Adam, G. Functional role of new aldosterone regulated gene products. 2008, University of Zurich, Faculty of Science.
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The kidney regulates the body’s fluid volume, mineral composition and acidity by excretion and reabsorption of electrolytes and water. The ability of the kidney to regulate salt homeostasis and extracellular fluid volume is crucial for blood pressure maintenance. The mineralocorticoid hormone aldosterone adjusts salt homeostasis largely by regulating sodium reabsorption across principal cells of the aldosterone-sensitive distal nephron (ASDN). The stimulatory action of aldosterone on sodium reabsorption in the distal nephron has been shown to depend mainly on transcriptional regulation that is mediated by the activation of the mineralocorticoid receptor, a member of the family of nuclear receptors. Early genomic effects on sodium reabsorption are thought to be mediated mostly by the induction or repression of elements of the regulatory pathways that control the function of preexisting epithelial sodium channel (ENaC), sodium potassium pump (Na+-K+-ATPase) and potassium channel (ROMK). Aldosterone regulates the cell-surface expression and function and subsequent internalization of ENaC in its target cells partly by the transcriptional induction of the serum- and glucocorticoid-regulated kinase 1 (Sgk1). This kinase prevents the ubiquitylation of ENaC by phosphorylating the ubiquitin ligase Nedd4-2. The aim of my project was to investigate the mechanism by which the early aldosterone-induced changes in gene expression impact on sodium and potassium transport in aldosterone target cells in the kidney. A microarray of RNA extracted from mouse epithelial cells of the ASDN had identified 22 significantly and more than two-fold early aldosterone-regulated RNA in vivo. The focus of this work was on three of these significantly up-regulated gene products: the ubiquitin-specific proteases Usp2 and Usp53 and the voltage gated proton channel Hv1. In exogenous gene expression systems like Xenopus laevis oocytes and cultured mouse cortical collecting duct cells their impact on channels and transporters involved in the sodium absorption in the collecting duct was investigated using electrophysiological techniques. A pronounced induction of ENaC mediated currents by the co-expression of the Usp2 isoform Usp2-45 was detected using two-electrode voltage-clamp recordings. The stimulatory effect on ENaC was not additive to that of Sgk1. This suggests that Usp2-45 acts on the same regulatory pathway as Sgk1, namely the ubiquitylation/deubiquitylation of ENaC. The stimulation of ENaC is further due to the catalytic action of Usp2-45 since substitution of Cys67 with Ala within the catalytic site of Usp2-45 abolished its effect. Further experiments performed in cultured human embryonic kidney cells showed that indeed Usp2-45 deubiquitylates and thereby activates ENaC. Another ubiquitin-specific protease, Usp53 was significantly up-regulated in the mouse distal nephron. Experiments aiming at investigating the possible impact of Usp53 on sodium transport molecules (ENaC, Na+-K+-ATPase) expressed in Xenopus laevis oocytes were inconclusive and the role of Usp53 in regulating salt reabsorption remains elusive. The third investigated gene product, a voltage gated proton channel (Hv1) structurally resembles the voltage sensor of typical voltage gated channels of the S4-family. Hv1 substantially decreases ENaC and ROMK function in oocytes which appears contradictory to the fact that it is up-regulated by aldosterone. However, early up-regulation of a voltage gated proton channel in the ASDN could be essential in the regulatory control of salt reabsorption fine tuning by aldosterone.
|Referees:||Verrey F, Staub O, Lutz T|
|Communities & Collections:||04 Faculty of Medicine > Center for Integrative Human Physiology
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
|Dewey Decimal Classification:||570 Life sciences; biology
610 Medicine & health
|Deposited On:||19 Dec 2008 11:11|
|Last Modified:||04 Apr 2012 12:27|
|Number of Pages:||115|
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