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

项目摘要/摘要 以前的研究已经评估了与疾病相关的单个器官内代谢通量的变化，但没有 试图同时检查共同控制全身的多个器官内的流量变化 营养新陈代谢。此外，目前的同位素示踪技术不具备解剖 单个器官内空间位置之间的代谢流量差异。这项提案的总体目标是 是开发一个多组织13C代谢通量分析(MFA)平台来评估肾脏代谢和其 与肝脏和心脏代谢的相互关系。这项技术的基本原理是它将使 研究人员将解决有关体内调节肾脏代谢的重要问题 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的基础上 已发表的研究中，肾脏和肝脏对糖异生(GNG)的贡献同时 在禁食小鼠身上进行了评估。据我们所知，这是13C MFA首次用于评估 活体动物肝脏和肾脏之间的相互作用。现在迫切需要的是，首先，扩大 这种通量测量技术包括糖酵解组织，第二，测试是否存在区域差异 可以在肾脏内区分代谢流。第一个目标是建立一个多器官13C MFA 同时量化肾脏糖异生组织和糖酵解组织体内代谢的平台， 肝脏和心脏。静脉和动脉导管的小鼠将在治疗期间接受13C-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得到优化，以精确确定新陈代谢 每种组织中的流量。然后，我们将应用这个新的平台来评估相关的代谢流量变化 糖尿病肾病动物模型：内皮型一氧化氮合酶基因敲除(eNOS−/−)BKS db/db小鼠。这个 第二个目标是扩大13C标记的代谢物测量，以评估空间分辨的代谢 肾脏。非靶向高分辨率LC-MS/MS图谱将应用于组织提取物和代谢物 富含~(13)C-葡萄糖的将使用新软件筛选KEGG 复合数据库。然后，基于MALDI的肾组织切片的成像质谱仪(IMS)将被 应用于定位这些标记的代谢物特征，确定其13C浓缩的空间模式，以及 进行~(13)C MFA以评估不同肾脏区域的代谢流量。拟议的研究是 创新，因为它将建立量化整合的多组织的流量表型的新技术 活体动物的新陈代谢网络。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 意义重大，因为它将使未来的研究能够评估新陈代谢是如何在进展过程中失调的 肾脏疾病的风险，以及针对肾脏途径的治疗如何影响全身代谢健康。

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.