Date of Award
PhD (Doctor of Philosophy)
Prof. Brian Harvey
Dr. Warren Thomas
Aldosterone, ENaC, Sodium Reabsorption, Protein Kinase D, PKD, Steroid Biology
Aldosterone is the key regulating hormone of whole body fluid and electrolyte balance. Perturbations in aldosterone synthesis and activation of its receptor, the mineralocorticoid receptor (MR), can lead to hypertension. Consequences of impaired sodium handling, for example hypertension, are a key risk factor in both renal and cardiovascular disease and indicative of impaired electrolyte homeostasis in the body. Renal sodium homeostasis can be modulated in several ways for example, by increasing aldosterone synthesis and activation of MR. Hypertension can also occur due to a dysregulation of aldosterone signalling which can often result in chronic pathologies of the kidney such as nephropathy and renal fibrosis. Aldosterone regulates sodium reabsorption in the kidney through its actions on the epithelial sodium channel (ENaC). ENaC abundance and activity are the major determinants of the rate of sodium reabsorption within the renal cortical collecting duct. We have previously reported a new and important role for the protein kinase D isoform PKD1 in regulating renal sodium reabsorption and here we report novel mechanisms by which aldosterone regulates the subcellular trafficking of ENaC subunits through the activation of protein kinase D2 (PKD2). Aldosterone (10nM) produced a rapid phosphorylation of PKD2 (within 10 min) which was sustained over several hours in M1-CCD cells. This rapid response was accompanied by the subcellular redistribution of PKD2 from the apical membrane into the cytosol. The activation of PKD2 was correlated with an increased abundance and stability of ENaC subunits at the apical membrane, an increase in the phosphorylation of the E3 ubiquitin ligase Nedd4-2 and a 3-fold stimulation of the amiloride-sensitive short circuit current (ISC). Suppression of PKD2 expression in M1-CCD cells using shRNA resulted in an increased expression of ENaC at the apical membrane and an 8-fold stimulation of the ISC. Conversely, aldosterone treatment resulted in a paradoxical 4-fold decrease in ISC in the M1-CCD PKD2 knock-down epithelium. In the case of the third isoform, protein kinase D3 (PKD3), aldosterone did not alter the subcellular localisation of this kinase. Additionally, increasing levels of aldosterone in Sprague-Dawley rats did not affect the abundance of PKD3. CRISPR knock-out of PKD3 in M1-CCD cells was ineffective in altering the abundance or the trafficking of ENaC either under basal conditions or in response to aldosterone. Aldosterone signalling has been implicated in autosomal dominant polycystic kidney disease (ADPKD) signalling. WT 9-12 cyst derived cells were used as a model for ADPKD and this work has determined that these cells express the PKD family isoforms, ENaC and MR. In conclusion, our results indicate that PKD2 has two opposing actions on ENaC activity. PKD2, in its basal state, normally suppresses ENaC activity and aldosterone releases this tonic inhibition by phosphorylating and removing PKD2 from the apical membrane. The removal of PKD2 from the membrane activates ENaC possibly by de-ubiquitination via PKD2- dependent phosphorylation of Nedd4-2. Knocking down PKD2 releases the basal inhibitory effect of PKD2 on ENaC, which in turn, revealed an inhibitory action of aldosterone on sodium reabsorption, exposing a previously unknown pleiotropic effect of aldosterone on ENaC membrane stability or channel conductance. We propose that protein kinase D isoforms, particularly PKD1 and PKD2, are important signalling molecules controlling the membrane localisation, stability and activity of ENaC and modulate the aldosterone-stimulated regulation of renal sodium reabsorption.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.
Quinn S. Aldosterone regulation of protein kinase D and sodium reabsorption in the renal cortical collecting duct [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2018.