Interrelationship between cell proliferation, actin disruption, and cytosolic calcium in cilia -deficient collecting duct cells

Identification of the genes involved in inherited cystic kidney diseases (CDKs) and their protein products has provided key insights into the cellular processes that underlie cyst development. The proteins implicated in these disorders (such as polycystin-1 and 2) localize to the apical membrane primary cilium of epithelial cells, focusing recent research on the role of this cellular structure in cystogenesis. Primary cilia have been reported to function as cellular mechanosensors, translating changes in renal tubular fluid flow into intracellular signaling cascades by activating calcium transients. Dysfunction of this process is thought to interrupt normal cell signaling and contribute to cellular de-differentiation and hyperplasia which initiate cyst formation. This dissertation work focused on determining the effects of cilia disruption on intracellular calcium regulation in renal epithelial cells, and identifying the consequences of this calcium regulatory defect on cellular processes that contribute to cystogenesis. The first set of studies examined regulation of apical calcium permeability in ciliadeficient cortical collecting duct principal cells form the Oak Ridge Polycystic Kidney (orpk) mouse. It was determined that cilia loss or dysfunction prevented a flow-induced calcium signal, but resulted in increased apical membrane calcium entry. The increased apical calcium flux was due to altered distribution of apically located polycystin-2, a calcium channel, and resulted in elevated steady-state sub-apical calcium levels. The second set of studies focused on the relationship between calcium dysregulation (due to cilia disruption) and cell proliferation. Cilia-deficient cells displayed increased cell proliferation, which was corrected when cytosolic calcium was "reset" to more normal levels. It was further demonstrated that the effects of elevated cytosolic calcium levels on proliferation were mediated by activation of protein kinase C alpha (α-PKC), a calcium-activated isozyme, and disruption of actin cytoskeletal architecture in ciliadeficient cells. This work provides novel information regarding primary cilia control of apical Ca²⁺ entry, and describes a potential cystogenic mechanism in cilia-deficient cells that interrelates dysregulated cytosolic Ca²⁺ with disrupted actin cytoskeletal stability and altered α-PKC signaling.