The chronic hyperglycemia of diabetes leads to serious complications, including neuropathy, retinopathy, microvascular disease, atherosclerosis, and diabetic nephropathy, the leading cause of end-stage renal disease. The severity of diabetic complications correlates with the severity of hyperglycemia, supporting the hypothesis that the elevated glucose levels trigger these complications. One of the major consequences of hyperglycemic conditions is acceleration of glucose modifications of proteins forming protein-Amadori intermediates that convert to a variety of advanced glycation endproducts, called AGEs. The AGEs have been implicated in the pathogenesis of diabetic complications, particularly diabetic nephropathy. Pyridoxamine (PM), a natural intermediate of vitamin B6 metabolism, has been shown to inhibit the conversion of Amadori intermediate to AGEs. Moreover, PM prevents development of early renal disease and retinopathy as well as formation of protein-AGEs in streptozotocin rat and db/db mouse model of diabetes. However, the mechanism of inhibition by PM is unknown, as are the mechanisms for the conversion of protein-Amadori intermediate to AGEs. The central hypothesis of this proposal is that: glucose modification of proteins, through the conversion of Amadori intermediates to AGEs, cause diabetic renal disease. It follows that by inhibiting this conversion; the development of diabetic complications can be prevented. This hypothesis can now be tested because we have developed a novel method for trapping the Amadori-intermediate under physiological conditions that mimic the diabetic state. Using this intermediate, the central hypothesis will be tested by the pursuit of four specific Aims: 1) to: determine the kinetics of post-Amadori reactions and structures of transient intermediates in the pathways of AGE formation; 2) to determine the mechanisms by which pryidoxamine inhibits protein post-Amadori pathways and formation of AGEs; 3) to determine the pathogenicity of albumin-Amadori and albumin-AGE when injected into rats; and 4) to elucidate specific pathogenic mechanisms of Amadori-AGE conversion in kidney basement membranes. The achievement of these aims requires the use of powerful methods of NMR spectroscopy, mass spectrometry, and classical protein chemistry coupled with animal studies and tissue analysis. We anticipate that the studies will yield novel insights into mechanisms of Amadori to AGE conversion and the role of AGEs in diabetic renal disease.
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