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1.
J Am Soc Nephrol ; 30(5): 795-810, 2019 05.
Article in English | MEDLINE | ID: mdl-30988011

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.


Subject(s)
Aquaporin 2/metabolism , Biological Transport/genetics , Colforsin/pharmacology , Diabetes Insipidus, Nephrogenic/drug therapy , Fluconazole/pharmacology , rhoA GTP-Binding Protein/drug effects , Analysis of Variance , Animals , Cell Membrane/metabolism , Cells, Cultured , Diabetes Insipidus, Nephrogenic/metabolism , Disease Models, Animal , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/drug effects , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Phosphorylation/genetics , Random Allocation , Signal Transduction , Statistics, Nonparametric
2.
PLoS One ; 13(1): e0191423, 2018.
Article in English | MEDLINE | ID: mdl-29373579

ABSTRACT

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.


Subject(s)
A Kinase Anchor Proteins/metabolism , Aquaporin 2/metabolism , Cell Membrane/metabolism , Kidney Tubules, Collecting/cytology , Minor Histocompatibility Antigens/metabolism , Proto-Oncogene Proteins/metabolism , Small Molecule Libraries/pharmacology , rhoA GTP-Binding Protein/metabolism , Cell Membrane/drug effects , HEK293 Cells , Humans , Protein Binding/drug effects , Protein Transport/drug effects , Small Molecule Libraries/chemistry , Structure-Activity Relationship
3.
Front Pharmacol ; 7: 23, 2016.
Article in English | MEDLINE | ID: mdl-26903868

ABSTRACT

Arginine-vasopressin (AVP) stimulates the redistribution of water channels, aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. By this AVP directs 10% of the water reabsorption from the 170 L of primary urine that the human kidneys produce each day. This review discusses molecular mechanisms underlying the AVP-induced redistribution of AQP2; in particular, it provides an overview over the proteins participating in the control of its localization. Defects preventing the insertion of AQP2 into the plasma membrane cause diabetes insipidus. The disease can be acquired or inherited, and is characterized by polyuria and polydipsia. Vice versa, up-regulation of the system causing a predominant localization of AQP2 in the plasma membrane leads to excessive water retention and hyponatremia as in the syndrome of inappropriate antidiuretic hormone secretion (SIADH), late stage heart failure or liver cirrhosis. This article briefly summarizes the currently available pharmacotherapies for the treatment of such water balance disorders, and discusses the value of newly identified mechanisms controlling AQP2 for developing novel pharmacological strategies. Innovative concepts for the therapy of water balance disorders are required as there is a medical need due to the lack of causal treatments.

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