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2002 Global Researcher Conference Proceeding

April 26 - 28, 2002

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Conference: 2002 Global Researcher Conference
Title: Osmolality and solute composition regulate aquaporin-2 expression in primary cultured renal principal cells
Authors: Storm, Robert; Maric, Kenan; Klussmann, Enno; Geelhaar, Andrea; Rosenthal, Walter
Institutions: Forschunginstitut fur Molekulare Pharmakologie, German Federal Ministry of Health and Social Security, Forschungsinstitut fur Molekulare Pharmakologie, Charite - Universitatsmedizin Berlin
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Storm

The water channel aquaporin-2 (AQP2) mediates the arginine vasopressin (AVP)-induced water reabsorption along the inner medullary collecting duct. The expression of this water channel is almost exclusively restricted to collecting duct principal cells which are subjected to high osmolality in the renal inner medulla. The biosynthesis of AQP2 is stimulated by the binding of AVP to the vasopressin V2 receptor which activates the Gs/adenylyl cyclase system. The subsequent formation of cAMP leads to activation of protein kinase A which in turn phosphorylates the transcription factor cAMP response element binding protein (CREB). The binding of the phosphorylated transcription factor to its recognition site in the AQP2 promoter initiates AQP2 biosynthesis.

We used our primary cultured inner medullary collecting duct cell model (Maric et al. 1998) to investigate the role of osmolality and solute composition on the expression of AQP2. These cells endogenously express AQP2 when the culture medium is supplemented with the membrane permeable cAMP-anologon dibuturyl-cAMP (dbcAMP; 500 µM). Cells were cultured for six to seven days in media with different osmolalities and solute compositions under the continued presence of dbcAMP. AQP2 expression was then analysed by Western and northern blotting.

Elevated osmolality was found to be necessary for high-level AQP2 expression. However, the AVP-elicited shuttling of AQP2 appeared to be unaffected by osmolality. High-level expression of AQP2 was induced more strongly by sodium chloride than by sorbitol, whereas the membrane permeable solute urea failed to maintain AQP2 expression. The upregulation of the AQP2 protein in response to hypertonicity as well as its rapid and severe downregulation by hypotonicity were found to be reversible. Changes in osmolality did not per se alter the activation of CREB. Within the rat AQP2 5´ promoter region (1058 bp; Rai et al. 1996) we found a region matching the tonicity responsive enhancer (TonE) consensus site (TGGAAANNYNY, Kwon et al. 1998). The TonE is recognized by the TonE binding protein (TonEBP), which regulates the expression of osmoprotective genes such as aldose reductase, betaine transporter and sodium-myo-inositol cotransporter. We conclude that in addition to the AVP-triggered signalling cascade, a further pathway triggered by elevated osmolality is crucial for proper expression of AQP2 in IMCD cells. The effect of elevated osmolality on AQP2 expression is likely to be mediated via TonEBP. To our knowledge a direct correlation between osmolality and AQP2 expression in a non-transfected system has not been shown yet. Thus, these findings may prove useful in the understanding and treatment of diuretic states.

Osmolality is a measure of the concentration of particles in a solution that are active in the osmosis process. A solution can be highly concentrated, highly dilute or anywhere in between. Storm, et al., wanted to know if osmolality and solute composition (the number and type of particles in a solution) played a part in determining the number of AQP2s in the inner medullary collecting duct (IMCD) cells of the kidney.

Their experiments with laboratory cell cultures revealed that elevated osmolality is crucial for a high number of AQP2s to be present in IMCD cells. The solute sodium chloride induced a higher number of AQP2s than sorbitol, and the solute urea failed to maintain an AQP2 expression.