The signalling pathways allowing hormonal regulation of Na+ transport in murine collecting duct cells
type: CreativeWork
description: The collecting duct of the distal nephron marks the final location where adjustments to Na+ excretion can be made, therefore determining the final concentration of Na+ conserved in the extracellular fluid which plays a role in governing overall blood volume and pressure. This transport of Na+ is subject to hormonal regulation but the signalling pathways underpinning this regulation however, are not fully understood. In this thesis the signalling pathways allowing both basal and insulin-stimulated Na+ absorption were explored in the murine collecting duct cell line, mpkCCDcl4. The effects of two insulin-sensitizing drugs, TZDs, on ENaC-mediated Na+ transport were investigated and the signalling pathways underlying two other hormonal regulators of ENaC, dexamethasone and vasopressin, were also examined. Unstimulated monolayers of mpkCCDcl4 cells generated spontaneous Na+ absorption which was quantified by measuring equivalent short circuit current (Ieq). Selective inhibition of PI3-kinase, mTORC2 and SGK1 left ~80 % of the current intact, indicating these signalling molecules are not required for basal Na+ transport. Acute addition of insulin stimulated Ieq and this occurred with a concomitant increase in mTORC2, SGK1 and Akt activity. Inhibition of PI3-kinase abolished the insulin-stimulated response as well as phosphorylation of downstream substrates, indicating a crucial role of PI3-kinase. Inhibition of mTORC1 with rapamycin did not alter basal or insulin-stimulated Na+ transport. The mTOR inhibitors TORIN1 and PP242 could therefore be used to evaluate the role of mTORC2. These inhibitors greatly reduced insulin-stimulated ENaC-mediated Na+ transport and also abolished SGK1 and mTORC2 activity, indicating a novel role of mTORC2. An inhibitor of SGK1, GSK650394A abolished insulin-stimulated Na+ transport and specifically inhibited SGK1 acitivty demonstrating the importance of SGK1 in insulin signalling. The inhibitor Akti-1/2 also abolished insulin-mediated Na+ tra…
abstract: The collecting duct of the distal nephron marks the final location where adjustments to Na+ excretion can be made, therefore determining the final concentration of Na+ conserved in the extracellular fluid which plays a role in governing overall blood volume and pressure. This transport of Na+ is subject to hormonal regulation but the signalling pathways underpinning this regulation however, are not fully understood. In this thesis the signalling pathways allowing both basal and insulin-stimulated Na+ absorption were explored in the murine collecting duct cell line, mpkCCDcl4. The effects of two insulin-sensitizing drugs, TZDs, on ENaC-mediated Na+ transport were investigated and the signalling pathways underlying two other hormonal regulators of ENaC, dexamethasone and vasopressin, were also examined. Unstimulated monolayers of mpkCCDcl4 cells generated spontaneous Na+ absorption which was quantified by measuring equivalent short circuit current (Ieq). Selective inhibition of PI3-kinase, mTORC2 and SGK1 left ~80 % of the current intact, indicating these signalling molecules are not required for basal Na+ transport. Acute addition of insulin stimulated Ieq and this occurred with a concomitant increase in mTORC2, SGK1 and Akt activity. Inhibition of PI3-kinase abolished the insulin-stimulated response as well as phosphorylation of downstream substrates, indicating a crucial role of PI3-kinase. Inhibition of mTORC1 with rapamycin did not alter basal or insulin-stimulated Na+ transport. The mTOR inhibitors TORIN1 and PP242 could therefore be used to evaluate the role of mTORC2. These inhibitors greatly reduced insulin-stimulated ENaC-mediated Na+ transport and also abolished SGK1 and mTORC2 activity, indicating a novel role of mTORC2. An inhibitor of SGK1, GSK650394A abolished insulin-stimulated Na+ transport and specifically inhibited SGK1 acitivty demonstrating the importance of SGK1 in insulin signalling. The inhibitor Akti-1/2 also abolished insulin-mediated Na+ tra…
author: Mansley, Morag K.
language: eng
keywords: Insulin, Kinase inhibitors, Epithelial Na+ Channel, Cortical Collecting Duct, SGK1
date published: 2010
date modified: 2026-07-24T02:07:31Z
provider: University of Dundee
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Dundee