Probing the Molecular Basis of Glomerular Injury in Diabetic Nephropathy

Vanderbilt University, Nashville TN

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Abstract

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.