2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

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Conference: 2004 Global Researcher Conference
Title: Modulation of vasopressin-dependent AQP2 expression in cultured mpkCCDc14 collecting duct principal cells by aldosterone, insulin, and hypertonicity
Authors: Hasler, Udo; Bustamante, Mauro; Mordasini, David; Vandewalle, Alain; Feraille, MD, PhD Privat Docent, Eric; Martin, Pierre-Yves
Institutions: Fondation pour Recherches Medicales, INSERM U773, University Hospital of Geneva
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Hasler While aquaporin-2 (AQP2) abundance is chiefly controlled by [8-arginine]vasopressin (AVP) through cAMP-inducible AQP2 gene transcription, other factors largely contribute to adjust the levels of AQP2 protein, and thus water excretion, to meet the organism’s daily needs. The mpkCCDcl4 immortalized collecting duct (CD) principal cell line exhibits many major functional properties of CD principal cells including AVP-inducible expression of endogenous AQP2 mRNA and protein. RNase protection assay, Real-time PCR analysis and Western blot analysis of whole cell AQP2 expression in this cell line revealed that both AQP2 transcription and mRNA translation provide a means by which AQP2 expression may be controlled. Protein degradation analysis revealed that AQP2 protein is quickly degraded via both lysosomal and proteasomal degradation pathways. In addition to the stimulatory effect mediated by AVP on AQP2 transcription, AVP was found to buffer the increase in AQP2 protein content by negatively-acting on AQP2 protein turn-over. The influence of several AVP-independent factors were also investigated. Aldosterone was found to decrease AQP2 mRNA expression levels but increased AQP2 protein abundance by increasing protein turnover. Insulin was also found to significantly increase AQP2 protein content by potentiating AVP-inducible AQP2 mRNA expression. Expression levels of both AQP2 mRNA and protein were found to decrease shortly (< 3h) following osmotic shock and to increase after longer exposure times (> 8 h) in response to hypertonicity. Like insulin, neither osmotic shock nor hypertonicity were found to alter AQP2 protein turn-over. The identification of molecular factors that modulate AQP2 abundance in response to extracellular factors would lend important support to our understanding of water handling. Among other candidate factors, investigation of the tonicity-responsive enhancer binding protein (TonEBP) in hypertonic-upregulation of AQP2 mRNA is currently under way.

The amount of AQP2 found in principal cells of the kidney collecting duct is principally controlled by the hormone arginine vasopressin (AVP). Using a laboratory cell culture, Hasler et al., found that AVP influences two processes central to the development of balanced AQP2 expression. While AVP is the principal factor controlling AQP2 gene transcription it also plays a role downstream of this event by influencing the rate of AQP2 mRNA translation and/or the rate of AQP2 protein degradation.

Besides AVP, other factors also influence AQP2 expression including the hormones aldosterone and insulin which both increase AQP2 levels. Other conditions in the principal cell environment such as osmotic shock and hypertonicity also affect AQP2 expression. The researchers state that exploring intracellular molecular factors that change the amount of AQP2 in the cell in response to extracellular factors would add to our understanding of how the kidney balances body water.