Among mammalian species, mice are unusually resistant to the development of diabetic complications. Our studies of the structure and enzymatic mechanisms of aldose reductase (AR) and recent data obtained in our laboratory on the murine aldose reductase enzyme suggest a probable explanation. The mouse AR activity is 1/3 of that of the human enzyme and our studies have enabled us to develop bio-engineered mouse models that will test the role of AR in the development of the diabetic complications of cataracts, neuropathy, retinopathy and nephropathy. Our models introduce single amino acid mutations into the murine aldose reductase gene and the germline by homologous recombination in embryonic stem cells (ES). These mutations restore mouse AR activity to levels comparable to the human enzyme. The mouse models will enable us to investigate the normal role of the enzyme in tissues and do complex studies of the etiology and pathogenesis of diabetic complications. The investigator will evaluate the effects of diabetes induction and galactose-feeding on the development of these complications in several tissues of several bio-engineered mouse models. These are models to study: 1) the pathogenic role of aldose reductase in the development of diabetic complications by restoring the activity of the murine aldose reductase to levels comparable to those in the human enzyme (""""""""humanized mouse""""""""); and 2) aldose reductase activity knockout model without disruption of the gene by a single amino acid mutation (Y48F) which inactivates the enzyme. By preserving gene structure, these models will also allow us to study feedback control and enhancer/promoter control of expression and transcription in the presence of enhanced enzyme activity as well in the total absence of enzyme activity. Each of the models will be characterized and evaluated by detailed quantitative studies for the development of cataracts, retinopathy, neuropathy and nephropathy after the induction of diabetes or galactose-feeding. They will also determine if the expression of aldose reductase is pathologically altered in diabetic tissues by comparing mRNA abundance, protein expression of enzyme activity in the tissues of the three mouse models: wild-type, high activity (""""""""humanized mouse""""""""), and a point mutation activity knockout model (Y48F). The effects of diabetes, insulin therapy and/or aldose reductase inhibitors on the regulation of aldose reductase levels in these tissues, and the activity of the p38 kinase cascade pathway which regulates the osmotic response element (ORE) of the aldose reductase gene will also be assessed.
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