Project Summary/Abstract Polycystic kidney disease (PKD) is the leading genetic cause of renal failure, affecting an estimated 500,000 Americans. There is currently no cure for the disease, as we do not know what pathway drives disease pathogenesis. What we do know is that the driver of this disease localizes to an organelle known as the primary cilium. Our preliminary data using a novel genetic mouse model provides strong evidence that ARL13B, a regulatory GTPase that localizes to the primary cilium, drives cyst formation. This proposal aims to uncover the mechanism of ARL13B action in the cilium, by specifically dissecting its interaction with three effector proteins using a combination of biochemical and genetic approaches. We prioritized a list of 30+ candidate interacting proteins based on their links to renal cystogenesis, ARL13B interaction, and known pathways in other tissues. From this list, we are bringing the top three candidates–CDK1, ARL3, and INPP5E– forward for investigation in this proposal. These top candidates will provide knowledge how they are involved in kidney cystogenesis and provide Dr. Van Sciver with a framework in which to test other putative interactors of ARL13B. In Aim 1, we identify critical amino acid residues required for interaction between ARL13B and each effector. In Aim 2, we directly test ARL13B’s enzymatic activation of ARL3 in driving kidney cystogenesis using a point mutant ARL13B in vivo mouse model. In Aim 3, we directly test the enzymatic activity of INPP5E, which requires ARL13B for its localization to cilia, in driving kidney cystogenesis using a point mutant INPP5E mouse model. Aim 1 will be performed under the co-mentorship of Dr. Rick Kahn, a renowned expert in GTPase biochemistry, specifically in ARL and ARF family GTPases, and Dr. Tamara Caspary, an accomplished mouse geneticist. Dr. Van Sciver will receive additional training through didactic lectures in kidney physiology and purification and analysis of protein complexes. In addition, the Atlanta Network Training in KUH Scientific Research (ATLANTIS) program offers Dr. Van Sciver opportunities through three cores: research training, professional development, and networking. The detailed training plan coupled with this proposal will contribute to the success of Dr. Van Sciver’s overarching goal of becoming a tenure-track faculty member at an academic or research institution. Long-term outcomes from this proposal will define precise mutations mediating ARL13B’s interaction with each effector molecule, providing fundamental knowledge with a high probability of dissecting ciliary ARL13B’s pro-cystic mechanisms in part or in whole. In addition, this proposal will determine whether ARL13B functions via ARL3 and INPP5E and set up Dr. Van Sciver to test whether ARL13B specifically acts via CDK1 by generating a mouse in analogous experiments to Aims 2 and 3 in the future. This 5-year research and training plan will allow Dr. Van Sciver to establish an independent research program identifying mechanisms driving PKD.

Project Narrative Polycystic kidney disease, in which the kidney accumulates fluid-filled sacs, is the leading genetic cause of renal failure, yet the primary driver of this disease remains unknown. We used a unique mouse model as an entry point to dissect disease-driving mechanisms uncovering a protein, ARL13B, as a main driver of renal cysts. Expanding upon this foundational knowledge, we propose to further examine ARL13B interacting proteins and unravel the pathways leading to kidney cysts.