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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040698-11
Application #
2684187
Study Section
Pathology A Study Section (PTHA)
Program Officer
Hirschman, Gladys H
Project Start
1989-01-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
2000-03-31
Support Year
11
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
114400633
City
New York
State
NY
Country
United States
Zip Code
10029
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Lasker, J M; Chen, W B; Wolf, I et al. (2000) Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of Cyp4F2 and Cyp4A11. J Biol Chem 275:4118-26
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Hartz, P A; Wilson, P D (1997) Functional defects in lysosomal enzymes in autosomal dominant polycystic kidney disease (ADPKD): abnormalities in synthesis, molecular processing, polarity, and secretion. Biochem Mol Med 60:8-26
Kuo, N T; Norman, J T; Wilson, P D (1997) Acidic FGF regulation of hyperproliferation of fibroblasts in human autosomal dominant polycystic kidney disease. Biochem Mol Med 61:178-91
Wilson, P D (1997) Epithelial cell polarity and disease. Am J Physiol 272:F434-42
Du, J; Wilson, P D (1995) Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD. Am J Physiol 269:C487-95
Wilson, P D; Du, J; Norman, J T (1993) Autocrine, endocrine and paracrine regulation of growth abnormalities in autosomal dominant polycystic kidney disease. Eur J Cell Biol 61:131-8

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