The overall hypothesis behind this proposal is that diabetic complications in renal cells are due to specific malfunctions in signal transduction mechanisms induced by hyperglycemia and abnormal glucose metabolism. Prolonged hyperglycemia has two major consequences in affected tissues. One is that there is an elevated level of diacylglycerol by de novo synthesis from glucose, while the second is an accumulation of advanced glycation end products (AGEs). These changes cause abnormal activation of protein kinase C and an expansion of the extracellular matrix. The effects of high glucose and AGE products on signaling pathways activated in response to mitogenic and non-mitogenic agonists will be correlated with functional changes in glomerular endothelial and mesangial cells in solo or co-culture conditions. Powerful new techniques involving microinjection of reagents and digital Ca2+ image analysis will be applied to study the changes in cell signaling at the single cell level. Effects of two classes of hormones, those that interact with receptors coupled to G-proteins and those that contain or recruit tyrosine kinases, will be investigated by using intracellular free Ca2+, PLA2 activity, DNA synthesis and secretion of type IV collagen as functional parameters. Steps in receptor-activated signal transduction pathways will be probed by microinjection or use of fluorescent indicators, caged compounds, antibodies and specific inhibitors. The following specific aims will be addressed, l) the specificity of receptor-G-protein coupling will be determined in glomerular endothelial and mesangial cells, 2) the mechanism and regulation of hormone-induced Ca2+ oscillations will be investigated in endothelial and mesangial cells, 3) the effects of high glucose and AGE products will be measured on agonist-induced Ca2+ mobilization, DNA synthesis, phospholipase A2 activity and secretion of type IV collagen, and 4) effects of high glucose and AGE products on cell-cell signaling mechanisms will be assessed in co-cultures of endothelial and mesangial cells An important goal of the present work will be to define the interplay of autocrine and paracrine factors between endothelial and mesangial cells under conditions simulating the diabetic state. This information will be critical for the development of future pharmacological approaches for the prevention and treatment of diabetic nephropathy.
