The broad, long term objective of this research is to identify intracellular mechanisms that mediate cyst formation in the human developmental renal disease known collectively as polycystic kidney disease (PKD). Although progress has been made in identifying genetic loci associated with inherited PKD, little is known about intracellular mechanisms that mediate cyst formation and progressive enlargement. In human autosomal recessive PKD (ARPKD) and autosomal dominant PKD, and in congenital murine models of PKD, quantitative and qualitative abnormalities of epidermal growth factor (EGFR) expression are consistent features of the cystic phenotype. Studies of cystic maldevelopment, using experimental models of cyst formation, may provide information about both cystogenesis and aberrations in normal renal developmental processes. This proposal will focus on (a) cyst formation and progressive enlargement in a murine model of ARPKD, the C57BL/6J cpk/cpk (CPK) mouse, and (b) tubulogenesis and cystogenesis in Madin Darby Canine Kidney (MDCK) cells in culture. The hypothesis that cellular consequences of altered EGFR expression lead to cyst formation and enlargement in PKD will be tested by the following Specific Aims: (1) Determine the mechanisms for EGFR mislocation and overexpression in CPK kidney; (2) Determine the functional intracellular consequences of over expressed, apical EGFRs and if overexpressed EGFRs are responsible for a """"""""cystic"""""""" phenotype; (3) Determine if distinct protein kinase systems mediate tubule and cyst formation in novel MDCK cell clones; (4) Determine if aberrations in pathways found to be crucial for morphogenic transformation in MDCK cells contribute to cyst formation in CPK kidney. Studies will be performed on whole kidneys and cell cultures of distinct cystic and non cystic tubular epithelia at specific developmental stages; organ culture models of tubular cyst formation; and on an established cell line. CPK mice will be used to characterize cystic EGFR gene and protein expression to determine if a mutation in the EGFR itself is responsible for EGFR mislocation and overexpression. Human or murine EGFR cDNA expression vectors will be used to determine if EGRF overexpression is sufficient to confer a cystic phenotype on a cultured organ or cell system. Protein kinase activities that appear to be involved in tubule and cyst formation in MDCK clones will be manipulated both pharmacologically and by recombinant DNA technology to determine the role of these kinases in morphogenesis. Pathways found to be crucial for morphogenic transformation in MDCK clones will be evaluated in the murine model. Molecular characterization of distinct cystic phenotypes in experimental and human PKDs, and identification of specific cystic genes and gene products, may provide insight into the pathogenesis of renal cyst formation and may aid the development of disease-specific therapy for human PKD.

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Case Western Reserve University
Schools of Medicine
United States
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Orellana, Stephanie A; Quinones, Andrea M (2003) The absence of prostaglandin e1 returned confluent cultures of highly proliferative murine polycystic kidney principal cells to a normal proliferation level. In Vitro Cell Dev Biol Anim 39:199-203
Orellana, Stephanie A; Quinones, Andrea M; Mandapat, Marylou L (2003) Ezrin distribution is abnormal in principal cells from a murine model of autosomal recessive polycystic kidney disease. Pediatr Res 54:406-12
Marfella-Scivittaro, Carmela; Quinones, Andrea; Orellana, Stephanie A (2002) cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia. Am J Physiol Cell Physiol 282:C693-707
Orellana, S A; Marfella-Scivittaro, C (2000) Distinctive cyclic AMP-dependent protein kinase subunit localization is associated with cyst formation and loss of tubulogenic capacity in Madin-Darby canine kidney cell clones. J Biol Chem 275:21233-40
Orellana, S A; Avner, E D (1998) Cell and molecular biology of kidney development. Semin Nephrol 18:233-43