Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C95

Oral Communications

Protein Kinase D Modulates Aldosterone-induced ENaC Activity in Renal Cortical Collecting Duct Cells through the Regulation of Subcellular Trafficking and MR-dependent Gene Expression

R. Dooley1, V. McEneaney1, B. J. Harvey1, W. Thomas1

1. Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.

Aldosterone treatment stimulates the phosphorylation and activation of Protein Kinase D 1 (PKD1), in murine cortical collecting duct cells (M1CCD), through the transactivation of the epidermal growth factor receptor (EGFR) [1]. PKD1 belongs to a family of serine/threonine kinases known to be important modulators of subcellular trafficking, through the regulation of vesicle fission from the Golgi [2]. The epithelial sodium channel (ENaC) is a major effector of aldosterone action in the kidney and plays a crucial role in the maintenance of whole body sodium homeostasis. Active ENaC is believed to be a heterotrimer composed of one each of the alpha, beta and gamma subunits. ENaC activity is dynamically regulated by aldosterone through multiple mechanisms. The nuclear mineralocorticoid receptor (MR) in complex with aldosterone behaves as a ligand-dependent transcription factor which stimulates the tissue-specific upregulation of ENaC subunit expression. In the distal nephron, the ENaC alpha subunit is under the transcriptional control of ligand-bound MR, while beta and gamma are constitutively expressed [3]. Using M1CCD cells where endogenous PKD1 was stably knocked down, we examined the role PKD1 plays in the aldosterone-mediated regulation of ENaC activity via transcription and/or trafficking of pre-expressed ENaC subunits, over an extended period of aldosterone treatment. The amiloride-sensitive, trans-epithelial current (ITE) was measured in wild-type (WT) and PKD1 suppressed M1CCD cells, grown to confluency on semi-permeable supports. A detectable rise in ITE was observed in WT cells within 2h of aldosterone treatment, an effect which was maximal after 24h. The induction of ITE by aldosterone was inhibited in PKD1 suppressed cells. Using immunocytochemistry and laser scanning confocal microscopy, we observed a stark increase in ENaC alpha expression in WT cells treated with aldosterone for 24h, an effect which was absent in the PKD1 suppressed cells. Moreover, using a specific plasma membrane marker, we observed an aldosterone-mediated induction of apical membrane insertion of the constitutively expressed ENaC beta subunit in WT cells. Aldosterone treatment failed to affect the subcellular localization of ENaC beta in PKD1 suppressed cells. Overall, PKD1 plays a central role in the aldosterone-mediated regulation of ENaC activity, through both transcriptional control and subcellular trafficking.

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