Cysts are "building blocks" for epithelial organs, such as the kidney, and abnormal regulation of cystogenesis results in disorders such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD, affecting 500,000 Americans, is the most common potentially lethal genetic disease. The primary cilium, an organelle projecting from the apical surface, has been strongly implicated in ADPKD pathogenesis and is thought to act as a sensory antenna that transmits information regarding flow dynamics to the cell. When primary cilia of renal tubule cells are disrupted, the cells, through a still unclear mechanism, misinterpret this as a signal to dedifferentiate and proliferate, resulting in the formation of lare cysts that destroy the kidney. The goal here is to understand the role of the highly conserved eight-protein exocyst complex in kidney development, especially how the exocyst interacts with Wnt proteins. The exocyst is a critical component of the secretory pathway, shuttling vesicles containing membrane proteins from the trans-Golgi network to targeted subcellular locales, including primary cilia. Sec10, in turn, is a critical component of the exocyst. The Lipschutz Lab (the mentor's lab) has shown through in vitro studies of renal tubule cells that Sec10 is critical for proper formation of both primary cilia and cysts. In addition, it was shown in human ADPKD cells that the exocyst is mislocalized intracellularly, thereby establishing a link between exocyst location and ADPKD. Wnt genes encode a large, highly conserved family of secreted glycoproteins that play essential roles in controlling tissue patterning, cell fate, and proliferaton from drosophila to humans. Vertebrate Wnts have been divided into two functional groups by reference to their downstream signaling pathways. The canonical Wnts signal through nuclear 2-catenin/TCF-LEF while the noncanonical Wnts function through alternate signaling cascades that include Ca2+/PKC and RhoA/JNK. The canonical Wnt pathway is necessary for adult kidney homeostasis, and abrogation of this signaling leads to cystic kidney disease. The Lipschutz Lab showed that Rho GTPases regulate the exocyst, and Preliminary Studies show Wnt and exocyst mRNA co-expression during kidney development, as well as differential Wnt expression following Sec10 knockdown in vitro. The hypothesis we propose testing is that Wnt genes work with the exocyst to regulate kidney development, especially ciliogenesis and cystogenesis. Accordingly, we will use embryonic mouse kidneys to examine spatial-temporal exocyst and Wnt expression patterns during development (Aim 1). We will then use in vitro renal epithelial cell models to characterize the molecular pathways by which the exocyst and Wnts interact (Aim 2). Finally, we will study the role of the exocyst in regulating kidney development and Wnt signaling in vivo utilizing kidney- specific Sec10-overexpressing, Sec10 knockout, and Pkd2WS25/WS183 mice, representing an outstanding animal model of ADPKD (Aim 3). Successful completion of these experiments will result in the identification of novel candidate targets for therapeutic intervention in ADPKD, a disease for which no approved treatments exist.
Autosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic disorder, is characterized by cystic overgrowth that destroys the kidney, and a cellular organelle called the primary cilium has been strongly implicated in the pathogenesis of ADPKD. The eight-protein exocyst complex is necessary for cilia and cyst formation and likely interacts with Wnt proteins, which are also involved in kidney development and cystogenesis. The proposed experiments are designed to determine how exocyst and Wnt proteins interact to form cilia and generate cysts, with the successful completion of these experiments resulting in the identification of novel candidate targets for therapeutic intervention in ADPKD, a disease for which no treatments currently exist.
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