We propose to study changes in the glomerulus leading up to sclerosis in the diabetic kidney. We hypothesize that alterations in the regulation of the expression and activity of a8B1 integrin are responsible for the microvascular changes in the early stages of diabetic nephropathy. Digital imaging microscopy will be used to investigate changes in the pattern of distribution and expression of this integrin in the glomeruli of a rodent model of type II diabetic nephropathy. Northern blot and Western immunoblot analysis will examine expression changes at the mRNA and protein level. In addition, a subset of the rodents will be used to establish cultures of mesangial cells where in vitro integrin expression and distribution can be studied under normoglycemic, hyperglycemic, and an osmotic control condition. All studies will utilize monoclonal antibodies raised against an ectodomain of a8B1 integrin, which we will develop using standard antibody production procedures. The results of all studies will also be compared to that of other integrins known to be in the glomerulus, particularly in the mesangial cells. Finally, manipulation of the expression of a8B1 integrin in the mesangial cells (and endothelial cells, if time allows) will be used to explore the role of a8B1 in altering the strength of attachment of these cells to the mesangial matrix. Together, these studies should aid in revealing the role of a8B1 in diabetes.
