THE EXOCYST, ADPCKD, AND RENAL ORGANOGENESIS

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DESCRIPTION (adapted from the application) Epithelial cyst and tubule formation represent complex, poorly understood processes that are crucial for organ development, regeneration following injury, and disease states such as autosomal dominant polycystic kidney disease (ADPCKD). In an in vitro model system, Madin-Darby canine kidney (MDCK) cells form cysts when grown in a collagen matrix and tubulate in response to hepatocyte growth factor, also known as scatter factor (HGF/SF). During tubulogenesis cells transiently lose polarity as they invade the collagen and regain polarity as new tubules form. Factors regulating this modulation of polarity are largely unknown. One candidate factor is the exocyst complex, which is a determinant of polarized secretion in budding yeast. The mammalian exocyst is highly conserved and is involved in polarized membrane traffic in epithelial cells. Preliminary data indicates that the exocyst complex is centrally involved in cyst and tubule formation. The exocyst complex localized to the tight junction of polarized MDCK cells and, during tubulogenesis, relocalized along the growing tubules in a pattern consistent with the changes in polarity. When a critical component of the exocyst, hSec10, was overexpressed in MDCK cells, more efficient and rapid cyst formation occurred along with greatly increased tubule formation upon stimulation with HGF/SF. In this R03 supplement application for NIH Grant K08DKO2509, the creation of transgenic mice overexpressing hSec10 under the control of a renal cell-specific promoter is proposed in order to evaluate the in vivo role of hSec10 in tubulogenesis. The expression pattern of exocyst complex members in developing kidneys and in ADPCKD tissue will also be studied. A probable dominant negative form of hSec10 will be cloned into an adenovirus vector and used to infect control and MDCK cells overexpressing full-length hSec10 in order to generate preliminary data for a more extensive study involving gene therapy. Finally, the groundwork will be laid for the generation of mice with targeted disruptions in the hSec10 gene. In summary, the exocyst complex has not previously been implicated in the process of cyst and tubule formation. Our preliminary data shows this complex is involved in cystogenesis and tubulogenesis in an in vitro model system. I will test the hypothesis that overexpression of the hSec10 component of the exocyst complex increases cystogenesis and tubulogenesis in vivo in a murine model and in tissue samples from patients with ADPCKD and is, therefore, a novel therapeutic target.