In diabetes, hyperglycemia disturbs many metabolic pathways and each may lead to diabetic complications. One of the major alterations is the non-enzymatic glucosylation of proteins; it occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of endproducts). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, among the most important alterations are the thickening and the increased permeability of the glomerular basement membrane. Our long term objective is to understand at the molecular level the pathogenesis of those alterations. Laminin is one of the most important basement membrane components; it plays a key role in their structural organization, by interacting with most other basement membrane macromolecules and cell surfaces. Therefore, we propose to investigate the effect of non-enzymatic glucosylation on several biological functions of laminin and the role of aspirin (known to inhibit the reversible step) and aminoguanidine (known to inhibit the irreversible step) in restoring affected functions. For our studies, two in vitro model systems will be used: first, laminin extracted from a basement membrane-producing murine tumor (EHS) will be glucosylated; second, isolated, intact glomerular basement membranes from rats will be glucosylated and then laminin will be extracted. Glucosylation protocols will include addition of aspirin or aminoguanidine. After these treatments laminin will be tested for its structural appearance and in functional assays to examine the effect of non-enzymatic glucosylation on its properties. The functional assays will include: a) dimer formation, b) polymerization, c) binding to type IV collagen, d) binding to heparin, e) binding to heparan sulfate proteoglycan. A biophysical technique (turbidometry) and biochemical one (solid-phase assays) and a morphological one (electron microscopy of rotary shadowed samples) will be used for these assays. The proposed studies will a) broaden our knowledge on structural and functional modifications of basement membranes under diabetic conditions, b) enable us to test for functional implications and treatment of each stage of the modifications due to non-enzymatic glucosylation, and c) allow us to determine possible side effects of compounds that appear promising for preventing progression of diabetic complications.
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| Tsilibary, E C; Reger, L A; Vogel, A M et al. (1990) Identification of a multifunctional, cell-binding peptide sequence from the a1(NC1) of type IV collagen. J Cell Biol 111:1583-91 |
