛素 - 糖类血糖夹。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确定代谢每个组织中的通量。然后，我们将应用这个新颖的平台评估相关的代谢通量改变糖尿病肾脏疾病的动物模型：具有内皮一氧化氮敲除（eNOS - / - ）的BKS DB/DB小鼠。这第二个目标将扩大13C标记的代谢物测量，以评估空间分辨的代谢中的代谢肾。未靶向的高分辨率LC-MS/MS分析将应用于组织提取物和代谢物将使用新型软件来鉴定由13C-葡萄糖富集的筛选，该软件将筛选出针对KEGG的命中复合数据库。然后，肾脏组织切片的基于MALDI的成像质谱（IMS）将是应用于定位这些标记的代谢物特征，确定其空间模式的13c富集和执行13C MFA以评估不同肾脏区域内代谢通量。拟议的研究是创新性，因为它将建立用于量化综合多组织的通量表型的新技术活动物中的代谢网络。分析平台将作为核心服务实施 Vanderbilt Mouse代谢表型中心和Vanderbilt O’Brien肾脏中心。研究是意义重大，因为它将使未来的研究能够评估代谢在进展过程中的失调肾脏疾病以及针对肾脏途径的治疗方法如何影响全身代谢健康。