The primary events underlying the pathogenesis of cyst formation in human autosome domininant adult polycystic kidney disease (APKD) are not known. An in vitro model of human APKD has been established and characterized by this investigator and involves the individual microdissection of normal human renal tubules and individual renal cysts and epithelial cell growth in monolayer culture in defined media. Previous studies have shown APKD epithelia have a growth defect since epithelia proliferate more rapidly and extensively, that their mitogenic response to growth factors is abnormal and that they synthesize and secrete a grossly abnormal basement membrane. The present proposal addresses the question of the basis of this growth defect of APKD epithelia and aims to elucidate: 1. whether there is an altered sensitivity to normally circulating endocrine or paracrine peptide growth factors such as insulin, insulin-like growth factor 1 (IGF-l), epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and TGFbeta; 2. whether an autocrine growth factor is synthesized and secreted by APKD cells which serves to stimulate cell growth and 3. to determine whether there are modified growth factor- extracellular matrix interactions which account for increased proliferative potential. The methods to be used include the use of the defined tissue culture system of primary cultures, the measurement of growth by a mitogenic bioassay in which H3-thymidine incorporation into nuclei is quantitated, after application of exogenous growth factors, autocrine factors, conditioned media or cyst fluid. Inhibition of growth by growth factor antibodies will also be determined using this bioassay. Specific protein alterations will be determined by metabolic labelling and analysis by one and two-dimensional SDS-PAGE. To determine the basis of enhanced sensitivity, binding studies will be carried out with I - labelled growth factors and tissue levels of known growth factors will be measured and localized by radioimmunoassay and immunolocalisation techniques at the light and electron microscope levels. Purification of a putative 45kD autocrine factor will be performed using chromatographic techniques. Finally, basement membrane interactions with growth factors will be determined by the mitogenic bioassay, biosynthetic labelling and antibody localization. These studies will lead to the elucidation of the role of peptide growth factors and epithelial -extracellular matrix interactions in the pathogenesis of APKD.
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