Aldosterone regulates ion transport across high resistance epithelia such as that of the distal nephron and is released by the adrenal gland in response to activation of the renin/ angiotensin/ aldosterone system (RAAS) or hyperkalaemia. In the renal collecting duct (CD), the net effect of aldosterone action is to promote K+ secretion and Na+ re-absorption, depending on the activating stimulus. The membrane ion transporters mediating these processes to aldosterone are regulated directly at the level of transcription by the mineralocorticoid receptor (MR) and indirectly by modulating the expression of regulatory kinases such as serum and glucocorticoid regulated kinase-1 (SGK-1) by MR. The abundance and activity of the epithelial Na+ channel (ENaC) at the apical surface of the principal cells of the renal CD is rate-limiting for aldosterone-induced Na+ re-absorption from the renal ultra-filtrate. In the distal nephron, the ENaCα subunit is under the transcriptional control of MR, while the β and γ channel subunits are constitutively expressed. Aldosterone also promotes the rapid activation of protein kinase signalling cascades and the mobilization of secondary messengers such as Ca2+ and cAMP to modulate its physiological effects. Protein kinase D1 (PKD) belongs to a family of serine/ threonine kinases known to be important modulators of sub-cellular trafficking through the regulation of trans-Golgi vesicle fission. We found that aldosterone rapidly stimulated the autophosphorylation of PKD1 at residue Ser916 and its activation within 5 min of treatment in the murine M1 cortical collecting duct (M1-CCD) cell line, with a further phase of activation at 30 min (1). The rapid activation of PKD was sensitive to MR antagonism with spironolactone (10 mM) and coupled to an nPKC isoform signaling cascade involving the autophosphorylation and activation of both PKCδ and PKCε, via EGFR trans-activation and phosphorylation at Tyr845 by c-Src. PKD activation coincided temporally with the rapid translocation of CFP-tagged ENaC subunits expressed in M1-CCD cells to discrete sites within the cytoplasm following aldosterone treatment (2). PKD is implicated in the regulation of intracellular protein trafficking, and the stable suppression of PKD1 expression using a plasmid encoding a PKD1-specific siRNA, blocked the aldosterone-induced sub-cellular distribution of ENaC subunits. The role of PKD1 in the regulation of ENaC activity was investigated in M1-CCD cells grown on semi-permeable supports. Aldosterone treatment (10 nM) resulted in an increase in the amiloride-sensitive transepithelial current (ITE) from 1±0.21 μA/cm2 to 7±0.86 μA/cm2 (n=8, P< 0.005) in wild-type (WT) cells within 24 h, an effect which was inhibited in the PKD1-knockdown cells. Furthermore, using immuno-cytochemistry and confocal microscopy, an increase in ENaCα expression in WT cells treated with aldosterone for 24 h was observed, and this response was absent in PKD1 suppressed cells. Aldosterone treatment stimulated the apical membrane insertion of constitutively expressed ENaCβ in WT cells; this effect was not replicated in PKD1-suppressed cells. In common with other steroid receptors, MR is a ligand-activated transcription factor and interacts with specific GRE sequences in the promoter regions of target genes to regulate gene expression. In combination with ligand binding, the increased nuclear association of MR is modulated through its phosphorylation by extracellular signal regulated protein kinases 1 and 2 (ERK1/2). The activation of ERK1/2 by aldosterone is stabilized by activation of PKD1 (3). We found that suppression of ERK1/2 activation in response to aldosterone using the MEK inhibitor PD98059 blocked the translocation of MR in response to aldosterone treatment as did siRNA knockdown of PKD1 expression. Suppression of PKD1 expression also inhibited aldosterone-induced SGK-1 expression. PKD1 thus modulates aldosterone-induced ENaC activity through the modulation of sub-cellular trafficking, and also through the stabilization of MR nuclear localization; probably through the stabilization of ERK1/2 activity to phosphorylate MR.