DESCRIPTION (provided by applicant): Cystogenesis is critical for early kidney development; however, if renal cystogenesis is delayed and/or occurs in an incorrect context, the resulting cysts can destroy kidney structure, thereby promoting polycystic kidney disease (PKD) and renal failure. Virtually all forms of PKD in human patients and animal models are associated with perturbations in renal primary cilia structure and/or function. For example, mutations in genes encoding the ciliary proteins polycystins-1 and -2 cause autosomal dominant PKD (ADPKD), the most common potentially lethal monogenic disorder in humans, while mutations in multiple ciliary proteins lead to nephronophthisis, a form of PKD that is the most common genetic cause of renal failure in children and young adults. Although maintenance of primary cilia structure and function appears to be key to preventing/treating PKD, it is not entirely clear how aberrant ciliogenesis promotes disease. Moreover, since there are no approved treatments for any form of PKD, and even the most promising treatments have significant side effects and/or toxicities, a more thorough understanding of primary cilia formation/function is critical for the identification of future therapeutic targets for the treatment of PKD. The long-term goal of our laboratory is to identify the mechanistic basis for ciliogenesis, and determine how aberrations in this process contribute to PKD. Our laboratory was the first to show that the highly-conserved eight-protein exocyst complex, which promotes targeting and docking of vesicles carrying proteins from the trans-Golgi network (TGN), localizes to, and is necessary for formation of, primary cilia. Notably, mutations in an exocyst protein, identified in a family with nephronophthisis, support a role for the exocyst in prevention of PKD. The exocyst has been shown to be critical for targeting Rab8-positive vesicles bearing ciliary proteins to the nascent cilium. More recently, we demonstrated that both the small GTPase Cdc42 and Tuba, a Cdc42 ciliary-specific guanine nucleotide exchange factor (GEF), regulate targeting of the exocyst itself to the nascent cilium, where the Sec10 component of the exocyst directly interacts with the Par complex. Most notably, we recently demonstrated that cdc42 knockdown in zebrafish causes ciliary defects and Cdc42 kidney-specific knockout in mice prevents ciliogenesis, which leads to a PKD nephronophthisis phenotype, renal failure, and death. Preliminary data show that tuba knockdown in zebrafish results in a similar phenotype. Based on these data, we hypothesize that activation of Cdc42 by one or more ciliary-specific GEFs is required to recruit the exocyst to the nascent cilium, thereby promoting Sec10/Par6 stabilizing interactions that allow for the targeting of Rab8-positive vesicles bearing ciliary proteins to promote ciliogenesis (tested in Aims 1 and 2). We are in a unique position to undertake these experiments as we have all the necessary tools, techniques, and reagents. As proper cilia formation and function are critical for preventing PKD, further understanding of a major delivery route for ciliary proteins will likely identify a number of novel candidate targets and points of therapeutic intervention. We also showed that Cdc42 and Sec10 disruption in vitro and in vivo leads to MAPK activation and an increase in phosphorylated ERK (pERK). As MAPK activation occurs in the nephronophthisis pcy mouse, and pharmacologic normalization of pERK prevents cystogenesis, we further hypothesize that inhibition of exocyst localization/stabilization at the nascent cilium results in abnormal MAPK activation that, in turn, leads to PKD. In Aim 3, we will take a pharmacologic approach to test this hypothesis in our Cdc42 and Sec10 knockdown zebrafish and conditional murine knockout models, and will also exploit our recently described novel adeno-associated viral (AAV) vector, and retrograde delivery system, that allows for gene expression in murine kidney tubule cells. Taken together, our work opens up new avenues for the treatment of PKD: utilizing pharmacologic means to inhibit the MAPK pathway, and molecular biology to deliver wild-type genes to rescue inherited ciliopathies.

PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD), affecting approximately 1/500 Americans, is the most common potentially lethal genetic disease, and nephronophthisis, a form of PKD, is the most common genetic cause of kidney failure until age 30. Given 25 million Veterans and 45 million family members, there are about 100,000 Veterans, Active Duty Service members, and Dependents with PKD. As a kidney doctor at the VA, and in the Army National Guard, I frequently see patients with ADPKD, and, sometimes, nephronophthisis, and it is difficult to tell them there are no approved treatments, and all therapies in clinical trials have significant toxicity and/or side effects. PKD is characterized by cystic overgrowth that destroys the kidney. An organelle called the cilium has been strongly implicated in PKD pathogenesis and we showed that the exocyst complex and Cdc42, which regulates the exocyst, are necessary for cilia formation, both in cell culture and in animals. Here, we will determine how Cdc42 and the exocyst build cilia, and then test two possible treatments for PKD.