Aquaporin-2 Trafficking Research Articles

Prostaglandin receptor EP4 induces transient membrane targeting of aquaporin‐2 through a novel intracellular signaling pathway

In our previous studies, the prostaglandin receptor EP2 agonist butaprost increased aquaporin‐2 (AQP2) membrane targeting (MT) and phosphorylation at ser‐269 (pS269), but EP4 agonist CAY10580 (CA) increased only MT. Potential EP4 signaling pathways alongside EP2 vs EP4 mediated AQP2 trafficking events were examined in these studies. In AQP2‐MDCK cells natively expressing EP2 and EP4, the EP2 antagonist AH6809 (10μM) abolished prostaglandin E2 (1–10nM) induced cAMP and pS269, but not MT. Stimulation with 1μM CA (2 to 80 min) increased MT from 5–80 min, but did not increase pS269‐AQP2/biotin‐AQP2 at any time point. Pretreatment with the Gi inhibitor, pertussis toxin (18h, 100ng/ml), neither increased pS269 nor affected AQP2 MT during CA stimulation. 40 min agonist washout abolished CA stimulated AQP2 MT whereas butaprost (1μM) or forskolin (10μM) induced MT was upheld for at least 80 min of washout, although pS269 was strongly decreased after 20 min. In conclusion: 1) EP4 increases AQP2 MT without increasing cAMP and pS269. This is neither due to agonist functional selectivity, concomitant Gi protein signaling nor time dependent receptor desensitization 2) EP4 mediated membrane targeted AQP2 is rapidly internalized after washout, whereas the effect of forskolin or EP2 on AQP2 MT are upheld 3) the latter is not due to continued pS269 after washout. Funded by Aarhus University and The Lundbeck Foundation.

New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells

Maintaining tight control over body fluid and acid-base homeostasis is essential for human health and is a major function of the kidney. The collecting duct is a mosaic of two cell populations that are highly specialized to perform these two distinct processes. The antidiuretic hormone vasopressin (VP) and its receptor, the V2R, play a central role in regulating the urinary concentrating mechanism by stimulating accumulation of the aquaporin 2 (AQP2) water channel in the apical membrane of collecting duct principal cells. This increases epithelial water permeability and allows osmotic water reabsorption to occur. An understanding of the basic cell biology/physiology of AQP2 regulation and trafficking has informed the development of new potential treatments for diseases such as nephrogenic diabetes insipidus, in which the VP/V2R/AQP2 signaling axis is defective. Tubule acidification due to the activation of intercalated cells is also critical to organ function, and defects lead to several pathological conditions in humans. Therefore, it is important to understand how these "professional" proton-secreting cells respond to environmental and cellular cues. Using epididymal proton-secreting cells as a model system, we identified the soluble adenylate cyclase (sAC) as a sensor that detects luminal bicarbonate and activates the vacuolar proton-pumping ATPase (V-ATPase) via cAMP to regulate tubular pH. Renal intercalated cells also express sAC and respond to cAMP by increasing proton secretion, supporting the hypothesis that sAC could function as a luminal sensor in renal tubules to regulate acid-base balance. This review summarizes recent advances in our understanding of these fundamental processes.

Differential, Phosphorylation Dependent Trafficking of AQP2 in LLC-PK1 Cells

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture.

PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKCα plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKCα shRNA, PKCα A/25E, or PKCα scrambled shRNA. The expression levels of PKCα, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKCα was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. Treatment of AQP2-mIMCD3 cells with 100 μmol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKCα by transfection with PKCα A/25E plasmids resulted in de-polymerization of α-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCα by PKCα shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering α-tubulin distribution. Although 100 μmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCα by PKCα shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCα. Moreover, PKCα did not physically interact with AQP2 in the presence or absence of DdAVP. Our results suggested that PKCα regulates AQP2 trafficking induced by DdAVP via microtubule assembly.

Impairment of TRPC1–STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Fluvastatin modulates renal water reabsorption in vivo through increased AQP2 availability at the apical plasma membrane of collecting duct cells

X-linked nephrogenic diabetes insipidus (XNDI), a severe pathological condition characterized by greatly impaired urine-concentrating ability of the kidney, is caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene. The lack of functional V2Rs prevents vasopressin-induced shuttling of aquaporin-2 (AQP2) water channels to the apical plasma membrane of kidney collecting duct principal cells, thus promoting water reabsorption from urine to the interstitium. At present, no specific pharmacological therapy exists for the treatment of XNDI. We have previously reported that the cholesterol-lowering drug lovastatin increases AQP2 membrane expression in renal cells in vitro. Here we report the novel finding that fluvastatin, another member of the statins family, greatly increases kidney water reabsorption in vivo in mice in a vasopressin-independent fashion. Consistent with this observation, fluvastatin is able to increase AQP2 membrane expression in the collecting duct of treated mice. Additional in vivo and in vitro experiments indicate that these effects of fluvastatin are most likely caused by fluvastatin-dependent changes in the prenylation status of key proteins regulating AQP2 trafficking in collecting duct cells. We identified members of the Rho and Rab families of proteins as possible candidates whose reduced prenylation might result in the accumulation of AQP2 at the plasma membrane. In conclusion, these results strongly suggest that fluvastatin, or other drugs of the statin family, may prove useful in the therapy of XNDI.

Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus

In the kidney, the actions of vasopressin on its type-2 receptor (V2R) induce increased water reabsorption alongside polyphosphorylation and membrane targeting of the water channel aquaporin-2 (AQP2). Loss-of-function mutations in the V2R cause X-linked nephrogenic diabetes insipidus. Treatment of this condition would require bypassing the V2R to increase AQP2 membrane targeting, but currently no specific pharmacological therapy is available. The present study examined specific E-prostanoid receptors for this purpose. In vitro, prostaglandin E2 (PGE2) and selective agonists for the E-prostanoid receptors EP2 (butaprost) or EP4 (CAY10580) all increased trafficking and ser-264 phosphorylation of AQP2 in Madin-Darby canine kidney cells. Only PGE2 and butaprost increased cAMP and ser-269 phosphorylation of AQP2. Ex vivo, PGE2, butaprost, or CAY10580 increased AQP2 phosphorylation in isolated cortical tubules, whereas PGE2 and butaprost selectively increased AQP2 membrane accumulation in kidney slices. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with butaprost. In conclusion, EP2 and EP4 agonists increase AQP2 phosphorylation and trafficking, likely through different signaling pathways. Furthermore, EP2 selective agonists can partially compensate for a nonfunctional V2R, providing a rationale for new treatment strategies for hereditary nephrogenic diabetes insipidus.

Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.

Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation

AS160, a novel Akt substrate of 160 kDa, contains a Rab GTPase-activating protein (GAP) domain. The present study examined the role of Akt and AS160 in aquaporin-2 (AQP2) trafficking. The main strategy was to examine the changes in AQP2 translocation in response to small interfering RNA (siRNA)-mediated AS160 knockdown in mouse cortical collecting duct cells (M-1 cells and mpkCCDc14 cells). Short-term dDAVP treatment in M-1 cells stimulated phosphorylation of Akt (S473) and AS160, which was also seen in mpkCCDc14 cells. Conversely, the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002 diminished phosphorylation of Akt (S473) and AS160. Moreover, siRNA-mediated Akt1 knockdown was associated with unchanged total AS160 but decreased phospho-AS160 expression, indicating that phosphorylation of AS160 is dependent on PI3K/Akt pathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocytochemistry revealed that AS160 knockdown in mpkCCDc14 cells was associated with increased AQP2 density in the plasma membrane [135 ± 3% of control mpkCCDc14 cells (n = 65), P < 0.05, n = 64] despite the absence of dDAVP stimulation. Moreover, cell surface biotinylation assays of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of control mpkCCDc14 cells (n = 3), P < 0.05, n = 3]. Taken together, PI3K/Akt pathways mediate the dDAVP-induced AS160 phosphorylation, and AS160 knockdown is associated with higher AQP2 expression in the plasma membrane. Since AS160 contains a GAP domain leading to a decrease in the active GTP-bound form of AS160 target Rab proteins for vesicle trafficking, decreased expression of AS160 is likely to play a role in the translocation of AQP2 to the plasma membrane.

Expression of aquaporin‐2 tagged with photoconvertible fluorescent protein in mpkCCD cells

Photoconvertible fluorescence proteins are potential tools for investigating dynamic processes in living cells. Monomeric Eos-fluorescent protein (mEosFP) was used as an autofluorescent tag to monitor aquaporin-2 (AQP2) trafficking in mpkCCD cells grown on semipermeable filter. The emission of mEosFP can be switched from green to red upon UV irradiation. mEos2 (an mEOSFP variant) was fused to the amino-terminus of AQP2. mpkCCD was transfected with the chimeric construct and cultured without vasopressin to diminish endogenous AQP2 expression. Apical osmotic water permeability (Pf) of mpkCCD was measured with calcein fluorescence. Trafficking of mEOSFP-AQP2 was monitored using two-photon confocal microscopy. Expression of mEOSFP-AQP2 conferred Pf to the transfected cells as indicated by cell swelling when exposed to apical hypotonic solution. Pf of transfected cells was further augmented by 10 μM forskolin. No corresponding increase in cell volume and Pf were found in non-transfected cells. Forskolin treatment was also accompanied with apical targeting of mEOSFP-AQP2 in live cells, which was confirmed with immunohistochemistry. Rapid local photoconversion from green mEOSFP-AQP2 to red mEOSFP-AQP2 was successfully induced with a 405 nm diode laser. These results provide proof-of-concept to monitor the trafficking of two populations of AQP2-containing vesicles in live cells.

Large‐scale iTRAQ‐based quantification of phosphorylation changes during vasopressin signaling

Protein phosphorylation plays a critical role in the signaling pathways regulating water transport in kidney collecting duct. A central mediator in this process is the hormone arginine vasopressin (AVP), which regulates phosphorylation of the water channel aquaporin-2 (AQP2), although the exact mechanisms are not fully understood. Here we utilized a multiplexed, isotopic label-based quantitative phosphoproteomic approach in order to explore the temporal dynamics of phosphorylation events triggered by vasopressin across multiple timepoints. Briefly, rat inner medullary collecting duct (IMCD) samples were incubated in the presence or absence of 1nM AVP for 0.5, 2, 5, and 15 min (n=3). Each sample was labeled with a different iTRAQ reagent, mixed and processed for shotgun phosphoproteomic analysis. Of the 12,533 phosphopeptides identified, 3,298 were found in at least 2 out of 3 time courses and had quantifiable iTRAQ ratios. Clustering this subset of peptides using a mapping-based temporal pattern mining algorithm identified 30 clusters of phosphopeptides with distinct temporal profiles. Phosphopeptides for Rap1 GTPase activating protein, kinesin 13B, Bcl-2-related ovarian killer (Bok), Pigb, Lrrfip1, and Tbc1d1 were increased in abundance within 0.5 min and displayed similar temporal dynamics to those of AQP2 peptides phosphorylated at residue Ser-256, a site which is critical for AQP2 trafficking. Phosphopeptides for two pore calcium channel protein 1, septin 9, EGFR pathway substrate 8-like protein 1, Ahnak, and actin binding LIM protein 1 were decreased in abundance within 5–15 min of AVP exposure. These data provide the foundation for development of a network model for vasopressin signaling.

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.

Fluid-shear-stress-induced translocation of aquaporin-2 and reorganization of actin cytoskeleton in renal tubular epithelial cells

In vivo, renal tubular epithelial cells are exposed to luminal fluid shear stress (FSS) and a transepithelial osmotic gradient. In this study, we used a simple collecting-duct-on-a-chip to investigate the role of an altered luminal microenvironment in the translocation of aquaporin-2 (AQP2) and the reorganization of actin cytoskeleton (F-actin) in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. Immunocytochemistry demonstrated that 3 h of exposure to luminal FSS at 1 dyn cm(-2) was sufficient to induce depolymerization of F-actin in those cells. We observed full actin depolymerization after 5 h exposure and substantial re-polymerization within 2 h of removing the luminal FSS, suggesting that the process is reversible and the fluidic environment regulates the reorganization of intracellular F-actin. We demonstrate that several factors (i.e., luminal FSS, hormonal stimulation, transepithelial osmotic gradient) collectively exert a profound effect on the AQP2 trafficking in the collecting ducts, which is associated with actin cytoskeletal reorganization.

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V₂ receptor (V₂R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V₂R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.

Functional Vanilloid Receptor-1 in Human Submandibular Glands

Vanilloid receptor-1 (VR1) was originally found in the nervous system. Recent evidence indicates that VR1 is also expressed in various cell types. We hypothesized that VR1 exists in the human submandibular gland (SMG) and is involved in regulating salivary secretion. VR1 mRNA and protein were expressed in human SMGs and a human salivary intercalated duct cell line. VR1 was mainly located in serous acinar and ductal cells, but not in mucous acinar cells. Capsaicin, an agonist of VR1, increased intracellular free calcium, enhanced phosphorylation of extracellular signal-regulated kinase, and induced the trafficking of aquaporin 5 (AQP5) from the cytoplasm to the plasma membrane. These effects were abolished by pre-treatment with the VR1 antagonist capsazepine. Furthermore, capsaicin cream applied to the skin covering the submandibular area increased salivary secretion. These findings indicated that a functional VR1 is expressed in the human SMG and is involved in regulating salivary secretion by mediating AQP5 trafficking.

Vasopressin independent trafficking of aquaporin‐2 by prostaglandin E2

Trafficking of aquaporin‐2 (AQP2) to the apical plasma membrane (APM) of principal cells in the renal collecting duct is predominantly regulated by vasopressin (VP). We investigated whether prostaglandin E2 (PGE2) could regulate AQP2 trafficking via an alternative VP independent mechanism. In vitro studies were performed using MDCK cells stably transfected with either wild type AQP2 (WT) or a mutated form where serine‐256 is substituted with alanine (S256A) and therefore cannot be phosphorylated. To analyze APM abundance of AQP2 we used both a biochemical approach based on biotin cell surface protein labeling and immunocytochemistry. Compared to controls, 40 minutes of PGE2 (10−7M) increased APM abundance of AQP2 in WT cells, but not in S256A cells. Varying PGE2 concentrations, from 10−10M to 10−6M, resulted in a gradual increase in AQP2 APM abundance. A time course study of PGE2 stimulation for 5 to 120 minutes revealed that APM abundance of AQP2 increased until 80 minutes and that this effect was sustained at 120 minutes. AQP2 S256 phosphorylation increased after 40 min of PGE2 stimulation. The prostanoid receptor 4 (EP4) antagonist AH23848, 30μM, attenuated, but did not completely abolish the effect of PGE2 on AQP2 trafficking. We conclude that PGE2 induces trafficking and S256 phosphorylation of AQP2 and that trafficking may be mediated, at least in part, by the EP4 receptor.

Primary cilium is required for vasopressin mediated aquaporin‐2 trafficking

The collecting duct cell lines PCDNA (cilia (−)) and BAP2 (cilia (+)/rescue) derived from a PKD mouse model (orpk mouse) were used to determine the role of the primary cilium in vasopressin (AVP) mediated aquaporin 2 (AQP2) trafficking and fluid absorption. With apical AVP addition, cilia (−) cells grown on impermeable supports demonstrated an increased net fluid absorption as evident by the formation of epithelial domes, which were not seen in cilia (+) cells. Immunofluorescence studies showed AQP2 localization along the ciliary shaft and in subapical membrane compartment in cilia (+). Moreover, after apical AVP treatment, there was an increase in AQP2 expression at the apical membrane in cilia (−) but not in cilia (+) cells. This was confirmed by biotinylation and Western blot analysis, which demonstrated an AVP induced elevated glycosylated form of apically located AQP2 in cilia (−) vs. cilia (+) cells. Interestingly, in cilia (−) cells there was evidence for basolateral membrane insertion of AQP2. In the absence of cilia, there appears to be an AVP mediated targeting of the V2 receptor to the apical membrane and enhanced AQP2 localization at both apical and basolateral membranes. Thus, we propose AQP2 trafficking requires a normal cilium and when absent, the collecting duct cells becomes dedifferentiate leading to enhanced salt and water absorption which may contribute to the hypertension observed in PKD patients.

Dehydration-induced increase in aquaporin-2 protein abundance is blocked by nonsteroidal anti-inflammatory drugs

It is now well established that the antidiuretic response to vasopressin is modulated by changes in aquaporin-2 (AQP2) expression in response to hydration status. While vasopressin itself is one signal driving expression, other signals also play a part. In this study, we planned to investigate whether prostaglandins, known to modulate AQP2 trafficking, may play a role in this process. Male Wistar rats were kept in metabolic cages, with either free access to water and food, or were given 15 g of food gelled with water, such that they were fluid restricted or fluid loaded. The effects of oral administration of two structurally different NSAIDs, indomethacin and ibuprofen, and a COX-2-selective NSAID, meloxicam, on urine output and AQP2 expression were investigated in kidneys removed under terminal anesthesia. All the NSAIDs decreased AQP2 expression significantly in water-restricted rats but did not significantly alter PGE excretion. In water-loaded rats, the effects were less marked, and meloxicam had no significant effect. Consistent with this, ibuprofen prevented the increase in AQP2 expression seen in response to dehydration. These results demonstrate that NSAIDs decrease AQP2 protein abundance, particularly during adaptation during dehydration. This may be of particular significance in older and critically ill patients, who are prone to dehydration.