A major risk factor for renal disease is hypertension which is also indicative of impaired electrolyte homeostasis. Na+ homeostasis in the kidney can be affected by increasing synthesis of aldosterone and activation of the mineralocorticoid receptor (MR). A dysregulation of aldosterone signalling results in hypertension and chronic pathologies of the kidney such as renal fibrosis and nephropathy. Aldosterone signalling is transduced via binding to MR in segments of the distal nephron including the cortical collecting duct (CCD). Aldosterone can modulate ENaC activity at the levels of transcription, protein stability and subcellular trafficking (1). Here we report a novel mechanism by which aldosterone can regulate the subcellular trafficking of ENaC subunits through activation of protein kinase D2 (PKD2). We investigated the activation of PKD2 by aldosterone and its effects on ENaC in the rat distal nephron in vivo and in a murine cortical collecting duct (M1-CCD) cell line. ENaC current was measured as the amiloride-inhibitable transepithelial short-circuit current (IENaC). Intracellular accumulation and rapid activation of PKD2 (<10min) was induced by aldosterone (10nM) treatment. Expression of PKD2 under basal conditions was found to localise at the apical membrane and in the sub-apical cytosolic space. Longer treatment with aldosterone (30min) induced sub-cellular redistribution of PKD2 into the trans-Golgi network. PKD2 knock-down using shRNA in M1 cells resulted in an elevated basal ISC from 1.9 ± 0.2µA/cm2 in wild-type cells to 9.3 ± 1.4µA/cm2 in PKD2 knock-down M1 cells (n=10, p=0.002). PKD2 knock-down also resulted in an increased amiloride-sensitive ENaC current (IENaC) from 1.3 ± 0.3µA/cm2 in wild-type cells to 6.0 ± 1.0µA/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 ± 0.2µA/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). Student’s t-tests were performed alongside a one way ANOVA. The effect of aldosterone on both the basal ISC and IENaC is abolished with the knock-down of PKD2. The abundance of ENaCγ in the apical membrane showed an increase following knock-down of PKD2 in M1 cells. Studies in rats fed on a low Na+ diet showed increased expression of ENaCγ at the apical membrane accompanied by a subcellular redistribution of PKD2 from the apical membrane to the trans-Golgi network. The use of animals was subject to local ethical approval in compliance with national legislation.