Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of fluid-filled sacs called cysts in both kidneys, but key signals that cause cyst formation are unknown. Multiple downstream cellular pathways are dysregulated during cystogenesis. However, targeting these pathways has limited effects on ADPKD treatment. ADPKD is caused by mutations in genes encoding for polycystin-1 (PC1) and polycystin-2 (PC2). Both polycystins localize to primary cilia. The primary cilium instructs cellular decisions in response to extracellular inputs by compartmentalizing subcellular signaling. However, the role of primary cilium in kidney cystogenesis is inherently complex. Loss of polycystins causes severe cystogenesis, which is mostly cilia- dependent, while loss of cilia by itself causes smaller cysts. These results suggest that a complex interplay between counter-regulatory positive and negative signals in cilia inhibit cyst formation in normal renal tubules and promote cyst growth in ADPKD, respectively. Identifying these signals could have profound impacts on novel therapeutic targets and strategies for ADPKD. However, uncoupling ciliary signals causing cystogenesis from downstream signaling pathways affected during cystogenesis and the difficulty in identification of ciliary signals in absence of cilia have prevented their identification. Here, by studying the ciliary trafficking adapter, tubby family protein Tulp3, we aim to identify and target the key upstream ciliary signals that regulate cystogenesis. We previously showed that Tulp3 functions in ciliary trafficking of membrane proteins without affecting respective protein levels or disrupting cilia by coupling to the intraflagellar transport complex A (IFT-A). We recently showed that embryonic kidney-specific conditional knockouts of Tulp3 developed renal cystogenesis that was less severe than from polycystin loss. Concomitant Tulp3 loss did not inhibit cystogenesis upon PC1 loss, unlike ciliary disruption, but caused early lethality, suggesting accelerated loss of renal function. These results further reinforce the polycystin independent inhibitory role of ciliary proteins in cystogenesis. Other groups have reported suppression of cystogenesis in adult mouse models of PKD from Tulp3 or IFT-A loss. Thus, we hypothesize that Tulp3-regulated ciliary cargoes determine cilia-dependent cyst inhibition during development and PC1/2- inhibited cilia-dependent cyst activation in adults. Here by leveraging our expertise in ciliary trafficking and signaling and using novel mouse models to block trafficking of potential cargo subtypes, we propose to identify ciliary regulators of cystogenesis. In Aim 1, we will determine novel Tulp3 trafficked ciliary cargoes and signaling outputs in cilia relevant to cystogenesis. In Aim 2, we will determine Tulp3 regulated cyst inhibitory ciliary cargo subtypes that genetically synergize with PC1 during murine kidney development. In Aim 3, we will test Tulp3 cargo subtypes as promoters of cystogenesis in adult mouse models of PKD in vivo using genetic epistasis. Our approach will determine dual ciliary drivers impacting cystogenesis in vivo during adult-onset ADPKD and embryonic-onset cystic disease unraveling the molecular nature of cilium-generated signaling in either setting.

Public Health relevance/Narrative Defects in the primary cilia modify progression of cystogenesis in polycystic kidney disease models in mice, but the identity and role of ciliary regulators are unknown. This project addresses several key issues in signaling generated by primary cilium, including mechanisms underlying trafficking of proteins to the ciliary membrane, and will use orthologous mouse and cell culture models to evaluate the role of ciliary trafficking in pathogenesis of polycystic kidney disease. Testing the mechanisms of ciliary trafficking of potential regulators and role of cilium-generated signaling with respect to kidney cystogenesis could potentially provide molecular targets for ameliorating disease progression in humans.