Cyst formation and expansion in human autosomal dominant polycystic kidney disease (ADPKD) is associated with epithelial hyperplasia in vivo and is reflected by abnormal proliferative responses to exogenous growth factors in vitro. Previous studies from this laboratory have defined important roles for epidermal growth factor (EGF) as an autocrine regulator of increased cyst epithelial growth in ADPKD and acidic FGF (aFGF) as an autocrine regulator of interstitial fibroblast proliferation. In both cases, significant abnormalities in receptor tyrosine kinase activation and expression have been detected. EGF receptors are over-expressed and abnormally polarized to apical cell membranes while aFGF receptors show abnormal molecular weight forms by covalent cross-linking analysis. These and other results have led to the formulation of a unifying hypothesis that proliferative abnormalities associated with progression of ADPKD are the result of modifications in the normal renal signal transduction pathways mediated by ligand-induced occupancy and stimulation of membrane receptor tyrosine kinases and subsequent activation of the cytoplasmic serine/threonine kinase cascade, culminating in mitogenesis. This hypothesis will be tested in 2 days in the present proposal.
Aim 1 will determine whether there are ADPKD-specific abnormalities in receptor/ligand internalization, trafficking or sorting which would lead to reduced down-regulation and degradation, increased receptor recycling to or aberrant insertion in polarized plasmamembrane domains. Constitutive fluid phase and ligand-induced (EGF, aFGF) rates and routes of endocytosis will be analyzed biochemically and by light and electron .
Aim 2 will determine whether novel tyrosine or serine/threonine kinases of the FGFR, EGFR or PKC/RAC families exist in ADPKD epithelia or fibroblasts by degenerate PCR cloning. Recent cloning, partial sequencing and homology analysis of a novel serine/threonine kinase by this strategy validate the feasibility of this approach. RNase protection assays showing expression in ADPKD, ARPKD and fetal but not adult kidneys suggest this is PKD and proliferation-related, most closely resembling a Drosophila gene but related to both PKA and PKC subfamilies. Functional analysis will be used to fully asses its function in ADPKD cells. An additional PGR product in ARPKD with EGFR-related degenerate primers has also been detected and will be studied. Detection of such specific kinases would lead to the possibility of therapeutic intervention to slow cyst expansion and disease progression in ADPKD since designer synthetic drugs which might be specific, safe and effective and could be tested in vitro in our culture system of ADPKD and normal renal epithelia.
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