Cilia calcium dysregulation in polycystic kidney disease

ABSTRACT. The primary cilium is a Ca2+-privileged, antenna-like cellular organelle found in all organ systems of the human body. The importance of primary cilia are highlighted by the growing number of renal ciliopathies— many of which are caused by mutations in Ca2+ signaling effector genes. Autosomal dominant polycystic kidney disease (ADPKD) is a fatal renal ciliopathy that can be caused by mutations in the PKD2 Ca2+ channel. Despite 20 years since determining its genetic cause, we do not know how ADPKD mutations alter PKD2 channel function and if Ca2+ dysregulation in the primary cilium contributes to kidney cyst formation. These basic questions remain outstanding because PKD2 localizes to the enigmatic primary cilium—which requires innovative tools to study. To this end, our lab has developed novel assays to study ciliary Ca2+ signaling and PKD2 channel dysregulation caused by ADPKD mutations in real-time, at super- and atomic resolution. In the cilia of the collecting duct, PKD2 regulated by internal Ca2+ is bimodal. When ciliary Ca2+ is elevated to micromolar concentrations, open probability of PKD2 increases (CDM) and over time becomes desensitized (CDD). However, molecular mechanism for both processes are unknown. Recently, we published a refutation of the hypothesis that C-terminal EF hands of PKD2 is involved in channel regulation and ADPKD progression. In our unpublished preliminary data, we identified a new Ca2+ binding site in the voltage sensor domain (VSDCa- site), where several ADPKD-causing mutations aggregate. We hypothesize that mutations at this site may cause either a loss- or gain-of-channel-function, depending on their functional impact on CDM and CDD. We have devised specific aims to define the Ca2+-dependent molecular regulation of PKD2, while assessing the impact of ADPKD mutations. There is no drug cure for ADPKD. Thus, assessing mechanistic differences between mutations is essential for designing future ADPKD treatment strategies. We will also test the “ciliary Ca2+ hypothesis of cystogenesis” by assessing the impact of VSDCa-site mutations on cilia Ca2+ dynamics and downstream gene expression in ADPKD patient cells. We will then determine if corrective PKD2 gene editing (CRISPR/Cas9) can reinstate normal ciliary Ca2+ and gene transcription. The aims proposed will test our limited understanding of how channel dysregulation in the primary cilia initiates cyst development in the kidney. Beyond ADPKD, ciliopathies which primarily other impact other organ systems, frequently exhibit kidney cysts as comorbidities. Thus, the findings from this proposal may extend to other renal ciliopathies, where aberrant cilia-to-cell Ca2+ transduction is a possible unifying signaling mechanism.