Tanja Vukićević1, Christian Hinze1,2,3, Sandrine Baltzer1, Nina Himmerkus4, Catarina Quintanova4, Kerstin Zühlke1, Friederike Compton2,3, Robert Ahlborn5, Alessandro Dema1, Jenny Eichhorst6, Burkhard Wiesner6, Markus Bleich4, Kai M Schmidt-Ott7,2,3, Enno Klussmann7,8,9. 1. Max Delbrück Center for Molecular Medicine Berlin, (MDC), Research area Cardiovascular & Metabolic Disease, Berlin, Germany. 2. Department of Nephrology and Medical Intensive Care and. 3. Berlin Institute of Health, Berlin, Germany. 4. Institute of Physiology, Christian Albrechts University Kiel, Kiel, Germany. 5. Information Technology Department, Charité-Universitätsmedizin Berlin, Berlin, Germany. 6. Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Cellular Imaging, Berlin, Germany. 7. Max Delbrück Center for Molecular Medicine Berlin, (MDC), Research area Cardiovascular & Metabolic Disease, Berlin, Germany; enno.klussmann@mdc-berlin.de Kai.schmidt-ott@mdc-berlin.de. 8. German Centre for Cardiovascular Research (DZHK), Partner Site Berlin, Berlin, Germany; and. 9. Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Vegetative Physiology, Berlin, Germany.
Abstract
BACKGROUND: Arginine-vasopressin (AVP) binding to vasopressin V2 receptors promotes redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. This pathway fine-tunes renal water reabsorption and urinary concentration, and its perturbation is associated with diabetes insipidus. Previously, we identified the antimycotic drug fluconazole as a potential modulator of AQP2 localization. METHODS: We assessed the influence of fluconazole on AQP2 localization in vitro and in vivo as well as the drug's effects on AQP2 phosphorylation and RhoA (a small GTPase, which under resting conditions, maintains F-actin to block AQP2-bearing vesicles from reaching the plasma membrane). We also tested fluconazole's effects on water flow across epithelia of isolated mouse collecting ducts and on urine output in mice treated with tolvaptan, a VR2 blocker that causes a nephrogenic diabetes insipidus-like excessive loss of hypotonic urine. RESULTS: Fluconazole increased plasma membrane localization of AQP2 in principal cells independent of AVP. It also led to an increased AQP2 abundance associated with alterations in phosphorylation status and ubiquitination as well as inhibition of RhoA. In isolated mouse collecting ducts, fluconazole increased transepithelial water reabsorption. In mice, fluconazole increased collecting duct AQP2 plasma membrane localization and reduced urinary output. Fluconazole also reduced urinary output in tolvaptan-treated mice. CONCLUSIONS: Fluconazole promotes collecting duct AQP2 plasma membrane localization in the absence of AVP. Therefore, it might have utility in treating forms of diabetes insipidus (e.g., X-linked nephrogenic diabetes insipidus) in which the kidney responds inappropriately to AVP.
BACKGROUND:Arginine-vasopressin (AVP) binding to vasopressin V2 receptors promotes redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. This pathway fine-tunes renal water reabsorption and urinary concentration, and its perturbation is associated with diabetes insipidus. Previously, we identified the antimycotic drug fluconazole as a potential modulator of AQP2 localization. METHODS: We assessed the influence of fluconazole on AQP2 localization in vitro and in vivo as well as the drug's effects on AQP2 phosphorylation and RhoA (a small GTPase, which under resting conditions, maintains F-actin to block AQP2-bearing vesicles from reaching the plasma membrane). We also tested fluconazole's effects on water flow across epithelia of isolated mouse collecting ducts and on urine output in mice treated with tolvaptan, a VR2 blocker that causes a nephrogenic diabetes insipidus-like excessive loss of hypotonic urine. RESULTS:Fluconazole increased plasma membrane localization of AQP2 in principal cells independent of AVP. It also led to an increased AQP2 abundance associated with alterations in phosphorylation status and ubiquitination as well as inhibition of RhoA. In isolated mouse collecting ducts, fluconazole increased transepithelial water reabsorption. In mice, fluconazole increased collecting duct AQP2 plasma membrane localization and reduced urinary output. Fluconazole also reduced urinary output in tolvaptan-treated mice. CONCLUSIONS:Fluconazole promotes collecting duct AQP2 plasma membrane localization in the absence of AVP. Therefore, it might have utility in treating forms of diabetes insipidus (e.g., X-linked nephrogenic diabetes insipidus) in which the kidney responds inappropriately to AVP.
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