Probing the Molecular Basis of Glomerular Injury in Diabetic Nephropathy

描述（由申请人提供）：在以下研究培训计划中，我建议应用成像质谱法详细了解糖尿病肾病（DN）的分子机制。DN的特征在于许多病理变化，包括肾小球和肾小管基底膜增厚、系膜扩张、肾小球硬化、小动脉玻璃样变性、肾小管肥大和肾小管间质纤维化。虽然发生在DN的形态学变化已被很好地定义，这种病理学的分子机制的信息是缺乏的。许多生化事件已被认为在DN发病机制中发挥作用，包括内源性蛋白质的非酶糖氧化损伤。虽然糖氧化反应途径的化学性质已被充分理解，但体内发生的特定反应及其在DN中的作用尚未明确。 我们推测，早期糖氧化修饰细胞外蛋白诱导的高血糖在DN的发展中发挥作用。由于氧化损伤可能通过多种途径在DN中发生，因此重要的是追求能够监测体内多种生化变化的新型分析技术。MALDI成像质谱（IMS）是一种新技术，可以直接分析分子原位，特别适用于分析复杂的多组分疾病，如DN。 尽管对可溶性蛋白质、肽和脂质进行了大量研究，但迄今为止还没有报道ECM蛋白质的IMS分析。因此，所提出的研究的第一个目标将是开发用于组织中的肾ECM蛋白的IMS分析的新方案。一旦建立，这些方法将被用来确定特定的糖基化修饰伴随进行性DN，以获得更好地了解糖化反应如何有助于糖尿病肾功能衰竭的临床特征。IMS将允许在明显的形态学变化出现之前分析早期糖氧化修饰。最后，吡哆胺，一种已被证明的DN抑制剂，DN易感小鼠的管理将有助于确定特定的糖氧化途径，连接到DN的病理，提供显着的洞察到这种疾病的分子机制。 除了设计研究计划外，我还与我的赞助商比利哈德逊博士密切合作，为我的专业发展制定了一个培训计划，这将有助于我在学术机构中作为独立研究员的职业生涯。我相信，我所设计的研究培训计划将有助于我作为一名科学家的发展，我很高兴能运用我在奖学金期间获得的技能， 质谱法研究肾脏和血管疾病的分子基础。

DESCRIPTION (provided by applicant): In the following research training plan, I propose to apply imaging mass spectrometry to gain a detailed understanding of the molecular mechanisms underlying diabetic nephropathy (DN). DN is characterized by a number of pathological changes, including thickening of the glomerular and tubular basement membranes, mesangial expansion, glomerular sclerosis, arteriolar hyalinosis, tubular hypertrophy, and tubulointerstitial fibrosis. Although the morphological changes that occur in DN have been well defined, information about the molecular mechanisms governing this pathology is lacking. A number of biochemical events have been suggested to play a role in DN pathogenesis, including nonenzymatic glycooxidative damage of endogenous proteins. While the chemistries of glycooxidative reaction pathways are well understood, the specific reactions that occur in vivo and the roles that they play in DN are not well defined. We hypothesize that early glyco-oxidative modifications to extracellular proteins induced by hyperglycemia play a role in the development of DN. Because oxidative damage may occur via multiple pathways in DN, it is important to pursue novel analytical technologies that have the ability to monitor diverse biochemical changes in vivo. MALDI imaging mass spectrometry (IMS) is a new technology that allows for direct profiling of molecules in situ and is uniquely suited for analysis of a complex, multi-component disease like DN. Despite numerous studies of soluble proteins, peptides, and lipids, there are no reported IMS analyses of ECM proteins, to date. Therefore, the first goal of the proposed research will be to develop new protocols for IMS analysis of renal ECM proteins in tissue. Once established, these methods will be used to identify specific glycooxidative modifications that accompany progressive DN, in order to gain a better understanding of how glycation reactions contribute to the clinical features of renal failure in diabetes. IMS will allo for analysis of early glycooxidative modifications, before noticeable morphological changes appear. Finally, administration of pyridoxamine, a proven inhibitor of DN, to DN-prone mice will help to identify specific glycooxidative pathways that are connected to the pathology of DN, providing significant insight into the molecular mechanisms governing this disease. In addition to designing the research proposal, I have worked closely with my sponsor, Dr. Billy Hudson to develop a training plan for my professional development that will aid in my pursuit of a career as an independent researcher at an academic institution. I am confident that the research training plan that I have designed will aide in my development as a scientist and I am excited to apply the skills I will gain over the course of the fellowship towards a successful research career using mass spectrometry to investigate the molecular basis of renal and vascular diseases.