Destructive proliferation and sclerosis at sites underlying endothelium in the kidney microvasculature irreversibly impair renal function through luminal narrowing, destruction of glomerular architecture and reduction of filtration surface. The objective of these studies is to define mechanisms regulating renal endothelial cell production of growth factors that are implicated in proliferation and sclerosis subjacent to the endothelial layer. Especially relevant in destructive glomerular diseases are proliferation of mesangial cells, fibroblasts and matrix deposition, all of which are stimulated by platelet-derived growth factor (PDGF). Our data, using primary cultured human renal microvascular endothelial cells, have shown PDGF B/c-sis messenger RNA (mRNA) expression and activity release are regulated by agents relevant at vascular sites. Transcription of PDGF B/c-sis mRNA is induced by platelet (transforming growth factor beta) and coagulation products (thrombin) and suppressed by hormones which elevate cAMP levels (catecholamines). New data has shown renal endothelial expression of distinct structural forms of PDGF B mRNA. The proposed studies will determine the relative contribution of different PDGF B mRNA's to translation of active PDGF protein product, using in vitro and xenopus oocyte translation of isolated and synthesized structural forms of each PDGF B/c-cic mRNA. Additional experiments will determine if expression of different PDGF B/c-sis mRNAs is differentially regulated by transcriptional and stabilization mechanisms. Genomic PDGF B/c-cic sequences participating in regulation of renal endothelial expression will be identified by DNA footprinting techniques and transient expression of a series of flanking sequence deletion constructs. DNA sequences participating in cAMP-mediated repression of PDGF B/c-sis transcription will be identified and characterized by gel shift assays, and DNA binding proteins identified by UV cross-linking and as substrates for cAMP dependent kinase. Intact tissue roles for PDGF B/c-sis expression will be identified using in situ hybridization of PDGF B/c-sis mRNA in renal biopsy samples. These studies will define molecular mechanisms regulating microvascular endothelial production of PDGF and may provide potential targets for intervention in destructive subendothlial proliferative processes.
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