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Differentiation of epithelium induced by aldosterone


Kretz, Monika Lina. Differentiation of epithelium induced by aldosterone. 2012, University of Zurich, Faculty of Medicine.

Abstract

For all mammalians the homeostasis of the water and electrolyte metabolism is very essential. One of the main regulators of this system is the mineralocorticoid hormone aldosterone. It acts mainly on the so called aldosterone-sensitive distal nephron (ASDN). To increase our understanding of the specific mechanism of aldosterone action in this tubule segment, a former co-worker of our group identified several early aldosterone-regulated mRNAs by microarray in mouse distal nephron. One of these mRNAs that was unknown at that time could recently be identified as YPEL2 (Yippee-like protein 2). YPEL2 is an almost unknown gene with high similarity to the Drosophila Yippee gene, of which the function is essentially unknown. The aim of the present project was to test whether YPEL2 expression levels have an impact on the differentiation and function of aldosterone-sensitive distal nephron principal cells.
As a first approach YPEL2 was cloned into an inducible Lentivector, such that it could be expressed under the control of doxycycline in the cortical collecting duct principal cell line (mCCD). To measure the expression level of YPEL2 mRNA in these cells, mRNA-extraction followed by qPCR was performed as well as immunofluorescence stainings using an antibody against YPEL. mCCD cells have been transduced either with GFP as a control or with the KRAB repressor plasmid only or with YPEL2 with or without KRAB. Due to the fact that the available antibody was also recognizing the other YPEL proteins YPEL1, 3 and 4, the untransduced cells showed a strong staining similar to the cells in which YPEL2 was expressed. Quantitative RT-PCR performed on the mCCD cells transduced with YPEL2 and KRAB detected no difference in the expression level of YPEL2 with or without induction with doxycyline.
The possibility that endogenous YPEL2 was regulated in mCCD cells by aldosterone was also tested and revealed that the hormone aldosterone itself did not impact on YPEL2 mRNA expression but that the stress provoked by a medium change strongly transiently decreased YPEL mRNA levels. Based on the fact that the specificity of the induction of YPEL2 by aldosterone in distal nephron was questioned and that the YPEL2 transduced cells didn’t show an evident phenotypic change this project was discontinued.
Taken together, the function of YPEL2 remains elusive and its overexpression in differentiated cultures of cortical collecting duct cells did not reveal a role in the context of the aldosterone-sensitive distal nephron.

Abstract

For all mammalians the homeostasis of the water and electrolyte metabolism is very essential. One of the main regulators of this system is the mineralocorticoid hormone aldosterone. It acts mainly on the so called aldosterone-sensitive distal nephron (ASDN). To increase our understanding of the specific mechanism of aldosterone action in this tubule segment, a former co-worker of our group identified several early aldosterone-regulated mRNAs by microarray in mouse distal nephron. One of these mRNAs that was unknown at that time could recently be identified as YPEL2 (Yippee-like protein 2). YPEL2 is an almost unknown gene with high similarity to the Drosophila Yippee gene, of which the function is essentially unknown. The aim of the present project was to test whether YPEL2 expression levels have an impact on the differentiation and function of aldosterone-sensitive distal nephron principal cells.
As a first approach YPEL2 was cloned into an inducible Lentivector, such that it could be expressed under the control of doxycycline in the cortical collecting duct principal cell line (mCCD). To measure the expression level of YPEL2 mRNA in these cells, mRNA-extraction followed by qPCR was performed as well as immunofluorescence stainings using an antibody against YPEL. mCCD cells have been transduced either with GFP as a control or with the KRAB repressor plasmid only or with YPEL2 with or without KRAB. Due to the fact that the available antibody was also recognizing the other YPEL proteins YPEL1, 3 and 4, the untransduced cells showed a strong staining similar to the cells in which YPEL2 was expressed. Quantitative RT-PCR performed on the mCCD cells transduced with YPEL2 and KRAB detected no difference in the expression level of YPEL2 with or without induction with doxycyline.
The possibility that endogenous YPEL2 was regulated in mCCD cells by aldosterone was also tested and revealed that the hormone aldosterone itself did not impact on YPEL2 mRNA expression but that the stress provoked by a medium change strongly transiently decreased YPEL mRNA levels. Based on the fact that the specificity of the induction of YPEL2 by aldosterone in distal nephron was questioned and that the YPEL2 transduced cells didn’t show an evident phenotypic change this project was discontinued.
Taken together, the function of YPEL2 remains elusive and its overexpression in differentiated cultures of cortical collecting duct cells did not reveal a role in the context of the aldosterone-sensitive distal nephron.

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Additional indexing

Other titles:Inaugural-Dissertation zur Erlangung der Doktorwürde der Medizinieschen Fakultät der Universität Zürich
Item Type:Dissertation
Referees:Verrey François
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2012
Deposited On:18 Jan 2013 10:50
Last Modified:07 Dec 2017 18:38
Number of Pages:49

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