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Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

The involvement of soluble N-ethylmaleimide sensitive factor-attachment protein receptor (SNARE) proteins in the cAMP-induced exocytosis of aquaporin 2 (AQP2)-containing vesicles was investigated in AQP2-transfected renal CD8 cells. RT-PCR and western blot analysis confirmed the presence of the SNARE homologs VAMP/synaptobrevin-2, syntaxin-1, syntaxin-4 and SNAP-23 in CD8 cells. Tetanus neurotoxin (TeNT) was efficient in cleaving synaptobrevin-like protein both in vitro and in intact CD8 cells incubated with the toxin. TeNT treatment in intact CD8 cells completely abolished cAMP-stimulated AQP2 targeting to the plasma membrane, as assessed by quantification of cell-surface immunoreactivity to an anti-AQP2 antibody raised against a peptide reproducing the extracellular AQP2 C-loop. These results represent the first evidence for the functional involvement of VAMP-2 in cAMP-induced AQP2 exocytosis in renal cells.

Central hypovolemia induced by orthostatic loading causes reno-vascular changes that can lead to orthostatic intolerance. In this study, we investigated volume regulating hormonal responses and reno-vascular changes in male and female subjects as they underwent central hypovolemia, induced by graded lower body negative pressure (LBNP). Aquaporin-2 (AQP2) excretion was measured as a biomarker for the renal system response to vasopressin. 37 young healthy subjects (n = 19 males; n = 18 females) were subjected to graded LBNP until - 40 mmHg LBNP. Under resting conditions, males had significantly higher copeptin (a stable peptide derived from vasopressin) levels compared with females. Adrenocorticotropin (ACTH), adrenomedullin (ADM), vasopressin (AVP) and brain natriuretic peptide (BNP) were not affected by our experimental protocol. Nevertheless, an analysis of ADM and BNP with the data normalized as percentages of the baseline value data showed an increase from baseline to 10 min after recovery in the males in ADM and in the females in BNP. Analysis of BNP and ADM raises the possibility of a preferential adaptive vascular response to central hypovolemia in males as shown by the normalized increase in ADM, whereas females showed a preferential renal response as shown by the normalized increase in BNP. Furthermore, our results suggest that there might be a difference between men and women in the copeptin response to alterations in orthostatic loading, simulated either using LBNP or during posture changes. [ABSTRACT FROM AUTHOR]

Background: Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. Methods: Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. Results: FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. Conclusion: Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Human pendrin (SCL26A4, PDS) is a 780 amino acid integral membrane protein with transport function. It acts as an electroneutral, sodium-independent anion exchanger for a wide range of anions, such as iodide, chloride, formate, bicarbonate, hydroxide and thiocyanate. Pendrin expression was originally described in the thyroid gland, kidney and inner ear. Accordingly, pendrin mutations with reduction or loss of transport function result in thyroid and inner ear abnormalities, manifested as syndromic (Pendred syndrome) and non-syndromic hearing loss with an enlarged vestibular aqueduct (ns-EVA). Pendred syndrome, the most common form of syndromic deafness, is an autosomal recessive disease characterized by sensorineural deafness due to inner ear malformations and a partial iodide organification defect that may lead to thyroid goiter. Later, it became evident that not only pendrin loss of function, but also up-regulation could participate in the pathogenesis of human diseases. Indeed, despite the absence of kidney dysfunction in Pendred syndrome patients, evidence exists that pendrin also plays a crucial role in this organ, with a potential involvement in the pathogenesis of hypertension. In addition, recent data underscore the role of pendrin in exacerbations of respiratory distresses including bronchial asthma and chronic obstructive pulmonary disease (COPD). Pendrin expression in other organs such as mammary gland, testis, placenta, endometrium and liver point to new, underscored pendrin functions that deserve to be further investigated. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300μM Gd3+ and with 10μM of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+]i although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+]i while stimulation with Gd3+ induced transient intracellular Ca2+oscillations. PLC inhibition completely abolished any [Ca2+]i increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+]i oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca2+oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+]o is mainly due to PLC/IP3 pathway activation. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adenine transversion at position 450 (c.450T>A) in exon 2 and a guanine-to-thymine at nucleotide position 643 (c.643G>T) in exon 4. The double heterozygous 450T>A and 643G>T transversion causes the amino acid substitution D150E and G215C. Direct sequencing of exons 2 and 4 of the AQP2 gene from each of the parents revealed that the c.450T>A mutation was inherited from the father while the c.643G>T mutation was inherited from the mother. Analysis of AQP2 excretion demonstrated that no AQP2 was detectable in the urine of the proband, whereas normal AQP2 levels were measured in both parents. When expressed in renal cells, both proteins were retarded in the endoplasmic reticulum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aquaporin-2 (AQP2), when expressed in fully differentiated 3T3-L1 adipocytes, displays cAMP-dependent plasma membrane translocation in a manner similar to its behavior in renal epithelial cells. The translocation of AQP2 required phosphorylation at serine 256, as the expression of AQP2/S256D was constitutively plasma membrane localized, whereas AQP2/S256A was refractory to forskolin stimulation. Unlike GLUT4, this property is not inhibited by depolymerization of cortical actin. In addition, coexpression with the dominant negative form of TC10 (TC10/T31N) or inhibition of phosphatidylinositol 3-kinase did not abrogate the cAMP-mediated response. Under basal conditions, AQP2 is localized in both the perinuclear region and in punctate vesicles scattered within the periphery of the cell. Two- and three-dimensional confocal immunofluorescence microscopy demonstrated that the adipocyte AQP2 cAMP-responsive compartment was distinct from the GLUT4 insulin-responsive compartment. Consistent with this conclusion, insulin was an effective stimulator of GLUT4 translocation but had no effect on AQP2. Conversely, forskolin induced AQP2 translocation but not GLUT4. Colocalization studies with the early endosomal marker EEA1 and transferrin receptor suggested that the AQP2 compartment is mostly distinct from endosomal vesicles. Interestingly, however, the peripheral AQP2 vesicles significantly overlapped vesicle-associated membrane protein-2, underscoring the role of the latter in hormone-regulated exocytosis. To acquire insulin responsiveness following biosynthesis, GLUT4 undergoes a slow sorting step that requires 6–9 h. In contrast, AQP2 rapidly acquires forskolin responsiveness (3 h following biosynthesis) and directly enters the cAMP-regulated compartment without transiting the plasma membrane. Together, these data demonstrate that adipocytes display two different intracellular sorting mechanisms that direct distinct hormone-sensitive partitioning of GLUT4 and AQP2. [ABSTRACT FROM AUTHOR]

Background: Exposure to microgravity or immobilization results in alterations of renal function, fluid redistribution and bone loss, which couples to a rise of urinary calcium excretion. We recently demonstrated that high calcium delivery to the collecting duct reduces local Aquaporin-2 (AQP2) mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration and reducing calcium saturation. To investigate renal water balance adaptation during bed rest, a model to mimic the effects of microgravity on earth, the effect of changes in urinary calcium on urinary AQP2 excretion were assessed.Methods: Ten healthy men (aged 21-28 years) participated in the experiment. Study design included 7 days of adaptation and 35 days of continuous bed rest (days -6 to 0 and 1 to 35, respectively) under controlled diet. Food records and 24-hour urine samples were collected daily from day -3 to 35. Changes in blood hematocrit were used as an indirect index of plasma volume changes. AQP2 excretion was measured by ELISA.Results: Bed rest induced bone demineralization and a transient increase in urinary calcium followed by transient decrease in AQP2 excretion, which can reduce the urine concentrating ability causing plasma volume reduction. The return of calciuria to baseline was followed by a recovery of AQP2 excretion, which allows for a partial restoration of plasma volume.Conclusions: These results further support the view that urinary calcium can modulate the vasopressin-dependent urine concentration through a down-regulation of AQP2 expression/trafficking. This mechanism could have a key role in the prevention of urine super-saturation due to hypercalciuria. [ABSTRACT FROM AUTHOR]