Na+ homeostasis in the body is regulated by aldosterone actions in kidney cortical collecting duct (CCD) cells. Aldosterone signalling is transduced via binding to the mineralocorticoid receptor (MR) and has been shown to modulate the epithelial Na+ channel (ENaC) activity by rapid nongenomic and latent genomic actions at the levels of transcription, protein stability and subcellular trafficking (1). Protein kinases such as protein kinase D are important modulators of Na+ transporters in CCD cells (2). Here we report a novel mechanism for aldosterone regulation of ENaC activity via protein kinase D2 (PKD2) signalling by affecting sub-cellular trafficking of ENaC subunits in a murine cortical collecting duct (M1-CCD) cell line. ENaC current was measured as the amiloride-inhibitable transepithelial short-circuit current (IENaC) in M1-CCD cell monolayers mounted in Ussing chambers. Student’s t-tests were performed alongside a one way ANOVA. Aldosterone (10 nM) caused a rapid (<10 min) phosphorylation of PKD2 and its sub-cellular redistribution from predominantly apical membrane to the cytosol. PKD2 knock-down using shRNA in M1 cells resulted in an elevated basal ISC from 1.9 ± 0.2 µA/cm2 in wild-type cells to 9.3 ± 1.4 µA/cm2 in PKD2 knock-down M1 cells (n=10, p=0.002). PKD2 knock-down increased the amiloride-sensitive ENaC current (IENaC) from 1.3 ± 0.3 µA/cm2 in wild-type cells to 6.0 ± 1.0 µA/cm2 in PKD2 knock-down M1 cells (n=11, p=0.0001). Long- term treatment (24h) of wild-type M1 cells with aldosterone increased the basal ISC from 1.9 ± 0.2 µA/cm2 to 4.6 ± 0.7 µA/cm2 (n=7, p=0.008). The ENaC current showed an increase from 1.3 ± 0.3 µA/cm2 in wild-type M1 cells to 3.3 ± 0.5 µA/cm2 when treated for 24h with aldosterone (n=8, p=0.001). The effect of aldosterone on both the basal ISC and IENaC were abolished in the PKD2 knock-down cells. We also investigated the activation of PKD2 by aldosterone in an autosomal dominant polycystic kidney disease in vitro cell model (WT 9-12). Expression of PKD2 under basal conditions was found to localize at the apical membrane and in the sub-apical cytosolic space (n=3). ENaCγ localization was observed to be cytosolic/basolateral membrane while the Na+/K+ATPase subunits α3 and β1 were localized at the apical membrane. Aldosterone (10nM) treatment induced the intracellular accumulation of PKD2 (n=3) within 10 min. In conclusion, our results indicate that PKD2 normally suppresses ENaC activity and aldosterone releases this inhibition by phosphorylating and removing PKD2 from the apical membrane. We propose that protein kinase D isoforms are pivotal signalling molecules controlling both the steady-state and aldosterone-induced membrane localization and stability of ENaC and Na+ /K+ pumps. Modulation of PKD activity could have important consequences for autosomal dominant polycystic kidney disease.