title: Analysis of the role of the mineralocorticoid receptor in renal principal cell sodium reabsorption in vivo
creator: Ronzaud, Caroline
subject: ddc-570
subject: 570 Life sciences
description: Germline inactivation of the mineralocorticoid receptor (MR) gene in mice results in early postnatal lethality due to massive loss of sodium and water associated with impaired epithelial sodium channel (ENaC) activity in kidney and colon. The aim of the present study was the analysis of the role of renal principal cell MR, which is activated by aldosterone, in ENaC-mediated sodium reabsorption. For this purpose, mice deficient for MR in ENaC-expressing renal principal cells were generated using the Cre-loxP recombination system. A large fragment of genomic mouse DNA harbouring 156 kb of the aquaporin 2 (AQP2) gene was used to drive the expression of Cre recombinase in principal cells (AQP2Cre mice). Mice carrying a conditional MR allele were crossed with AQP2Cre mice to obtain mutant (MRAQP2Cre) mice. MRAQP2Cre mice developped normally under standard diet and exhibited unaltered renal sodium excretion, but strongly elevated plasma aldosterone levels. When challenged with a low-sodium diet, MRAQP2Cre mice showed increased renal sodium and water excretion resulting in a continuous loss of bodyweight. Immunofluorescence for MR and ?ENaC staining revealed that the loss of MR expression is restricted to principal cells of the collecting duct (CD) and late connecting tubule (CNT), and that MR is crucial for apical ?ENaC trafficking to the apical membrane. The early CNT that accounts for about 30% of the CNT was not targeted. Determination of the fractional excretion of sodium before and after treatment with the ENaC blocker amiloride showed that renal ENaC activity in the mutant mice was preserved. These data demonstrate that the late distal convoluted tubule (DCT) and early CNT, which were not targeted in MRAQP2Cre mice, can compensate to a large extent impaired ENaC-mediated sodium reabsorption in the late CNT and CD.  To overcome a possible postnatal lethality of the MRAQP2Cre mice, an inducible principal cell-specific gene inactivation system was established in parallel to induce MR inactivation in adult mice. The large genomic DNA fragment harbouring the AQP2 gene, used for the generation of the constitutive AQP2Cre transgene, was also used to drive the expression of the tamoxifen-inducible CreERT2 fusion protein (AQP2CreERT2 mice). Three AQP2CreERT2 transgenic lines containing 1, 4 and 9 copies of the transgene respectively were established and showed tamoxifen-induced recombination in most of the renal principal cells, but leakiness of the fusion protein in the papilla region of the kidney. To identify aldosterone-regulated genes potentially involved in the control of ENaC-mediated renal sodium reabsorption, gene expression profiling using microarrays was performed on a mouse renal cortical CD principal cell line (mpkCCDc14) in the presence or absence of aldosterone. Quantitative RT-PCR for the identified candidates on microdissected CDs from control mice fed with standard or low-sodium diet confirmed the induction by aldosterone of the genes encoding the serum- and glucocorticoid-regulated kinase 1 (Sgk1) and the ubiquitin-specific protease 2 (Usp2). Quantitative RT-PCR on microdissected CDs from control mice and principal cell MR-deficient (MRAQP2Cre) mice under low-sodium diet showed that the gene expression levels of Sgk1 and the serine/threonine protein kinase 3 (Pim3) were reduced in the mutant mice. Thus, these data identified Pim3 as a new MR-regulated gene involved in the control of ENaC-mediated sodium reabsorption. 
date: 2007
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/7291/1/Doktorarbeit_Ronzaud.pdf
identifier: DOI:10.11588/heidok.00007291
identifier: urn:nbn:de:bsz:16-opus-72919
identifier:   Ronzaud, Caroline  (2007) Analysis of the role of the mineralocorticoid receptor in renal principal cell sodium reabsorption in vivo.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/7291/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng