Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.     

Trafficking mechanism of water channel aquaporin-2

Targeted positioning of the water channel AQP2 (aquaporin-2) strictly regulates body water homoeostasis. Trafficking of AQP2 to the apical membrane is critical for the reabsorption of water in renal collecting ducts. In addition to the cAMP-mediated effect of vasopressin on AQP2 trafficking to the apical membrane, other signalling cascades can also induce this sorting. Recently, AQP2-binding proteins which could regulate this trafficking have been discovered; SPA-1 (signal-induced proliferation-associated gene-1), a GAP (GTPase-activating protein) for Rap1, and the cytoskeletal protein actin. This review summarizes recent advances related to the trafficking mechanisms of AQP2.     

The AQP2 water channel: Effect of vasopressin treatment,microtubule disruption,and distribution in neonatal rats

Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.     

Aquaporins of plasma membranes of epithelial cells

The early 90s have brought us a discovery of a new class of membrane proteins--aquaporins with a function of transmembrane water channels. Being genetically closed proteins aquaporins are members of a large family of channel-forming proteins called MIPs (major intrinsic proteins). All aquaporins, except AQP4, are mercury-sensitive. Many aquaporins have been cloned and identified. Polyclonal antibodies grown against some of them promoted numerous studies of aquaporin localization and distribution in animal and plant tissues. Up to the present, 10 and 2 aquaporins have been described in mammalian and amphibian epithelial tissues, respectively. One of described aquaporins, AQP2, whose localization is confined to kidney collecting duct principal cells, has been found to be a hormone-depending water channel. The insertion of apical vesicles bearing AQP2 was shown to be regulated by vasopressin, meanwhile all other aquaporins are inserted into the plasma membrane constitutively. There is a vast evidence showing that the integrity of microtubules is necessary for both pathways of aquaporin insertion. AQP2 is important for normal kidney functioning and AQP2 mutations cause water-balance disorders. On the contrary, the AQP1 mutations are not accompanied by any evident clinical pathology. This review is focused on a discussion of the data so far available on aquaporin distribution in different animal tissues.     

Electrophoresis in 2004: The Annual Meeting of the American Electrophoresis Society

Protein phosphorylation plays a critical role in the signaling pathways regulating water and solute transport in the distal renal tubule (i.e., renal collecting duct). A central mediator in this process is the antidiuretic peptide hormone arginine vasopressin, which regulates a number of transport proteins including water channel aquaporin-2 and urea transporters (UT-A1 and UT-A3). Within the past few years, tandem mass spectrometry-based proteomics has played a pivotal role in revealing global changes in the phosphoproteome in response to vasopressin signaling in the renal collecting duct. This type of large-scale ‘shotgun’ approach has resulted in an exponential increase in the number of phosphoproteins known to be regulated by vasopressin and has expanded on the established signaling mechanisms and kinase pathways regulating collecting duct physiology. This article will provide a brief background on vasopressin action, will highlight a number of recent quantitative phosphoproteomic studies in both native rat kidney and cultured collecting duct cells, and will conclude with a perspective focused on emerging trends in the field of phosphoproteomics.     

Phosphoproteomics of vasopressin signaling in the kidney

Protein phosphorylation plays a critical role in the signaling pathways regulating water and solute transport in the distal renal tubule (i.e., renal collecting duct). A central mediator in this process is the antidiuretic peptide hormone arginine vasopressin, which regulates a number of transport proteins including water channel aquaporin-2 and urea transporters (UT-A1 and UT-A3). Within the past few years, tandem mass spectrometry-based proteomics has played a pivotal role in revealing global changes in the phosphoproteome in response to vasopressin signaling in the renal collecting duct. This type of large-scale 'shotgun' approach has resulted in an exponential increase in the number of phosphoproteins known to be regulated by vasopressin and has expanded on the established signaling mechanisms and kinase pathways regulating collecting duct physiology. This article will provide a brief background on vasopressin action, will highlight a number of recent quantitative phosphoproteomic studies in both native rat kidney and cultured collecting duct cells, and will conclude with a perspective focused on emerging trends in the field of phosphoproteomics.     

Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel

In mammals, the regulation of water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which localizes to the basolateral and apical membranes of the early nephron segment, and AQP2, which is translocated from intracellular vesicles to the apical membrane of collecting duct cells after vasopressin stimulation. Because a similar localization and regulation are observed in transfected Madin-Darby Canine Kidney (MDCK) cells, we investigated which segments of AQP2 are important for its routing to forskolin-sensitive vesicles and the apical membrane through analysis of AQP1-AQP2 chimeras. AQP1 with the entire COOH tail of AQP2 was constitutively localized in the apical membrane, whereas chimeras with shorter COOH tail segments of AQP2 were localized in the apical and basolateral membrane. AQP1 with the NH₂ tail of AQP2 was constitutively localized in both plasma membranes, whereas AQP1 with the NH₂ and COOH tail of AQP2 was sorted to intracellular vesicles and translocated to the apical membrane with forskolin. These data indicate that region N220-S229 is essential for localization of AQP2 in the apical membrane and that the NH₂ and COOH tail of AQP2 are essential for trafficking of AQP2 to intracellular vesicles and its shuttling to and from the apical membrane. routing signals; chimera; Madin-Darby canine kidney cells; regulated trafficking     

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)-dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference-mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.     

Urinary excretion of aquaporin-2 and inappropriate secretion of vasopressin in hyponatremic patients after cerebral infarction.

Aquaporin-2, a water-channel protein, is known to increase water permeability due to vasopressin binding to V2 receptors at the renal collecting duct and is excreted into the urine. It is still unclear whether a hyponatremic state is caused by vasopressin-dependent aquaporin-2 in patients clinically diagnosed with the syndrome of inappropriate secretion of antidiuretic hormone. To determine this, we measured urinary aquaporin-2 and vasopressin by radioimmunoassay in normonatremic or hyponatremic patients after cerebral infarction and in healthy controls. In the normonatremia group, urinary aquaporin-2 and plasma AVP levels were higher than in controls. In the hyponatremia group, plasma AVP was relatively high despite low plasma osmolality in each patient. However, urinary aquaporin-2 in hyponatremia was significantly increased when compared with the other two groups. In conclusion, AQP-2 increment does not directly reflect non-osmotic AVP secretion in a hyponatremic state. This result indicates that the urinary excretion of AQP-2 is not only AVP-dependent in hyponatremic states.     

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.     

Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser(256), Ser(261), Ser(264), and Ser(269)) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser(269) and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)(269)-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser(256) phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser(256), but not Ser(269). Phosphorylation of AQP2 at Ser(269) did not occur when Ser(256) was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser(269) phosphorylation depends upon prior phosphorylation at Ser(256). Immunogold electron microscopy localized Ser(P)(269)-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D "phosphomimic" AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser(269):(a) depends on prior PKA-mediated phosphorylation of Ser(256) and (b) enhances apical plasma membrane retention of AQP2.     

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting (n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.     

Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium

Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H(+)-K(+)-ATPase but not with Na(+)-K(+)-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.     

Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.     

Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle.     

Microtubules are needed for the perinuclear positioning of aquaporin-2 after its endocytic retrieval in renal principal cells

Water reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principal cells, thereby increasing the osmotic water permeability. The redistribution of AQP2 to the plasma membrane is a cAMP-dependent process and thus a paradigm for cAMP-controlled exocytic processes. Using primary cultured rat inner medullary collecting duct cells, we show that the redistribution of AQP2 to the plasma membrane is accompanied by the reorganization of microtubules and the redistribution of the small GTPase Rab11. In resting cells, AQP2 is colocalized with Rab11 perinuclearly. AVP induced the redistribution of AQP2 to the plasma membrane and of Rab11 to the cell periphery. The redistribution of both proteins was increased when microtubules were depolymerized by nocodazole. In addition, the depolymerization of microtubules prevented the perinuclear positioning of AQP2 and Rab11 in resting cells, which was restored if nocodazole was washed out and microtubules repolymerized. After internalization of AQP2, induced by removal of AVP, forskolin triggered the AQP2 redistribution to the plasma membrane even if microtubules were depolymerized and without the previous positioning of AQP2 in the perinuclear recycling compartment. Collectively, the data indicate that microtubule-dependent transport of AQP2 is predominantly responsible for trafficking and localization of AQP2 inside the cell after its internalization but not for the exocytic transport of the water channel. We also demonstrate that cAMP-signaling regulates the localization of Rab11-positive recycling endosomes in renal principal cells. dynein; Rab11