Anks3 Mediates Cilia-Dependent Polycystin Signaling and Is Essential for Adult Kidney Homeostasis

Background: The cilia-dependent cyst activation (CDCA) pathway underlying autosomal dominant polycystic kidney disease (ADPKD) was identified by showing that intact primary cilia are crucial for cyst growth following loss of polycystins (PCs). We previously applied TRAP RNASeq on pre-cystic mouse kidneys to determine a translatome associated with CDCA and identified Glis2 as an early effector of PC signaling. Here, to discover additional components of CDCA, we used PC-dependent Glis2 expression as a functional readout and identified Anks3, from the CDCA translatome, as a cytosolic regulator of PC signaling. Methods: We used CRISPR gene editing to study the effect of Anks3 inactivation on PC-dependent Glis2 expression. We used LC-MS/MS to elucidate the PC-dependent phosphorylation of Anks3. We generated an Anks3fl conditional allele and crossed it with Pkd1fl/fl; Pax8rtTA; TetOcre mice to investigate the genetic interactions in early- and adult-onset ADPKD models. We did RNASeq on renal primary cells and kidney tissues to understand the transcriptomic changes in Anks3 inactivation and to uncouple the effect of Anks3 inactivation on CDCA signaling from other effects on kidney homeostasis. Results: Inactivation of Anks3 suppresses upregulation of Glis2 following Pkd gene inactivation in mRNA and protein expression both in vitro and in vivo. Anks3 also shows an increased phosphorylation state in cells and kidney tissues following loss of PCs. In keeping with its suppressive effect on Glis2 upregulation, inactivation of Anks3 suppresses cyst progression in early and late onset models of ADPKD. Beyond its effect on cyst progression, inactivation of Anks3 causes rapidly progressive kidney injury. RNASeq from primary cells and kidney tissues shows that Anks3 inactivation normalizes a significant portion of the PC-dependent CDCA translatome while also resulting in significant changes in ECM expression in vitro and a inflammatory responses in vivo. The ECM and inflammatory changes are related to the kidney injury and are not dependent on PCs. Conclusion: These findings define Anks3 as a central regulator of cilia dependent PC signaling (CDCA), functioning downstream of cilia and PCs and upstream of Glis2. They also show that Anks3 has broader functions in maintaining renal structural and functional homeostasis. Funding: NIDDK Support