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Aldosterone Regulates Rapid Trafficking of Epithelial Sodium Channel Subunits in Renal Cortical Collecting Duct Cells via Protein Kinase D Activation

Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Smurfit Building, Beaumont Hospital, Dublin 9, Ireland

Address all correspondence and requests for reprints to: Dr. Warren Thomas, Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Smurfit Building, Beaumont Hospital, Dublin 9, Ireland. E-mail: wthomas{at}rcsi.ie.

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKC. The activation of PKC and PKC coincided with PKD1 activation and while a complex was formed between PKD1 and PKC after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKC. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, , β, and ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKC /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.

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Ligands:   Spironolactone  |  Aldosterone

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