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Tonicity-Responsive Enhancer Binding Protein is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells

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Title: Tonicity-Responsive Enhancer Binding Protein is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells
Authors: Hasler, Udo; Jeon, Un Sil; Kim, Jeong Ah; Mordasini, David; Kwon, H. Moo; Feraille, MD, PhD Privat Docent, Eric; Martin, Pierre-Yves
Publisher: Journal of American Society of Nephrology
Date Published: June 01, 2006
Reference Number: 704
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Tonicity-responsive enhancer binding protein (TonEBP) plays a key role in protecting renal cells from hypertonic stress by stimulating transcription of specific genes. Under hypertonic conditions, TonEBP activity is enhanced via increased nuclear translocation, transactivation, and abundance. It was reported previously that hypertonicity exerted a dual, time-dependent effect on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCDcl4). Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge, it increased after 24 h of hypertonic challenge. This study investigated the role that TonEBP may play in these events by subjecting mpkCCDcl4 cells to 3 or 24 h of hypertonic challenge. Hypertonic challenge increased TonEBP mRNA and protein content and enhanced TonEBP activity as illustrated by both increased TonEBP-dependent luciferase activity and mRNA expression of several genes that are targeted by TonEBP. Irrespective of the absence or presence of vasopressin, decreased TonEBP activity in cells that were transfected with either TonEBP small interfering RNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNA and protein content under iso-osmotic conditions and blunted the increase of AQP2 abundance that was induced after 24 h of hypertonic challenge. Conversely, decreased TonEBP activity did not significantly alter reduced expression of AQP2 mRNA that was induced by 3 h of hypertonic challenge. Mutation of a TonE enhancer element located 489 bp upstream of the AQP2 transcriptional start site abolished the hypertonicity-induced increase of luciferase activity in cells that expressed AQP2 promoter-luciferase plasmid constructs, indicating that TonEBP influences AQP2 transcriptional activity at least partially by acting directly on the AQP2 promoter. These findings demonstrate that in collecting duct principal cells, TonEBP plays a central role in regulating AQP2 expression by enhancing AQP2 gene transcription.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The aquaporin-2 (AQP2) water channel protein plays an important role in the kidney’s ability to reabsorb water and therefore concentrate urine. AQP2s located in the interior of the principal cells of the kidney collecting duct are stimulated to move to the membranes of those cells when the hormone, arginine vasopressin (AVP) binds with the vasopressin 2 receptor (V2R). This initiates a chemical sequence that results in the AQP2s lodging in the cell membranes so that water outside the cell can enter the cell by passing through the AQP2. Besides initiating the urine concentrating mechanism in the kidneys, AVP plays a major role in determining the number of AQP2s found in the principal cells of the kidney collecting duct.

Research by Hasler, et al., and other researchers has documented that another factor can increase AQP2 numbers in the cell: extracellular hypertonicity. In extracellular hypertonicity, a hypertonic solution bathes body cells, causing water to flow from inside to outside the cell. In short, extracellular tonicity stimulates the process of osmosis where water flows from within the cell to outside the cell in order to help equalize solute distribution on either side of the cell membrane.

The purpose of Hasler, et al.’s, current study was to investigate the role of the Tonicity-responsive enhancer binding protein (TonEBP) on AQP2 abundance in laboratory cultures of mice kidney collecting duct principal cells (mpkCCD). TonEBP activity is enhanced by hypertonic conditions, and it functions to help stimulate the synthesis of proteins that help kidney cells respond to hypertonicity. Hasler, et al., previously had shown that extracellular hyperosmolarity increases AQP2 mRNA and the number of AQP2s in the cell by stimulating AQP2 gene transcription (the process by which AQP2 mRNA is synthesized). Now the researchers wanted to refine their understanding of this increase by seeing if TonEBP affected AQP2 gene transcription.

The researchers subjected mpkCCD cell cultures to hypertonic conditions. They found that the cell cultures subjected to hypertonic conditions for 24 hours increased their numbers of TonEBP mRNA and TonEBPs, and increased TonEBP activity. When the cell cultures were manipulated to inhibit TonEBP synthesis, the numbers of AQP2 mRNA and AQP2 were reduced significantly. This result occurred in both the presence and absence of AVP in the cell cultures.

Further, when the researchers experimented with the TonE enhancer element that was located on the DNA strip near the location of the AQP2 gene transcriptional start site, they found that TonEBP influences AQP2 synthesis, partly by acting directly on the AQP2 promoter, the segment of the DNA that acts as a controlling element in the synthesis of AQP2.

These findings refine the understanding of the AVP independent hypertonic increase of AQP2 in collecting duct principal cells by showing that TonEBP significantly determines the amount of AQP2 found in the cells. It does this through regulating AQP2 synthesis by enhancing that part of the synthesis process called AQP2 gene transcription.