The glomerular extracellular matrix (ECM) is a continuous structure including the glomerular basement membrane (GBM) and the mesangial matrix. The GBM contains mainly heparan sulfate (HS) PG, a member of the perlecan family of basement membrane PGs synthesized by glomerular epithelial cells, while glomerular mesangial cells in culture synthesize mainly CS PGs. This PG was immunolocalized to both the mesangial matrix and the GBM. While it is important in kidney development, the role of CS PG in mature glomerular structure and function is poorly understood. We hope to develop specific immunologic and cDNA tools to investigate CS PG distribution and processing in normal and diabetic tissues by isolating cDNAs with sequences unique to the CS PG gene. We will do this after determining the DNA sequence of mesangial cell CS PG cDNA, having isolated several CS PG cDNAs by immunoscreening a mesangial cell cDNA expression library using anti-mesangial cell CS PG protein core antibodies. GBM proteoglycans (PG) participate in the charge barrier to plasma ultrafiltration and by virtue of this property may play a role in the pathogenesis of proteinuria. While most glomerular ECM components accumulate in the diabetic mesangium, its relative PG content is decreased. This may result from alterations in PG structure or synthesis, in interactions with other ECM components, or in PG processing. We will investigate the latter possibility in experiments designed to determine the role of the metabolic milieu in the pathogenesis of ECM accumulation in diabetes. Mesangial cell CS PG and protein synthesis is stimulated by insulin-like growth factor I (IGF-I). High concentrations of glucose (HG) abrogate the stimulatory effect of IGF-I on PG synthesis while further enhancing protein synthesis, an ECM composition similar to that found in diabetes. We propose to continue these studies by investigating the long and short term effects of HG on IGF-I mediated ECM processing in label- chase experiments. We will investigate PG and ECM processing at the translational (by Northern analysis) and post-translational (by radioactive labelling) levels. Basement membrane CS PG processing by glomerular mesangial cells in culture will be compared to processing of perlecan by arterial endothelial cells exposed to similar culture conditions. The development of specific CS PG cDNAs and antibodies will allow us to learn how this novel basement membrane PG is processed in normal and diabetic tissues.
