The long term goal of our research proposal is to define in vitro and in vivo the membrane, cytoplasmic and nuclear signal transduction mechanisms by which cytokines activate mesangial and other glomerular cells. Expression of growth factors and immune-modulatory cytokines and their receptors is upregulated in renal tissue of patients and experimental animals with proliferative glomerular diseases. This proposal will explore the role of tyrosine phosphorylation/dephosphorylation mechanisms in activating mesangial cells. Our hypothesis is that the sustained activation of certain signals is responsible for changes in behavior of glomerular cells during disease. Thus, understanding the regulation of cell signaling and gene expression transduced by activated cytokine receptors provides the best chance to identify the most relevant targets for therapeutic intervention. We propose to carry out in vivo and in vitro studies to define mechanisms and pathways that platelet-derived growth factor (PDGF) and immune-modulary cytokines such as interferon- gamma, interleukin-1 (IL-1), IL-6 and tumor necrosis factor utilize to elicit their biologic effects in cultured mesangial cells and in experimental proliferative glomerulonephritis. Signals to be studied include tyrosine phosphorylation of membrane and cytoplasmic proteins that associate with the PDGF receptor or non-receptor tyrosine kinases stimulated by other cytokines. Signals to be explored include phosphatidyl inositol 3 kinase Janus kinases (JAKs). Immunoprecipitation and immunoblotting techniques will be used to identify and characterize tyrosine-phosphorylated proteins. Cytoplasmic and nuclear transcription factors that recognize specific DNA sequences present on target genes will be identified by gel retardation using labeled specific oligonucleotides. Activation of cytoplasmic transcription factors including members of the signal transducers and activators of transcription (STATS) will be explored. To define the mechanisms of cytokine interaction, we will periodically examine the effect of selected relevant cytokines interaction in glomerular cells as well as after in vivo administration. To deactivate signals, gene therapy approach will be utilized to deliver tyrosine phosphatases to the kidney using retroviral vectors. These studies should define signals involved in mediating glomerular pathology and establish effective therapeutic modalities to treat inflammatory forms of glomerulonephritis.
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