Probing the Molecular Basis of Glomerular Injury in Diabetic Nephropathy

描述（由申请人提供）：在以下的研究培训计划中，我建议应用成像质谱法来详细了解糖尿病肾病（DN）的分子机制。DN以多种病理改变为特征，包括肾小球和小管基底膜增厚、系膜扩张、肾小球硬化、小动脉透明质症、小管肥大和小管间质纤维化。虽然在DN中发生的形态学变化已经被很好地定义，但关于控制这种病理的分子机制的信息是缺乏的。许多生物化学事件被认为在DN发病机制中起作用，包括内源性蛋白质的非酶糖协同氧化损伤。虽然糖氧化反应途径的化学性质已被很好地理解，但体内发生的特定反应及其在DN中所起的作用尚未得到很好的定义。我们假设由高血糖引起的细胞外蛋白的早期糖氧化修饰在DN的发展中起作用。由于氧化损伤在DN中可能通过多种途径发生，因此寻求能够监测体内多种生化变化的新型分析技术是很重要的。MALDI成像质谱（IMS）是一种新技术，可以直接对分子进行原位分析，特别适合于分析像DN这样复杂的多组分疾病。尽管对可溶性蛋白、多肽和脂质进行了大量的研究，但迄今为止还没有关于ECM蛋白的IMS分析的报道。因此，提出的研究的第一个目标将是开发组织中肾ECM蛋白的IMS分析的新方案。一旦建立，这些方法将用于识别伴随进行性DN的特异性糖协同氧化修饰，以便更好地了解糖基化反应如何促进糖尿病肾衰竭的临床特征。IMS将允许分析早期糖协同氧化修饰，在明显的形态变化出现之前。最后，对DN易感小鼠给予pyridoxamine（一种已证实的DN抑制剂）将有助于确定与DN病理相关的特定糖协同氧化途径，从而为控制该疾病的分子机制提供重要见解。除了设计研究计划外，我还与我的赞助人比利·哈德森博士密切合作，为我的专业发展制定了一个培训计划，这将有助于我成为学术机构的独立研究员。我有信心，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，我很高兴将我在奖学金课程中获得的技能应用到成功的研究生涯中

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.