Cysts are basic "building blocks" for epithelial organs, such as the kidney, and abnormal regulation of cystogenesis results in potentially lethal disorders such as polycystic kidney disease (PKD). The primary cilium, an organelle that projects from the apical surface of epithelial cells and has been implicated in the pathogenesis of PKD, is thought to act as a sensory antenna for the cell. When the primary cilia of renal tubular epithelial cells are disrupted in form or function, the cells misinterpret this as a signal to dedifferentiate and proliferate, resulting in the formation of large renal cysts. The highly conserved eight-protein exocyst complex is a critical component of the secretory pathway, shuttling vesicles containing membrane proteins to targeted subcellular locales, including the primary cilium. The overall goal of this research proposal is to elucidate the role of the exocyst in kidney development and cystogenesis, in order to better understand the process of pathogenic cyst formation. We have shown, through in vitro studies of the Manine-Darby Canine Kidney (MDCK) tubular epithelial cell line, that the exocyst is critical both for the formation of primary cilia and normal cysts. Additionally, we showed the silencing of Sec10, a central component of the exocyst, resulted in decreased intracellular calcium and increased cellular proliferation, both hallmarks of PKD. Based on these findings, our overall hypothesis is that the exocyst is critical for ciliogenesis and cystogenesis, and when disrupted in vivo, will result in a polycystic phenotype similar to that seen in other ciliopathies. We will test this hypothesis through the following Specific Aims: (1) Determine if Sec10-knockdown MDCK cells fail to form cysts due to defective lumen-coalescing hollowing or defective apoptosis-induced cavitation. Since renal cyst formation can be accomplished through these two alternative pathways, we will use three-dimensional cultures of MDCK cells to identify the specific stage (or stages) of cystogenesis that is disrupted when the exocyst is absent: polarity establishment, apical vesicle delivery, or apoptosis. (2) Identify the proliferative pathways regulated by the exocyst in renal epithelial cells. In this aim, we will determine if the increased proliferation measured in Sec10-silenced MDCK cells is due to similar molecular mechanisms that have been observed in PKD. (3) Generate a kidney-specific murine knockout of Sec10 and compare the renal phenotype with a known mouse model of autosomal dominant PKD. Since germline disruption of the exocyst has led to early embryonic lethality, we will use a Cre/lox targeting strategy to knockout Sec10 only in renal tubular epithelial cells. This will allow us to test if the absence of the exocyst in vivo recapitulates our in vitro findings of disruption of ciliogenesis and increased cellular proliferation, and leads to a renal phenotype similar to PKD. In addition to the practical training received from performing the proposed research, the didactic and mentoring that the applicant will receive during this award will ensure the successful transition from a postdoctoral trainee to an independent researcher focused on understanding the molecular basis of renal diseases.

Public Health Relevance

Autosomal dominant polycystic kidney disease (ADPKD), affecting 500,000 Americans, is the most common potentially lethal genetic disease and is characterized by cystic overgrowth that destroys the kidney. A cellular organelle called the primary cilium has been implicated in the pathogenesis of ADPKD and our Preliminary Studies show that the eight-protein exocyst complex is necessary for both cilia and cyst formation. In this application, we propose experiments to analyze the exocyst in kidney development and cyst formation, with successful completion of these experiments laying the groundwork for development of novel therapies for ADPKD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK087852-04
Application #
8290581
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
2010-07-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$152,965
Indirect Cost
$11,331
Name
University of Hawaii
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
965088057
City
Honolulu
State
HI
Country
United States
Zip Code
96822
Zuo, Xiaofeng; Fogelgren, Ben; Lipschutz, Joshua H (2011) The small GTPase Cdc42 is necessary for primary ciliogenesis in renal tubular epithelial cells. J Biol Chem 286:22469-77
Fogelgren, Ben; Lin, Shin-Yi; Zuo, Xiaofeng et al. (2011) The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes. PLoS Genet 7:e1001361
Somponpun, S Jack; Wong, Brittany; Hynd, Thomas E et al. (2011) Osmoregulatory defect in adult mice associated with deficient prenatal expression of six2. Am J Physiol Regul Integr Comp Physiol 301:R682-9