PATHOLOGY OF BASEMENT MEMBRANE COMPONENTS IN DIABETES

Diabetes has profound microvascular complications which lead to multisystem abnormalities involving the basement membrane and extracellular matrix. The diabetic kidney manifests much pathology where: glomerular basement membrane (GBM) function is abnormal, the mesangium becomes expanded and there is a progressive thickening of GBM. The GBM and mesangial matrix are composed of: type IV, V collagens, proteoglycans, fibronectin (FN), laminin (LMN), and entactin, among other things. Among numerous molecular abnormalities in the glomerulus and plasma in diabetes are: 1) increased FN in the mesangium, 2) decreased heparan sulfate proteoglycan, 3) increased nonenzymatic glycation of type IV collagen, and 4) increased nonenzymatic glycation of FN. Nonenzymatic glycation of FN or collagen decreases the binding avidity of these molecules. The binding of fibronectin and collagen (gelatin or type IV) or laminin and type IV collagen, leads to positive cooperativity for 3H-heparin binding. Nonenzymatic glycation of FN, LMN or collagen(s) leads to a profound decrease in positive cooperativity for heparin binding. Glycation of laminin also produces a direct decrease in heparin binding to it. The proposed studies will perform binding assays with normal and nonenzymatically glycated FN, LMN, heparin, heparan sulfate proteoglycan derived from glomeruli and the EHS tumor, native collagens (IV, I and III), Clq and fibrin. We will define the domain of FN that binds type IV collagen and further define the collagen I (gelatin) binding domain. This will involve proteolytic and chemical cleavage and purification of various domains of fibronectin. We will next determine the deposition and turnover of radiolabeled FN and nonenzymatically glycated FN following I.V. administration of normal and diabetic animals. Next, we will determine if there is increased accumulation or turnover of fibronectin in glomerular cultures from normal and diabetic animals. Lastly, we will isolate plasma fibronectin from poorly controlled diabetic humans to determine whether it demonstrates the same abnormalities described above. Collectively these studies will allow us to investigate what may be a key biochemical abnormality in the diabetic GBM and mesangium, namely the perturbed molecular association of relevant molecules. Evaluation of a plasma protein which could serve as an indicator for the microangiopathic and nephropathic changes that are occurring in diabetes is very attractive. It seems clear that these studies may also shed light on the processes governing synthesis, turnover, and accumulation of basement constituents.