In addition to the role of hyperglycemia, it is clear that there are genetic and metabolic factors which predispose an individual to develop diabetic complications. Methylglyoxal (MG) is a powerful glycating agent and precursor of advanced glycation end products (AGEs) which appears to play an important role in the development of diabetic complications. In addition, high concentrations of glucose and lysine in the intracellular milieu leads inevitably to glucose-mediated Maillard reactions, the early and advanced products of which are also harmful to cell function. We have shown that MG production and oxidative stress are increased in accelerated diabetic nephropathy. This increased MG production could be secondary to genetic or acquired defects in downstream glyceraldehyde phosphate dehydrogenase (GAPDH). The purification and characterization of fructosamine-3-kinase (FN3K), which is capable of deglycating fructoslysine on glycated proteins, is the first indication of a potential system for controlling the Maillard reaction in higher organisms and may also play a role in susceptibility to nephropathy. The broad goal of this work is to examine factors that effect the production and detoxification of AGE precursors and to explore the role played by enzymatic mechanisms in chemical reactions that are non-enzymatic. The first objective is to elucidate the mechanisms responsible for increased MG production in nephropathy prone individuals. We will measure GAPDH activity, gene expression and mutations from two large groups who show biopsy proven progression or non-progression of diabetic nephropathy. To examine possible genetic factors we will do similar experiments in 40 sibling pairs who show similar degrees of nephropathy. The role of oxidative stress, glycation, and NAD+ levels, on GAPDH activity will also be evaluated. The second objective is to identify human FN3K isoforms at the protein, mRNA and genomic levels and to develop specific markers of in vivo enzyme activity, in complication-prone and resistant diabetic patients. This proposal utilizes unique study populations to provide a rigorous test of glycation mechanisms that determine resistance or susceptibility to the development of diabetic nephropathy. Increased understanding of the role played by enzymatic mechanisms that determine levels of dicarbonyls and Amadori products and their resulting AGEs is essential if we are to find effective strategies to prevent diabetic nephropathy.