Axoneme Hypoglutamylation in Primary Cilia Promotes Cystogenesis in ADPKD

Background: ADPKD is primarily caused by mutations in the PKD1 and PKD2 genes, which encode the proteins PC1 and PC2, respectively. PC1 and PC2 are hypothesized to form a receptor-channel complex on primary cilia membrane, which is crucial for maintaining the normal function and structure of renal tubules. PC1 and PC2 reciprocally regulate each other's ciliary localization by unclear mechanism. Loss of PC1 and PC2 in cilia significantly contribute to the progression of PKD. Therefore, increasing the levels of PC1/2 within cilia emerges as a potential therapeutic strategy for PKD. However, feasible methods or molecular targets to increase ciliary levels of PC1 and PC2 in the context of PKD are currently lacking. Polyglutamylation (PG) is a prominent post-translational modification of tubulin that occurs on the primary cilia axoneme. The established correlation between axoneme hypoglutamylation (reduced glutamylation) and human ciliopathies, which often present with cystic kidney phenotypes, suggests a plausible link between impaired axoneme PG and PKD pathogenesis. Our prior research has revealed that axoneme is crucial for ciliary localization of PC2. In this study, we foucs on the role of axoneme hypoglutamylation in renal cystogenesis in ADPKD. Methods: 1. 3D sephroid culture. 2. Immunofluoresent. 3. siRNA/shRNA-based knockdown. 4. Lentivirus-based overexpression. Results: Loss of PC1 leads to marked axoneme hypoglutamylation in PKD1 mutant renal epithelial cells. Remarkably, genetically or pharmacologically reducing axoneme PG significantly promotes 3D cystogenesis, while increasing axoneme PG suppresses it in PKD1 mutant renal tubular epithelial cells. Furthermore, reducing axoneme PG decreases, while increasing axoneme PG enhances the ciliary levels of PC2. These data suggest that PC1 regulates the ciliary localization of PC2 through modulating axoneme PG, and that axoneme hypoglutamylation significantly contributes to PKD progression. Our ongoing work focuses on investigating the role of axoneme hypoglutamylation in in vivo cystogenesis and testing whether increasing axoneme PG could attenuate PKD progression in Pkd1RC/RC mice. Conclusion: 1. PKD1 deletion leads to axoneme hypoglutamylation in renal epithelial cells. 2. Reducing axoneme PG aggrevates, while increasing axoneme PG suppresses cystogenesis in 3D culture. 3. Axoneme hypoglutamylation contributes to PC1-deficicency induced ciliary loss of PC2. Funding: NIDDK Support, Private Foundation Support