Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3’s ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.
Aquaporin‐2 (AQP2) is a water channel protein expressed in the kidney collecting duct principal cells responsible for water reabsorption. AQP2's ability to regulate water reabsorption depends on its phosphorylation states and trafficking to and from the apical plasma membrane regulated by the peptide hormone vasopressin. The mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking. However, the cells require a time‐consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non‐polarized AQP2‐overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cyclic AMP (cAMP), a second messenger of vasopressin. We found that forskolin induced AQP2 phosphorylation at serine 269 (S269) by 1.6 folds as observed in polarized mpkCCD and isolated rat inner medullary collecting duct cells treated with vasopressin. Before forskolin stimulation, most AQP2 was intracellular and was not phosphorylated at S269 by confocal immunofluorescence microscopy. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized mpkCCD cells and native collecting duct cells stimulated with vasopressin. As a proof of principle, we found that small hairpin RNA‐mediated protein kinase A knockdown reduced AQP2 phosphorylation at S269 and membrane localization in the non‐polarized mpkCCD cells treated with forskolin. Thus, non‐polarized AQP2‐overexpressing mpkCCD cells can be used in lieu of polarized mpkCCD cells when time is a limiting factor including transient small hairpin RNA‐mediated gene knockdown experiments.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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