Minireview: Aquaporin 2 Trafficking
http://www.100md.com
《内分泌学杂志》
Department of General and Environmental Physiology (G.V., G.P., G.T., M.C., M.S.) and Centro di Eccellenza in Genomica Comparata (G.V., M.S.), University of Bari, 70126 Bari, Italy
Abstract
In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.
Introduction
A MAJOR BREAKTHROUGH in understanding how the kidney regulates water transport was the discovery that vasopressin stimulates the translocation of a specific water channel protein, aquaporin-2 (AQP2), from intracellular vesicles to the cell surface. Elucidating how this process is regulated has remained a challenge because it represents a convergence of two different and complex fields of research, namely vesicle transport and signal transduction. AQP2 protein possesses the typical aquaporin fold observed in the aquaporin water channel family members, as recently confirmed by crystallization and structural characterization by atomic force microscopy (1).
The trafficking of the vasopressin-sensitive water channel AQP2, reviewed in several excellent publications (2, 3, 4, 5, 6, 7, 8), is the paradigm of how cells control the movement of membrane proteins through complex pathways in response to external stimuli and how, by doing so, they regulate their function. In response to an increase in vasopressin levels, AQP2, intracellularly localized in resting collecting duct principal cells, becomes exposed on the cell surface on which it facilitates water reabsorption and consequently urine concentration. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins by a clathrin-mediated mechanism (3, 9, 10) and their recycling to the original stores, in which they are retained. These complex mechanisms include the spatiotemporal modulation of several regulatory proteins. The role of some accessory proteins such as soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) proteins and small GTPases in AQP2 shuttle has been recently clarified. Moreover, actin cytoskeleton remodeling regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking.
In addition to a vasopressin-regulated pathway, it has been shown that AQP2 recycles constitutively in epithelial cells, and it can be inserted in different membrane domains. In epithelial cell lines, AQP2 has been shown to be targeted to the apical or basolateral surface, although the polarity signals are still to be clarified (3). Osmolality environment can influence the final membrane destination. Madin-Darby canine kidney cell growth in a hypertonic medium showed a basolateral localization of AQP2 on hormonal stimulation (11). Moreover, hypertonicity has been proved to modulate AQP2 expression in immortalized mouse collecting duct principal cells (12).
On the other hand, maintaining AQP2 membrane compartment identity and fusion specificity is of critical importance because mislocalization or promiscuous fusion of AQP2 vesicles could compromise vasopressin responsivity.
Defective AQP2 trafficking can lead to severe diseases like congestive heart failure, liver cirrhosis, and nephrogenic diabetes insipidus (NDI), a condition characterized by the kidney’s inability to produce concentrated urine due to the insensitivity of the distal nephron to vasopressin (2, 13, 14, 15, 16). NDI can be acquired or congenital. Congenital NDI can be due to either mutation of the V2 receptor gene or mutations in AQP2 gene. To date, 30 mutations in the AQP2 gene have been reported (17, 18, 19).
The acquired forms of NDI may occur at any time in life and are associated with urinary concentrating defect due to decreased expression of AQP2 or impaired targeting of these channels to the apical plasma membrane. Many of these forms are reversible and secondary to drug treatment and metabolic disturbances. For example, studies in rats have shown the reversible down-regulation of AQP2 after treatment with lithium and hypokalemia (20, 21). However, patients treated with lithium with an average exposure of 8 yr recovered only in a very small percentage, whereas most failed to subside after lithium cessation (16).
Whereas many aspects of AQP2 shuttle have been clarified in more than 10 yr of investigation, several features are still unclear.
This brief review discusses recent advances in some selected aspects of this research field.
Role of Phosphorylation in Regulated AQP2 Exocytosis
Binding of vasopressin to V2 receptors on the basolateral surface of principal cells stimulates the G protein Gs, which activates adenyl cyclase. The resulting increase in intracellular cAMP leads to activation of protein kinase A (PKA) that phosphorylates many substrates including the C-terminal Ser-256 of AQP2.
It has to be underlined that the AQP2 primary structure exhibits several potential consensus sequences for at least five kinases, namely PKA, protein kinase C (PKC), protein kinase G, casein kinase II and Golgi casein kinase. To date, however, the only site that has been demonstrated to be involved in AQP2 trafficking is Ser-256. This was first investigated by Kuwahara et al. (22) in 1995 using site-directed mutagenesis. In this study, the author demonstrated that oocytes expressing wild-type, but not Ser-256-mutated, AQP2 responded to cAMP injection by increasing membrane water permeability (Pf). In vitro phosphorylation studies also showed that mutating Ser-256 prevented AQP2 phosphorylation, indirectly implicating this site for PKA-mediated phosphorylation.
Whether AQP2 phosphorylation can regulate channel osmotic Pf was addressed by Lande et al. (23), who showed that Pf of endosomes containing phosphorylated or dephosphorylated AQP2 was not significantly different, suggesting that phosphorylation regulate AQP2 accumulation to the plasma membrane rather than gating the channel. Stopped-flow light-scattering in LLC-PK1 cells transfected with wild-type or S256A AQP2 demonstrated that Ser-256 substitution impaired the surface expression of AQP2 in response to forskolin (24). Moreover, phosphorylation experiments in intact cells demonstrated significant phosphorylation of wild-type AQP2 and only minimal phosphorylation of S256A AQP2 (24).
In a separate study, Katsura et al. (25) showed that vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of AQP2-expressing LLC-PK1 cells with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89).
Studies using intact rat renal tissue confirmed data obtained using cultured renal cells. Physiological serum levels of vasopressin were able to induce a time- and dose-dependent increase in AQP2 phosphorylation, and the same effect was obtained using the vasopressin V2 receptor agonist 1-desamino-8-D-arginine vasopressin or forskolin (26). Moreover, phosphorylation state-specific antiserum revealed an increase in AQP2 phosphorylation at Ser-256 after incubation of renal tissue with arginine vasopressin (AVP). Using the same antibody as a tool to determine the subcellular localization of phosphorylated AQP2 (p-AQP2), it was shown that a consistent amount of the water channel, localized in both the plasma membrane and intracellular vesicles, is constitutively phosphorylated, even in low circulating vasopressin states (27). Because AQP2 is expressed at the plasma membrane as a tetramer, coinjection in oocytes of different ratios of AQP2-S256D and AQP2-S256A mimicking p-AQP2 and non-p-AQP2, respectively, demonstrated that at least three monomers in an AQP2 tetramer should be phosphorylated for steady-state plasma membrane localization (28).
The possible role of the other putative kinase sites in the trafficking and hormonal regulation of AQP2 has been investigated by van Balkom et al. (29). Three putative casein kinase II (Ser-148, Ser-229, Thr-244), one protein kinase C [PKC (Ser-231)], and one PKA (Ser-256) site were mutated to mimic a constitutively AQP2 nonphosphorylated/phosphorylated state and expressed in Madin-Darby canine kidney cells (29). The subcellular localization and the forskolin responsiveness of all the proteins, except for the Ser-256 mutants, were no different from wild-type AQP2. Not surprisingly, AQP2-S256A was retained in intracellular vesicles, and AQP2/S256D was localized to the plasma membrane. Interestingly though, PKC activation after forskolin treatment, increased the endocytosis of all the mutants (including AQP2/S256D), without affecting phosphorylation at Ser-256. The authors concluded that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state (29).
In an apparent contradiction to these findings, Valenti et al. (30) demonstrated that AQP2 fusion with the plasma membrane might occur even in the absence of phosphorylation by PKA. Using the phosphatase inhibitor okadaic acid in an AQP2-transfected renal cell line, the author demonstrated that by increasing the phosphorylation state of intracellular proteins, it is possible to induce translocation of AQP2 and increase Pf independent of AQP2 phosphorylation state, even in the presence of the PKA inhibitor H89 (30). AQP2 phosphorylation, therefore, may not be responsible solely for water channel translocation in renal cells, and other pathways may exist to account for AQP2 membrane translocation. In agreement with this finding, it has been shown that AQP2 recycles constitutively and rapidly between intracellular storage compartments and the cell surface in renal cells using a phosphorylation-independent process (31).
Another signaling pathway that leads to AQP2 membrane insertion was demonstrated by Bouley et al. (32) in both transfected epithelial cells and kidney collecting duct. In this pathway, elevation of intracellular cGMP levels induced AQP2 redistribution, probably through a protein kinase G-dependent AQP2 phosphorylation at Ser-256 (32). This cGMP-mediated process does not involve V2 receptor signaling and intracellular cAMP elevation. More recently the same group showed that the cGMP phosphodiesterase type 5 inhibitor, sildenafil citrate (Viagra), caused plasma membrane accumulation of AQP2 mimicking vasopressin effect in LLC-PK1 cells (33). The results of this study provide the backbone for a promising approach in the treatment of several forms of NDI characterized by V2 receptor dysfunction. In a recent work (34), AQP2 phosphorylation dynamics during maturation from the endoplasmic reticulum to the vasopressin regulated compartment have been reported. Interestingly, AQP2 transition in the Golgi apparatus was associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256, possibly accounted for by the activity of a Golgi-localized serine kinase, the Golgi casein kinase (34). According to this hypothesis, the phosphorylation-defective E258K AQP2 mutant causing a dominant form of NDI would be routed from Golgi to lysosomes for degradation (34).
Despite this plethora of experimental evidence indicating phosphorylation as a key event regulating different steps in AQP2 intracellular trafficking, nobody has so far demonstrated a direct interaction of p-AQP2 with any motor or regulatory proteins. That would be the challenge of investigators in the field in the years to come.
PKA Anchoring Proteins (AKAPs)
Compartmentalization of signaling proteins through association with anchoring proteins ensures specificity in signal transduction. In this respect, the phosphorylation by PKA and also the tethering of PKA by AKAPs to subcellular compartments are prerequisites for AQP2 translocation. In IMCD cells, AQP2 trafficking required anchoring of PKA to AKAPs. Preincubation of cells with the synthetic peptide, S-Ht31, which prevents the binding between AKAPs and the regulatory subunit of PKA, significantly impaired hormonal-dependent AQP-2 trafficking (5, 35, 36). Klussman et al. (36) cloned and sequenced the full length of the rat ortholog Ht31, showing that it not only contains the anchoring domain but also directly interacts with RhoA in vivo, thus integrating PKA and RhoA, both of which are involved in the vasopressin-dependent water reabsorption.
A new splice variant of AKAP18, AKAP18, has been found to be colocalized with AQP2 in IMCD cells, and increases in cAMP produced the dissociation of AKAP18 and PKA, suggesting that AKAP18 plays a role during AQP2 trafficking (37).
Cytoskeletal Dynamics and Rho Signaling
Cytoskeleton remodeling at the cell cortex participates in several cellular processes including endocytosis and exocytosis. In amphibian bladder vasopressin-induced osmotic water flow decreases on colchicines and nocodazole treatment, indicating that microtubule might play a role in the regulation of water permeability (38, 39, 40). Moreover, it has been reported that the driving force to promote intracellular trafficking along microtubules is provided from several motor proteins like dynactin and dynein, which have been found in immunoisolated AQP2-bearing vesicles (41).
Actin cytoskeletal dynamics have also been shown to be essential in the trafficking of AQP2-bearing vesicles. It was already known from previous studies that in mammalian collecting duct and in the amphibian bladder vasopressin increased the apical sorting of water channels was associated with depolymerization of the actin cytoskeleton (42, 43, 44). Consistently, in toad bladder epithelial cells, cytochalasin D, in the presence of vasopressin, significantly increased the fusion rate of water channel-bearing vesicles, compared with vasopressin alone, likely indicating that actin filaments might retard fusion processes (45, 46). On the other hand, in renal collecting duct CD8 cells (47), okadaic acid, a specific phosphatase inhibitor, caused actin depolymerization at the cell cortex and increase in the osmotic water transport (30). Recent studies highlighted the importance of the small GTPase Rho as a key player regulating AQP2 trafficking through regulation of the actin network (48, 49). RhoA inhibition occurring in response to forskolin stimulation represents a physiological step in the signal transduction cascade activated by hormonal stimulation leading to a partial depolymerization of the actin cytoskeleton, which facilitates the apical targeting of AQP2 (50). Consistent with these observations, Rho-dependent actin polymerization observed either on prostaglandin EP3 receptor activation (51) or by selective activation of the sensing calcium receptor (CaR) significantly impaired vasopressin-stimulated AQP2 trafficking (52). These data indicate that RhoA exerts a bidirectional control in the regulation of AQP2 trafficking though the regulation of actin network organization.
Ezrin-Radixin-Moesin (ERM) Protein Involvement in AQP2 Trafficking
ERM proteins cross-link actin filaments with plasma membrane proteins and are important integrators at the cell cortex, where they participate in the reorganization of F-actin-containing cytoskeletal structures.
Rho activity is controlled by several regulators, and ERM proteins function both upstream and downstream of Rho GTPases, implying that there could be a feedback loop for Rho pathway autoregulation (36, 53, 54, 55). Recent data have provided the first evidence that AQP2 translocation to the plasma membrane requires the functional involvement of the ERM protein moesin (56). ERM may represent scaffolding proteins mediating AQP2 interaction with actin. Interestingly, immunoaffinity chromatography experiments in rat kidney papilla revealed that AQP2 interacts with not only several actin binding proteins but also - and -isoforms of actin (57, 58). Proteomic analysis of immunoisolated AQP2-bearing vesicles from renal inner medullary collecting duct suggested the existence of a dynamic complex including myosin IC, nonmuscle myosin IIA and IIB, myosin VI, and myosin IXB involved in the generation of force necessary to promote AQP2 trafficking to the plasma membrane (59). More recently Chou et al. (60) showed that Pf is partially due to the activation of calcium/calmodulin-dependent myosin light chain kinase. Moreover, consistent with these observations, AQP2-containing exosomes isolated from human urine also expressed several cytoskeletal proteins like ERM, many isoforms of actin, cofilin, dynein, and tubulin (61).
Role of Calcium in Vasopressin-Stimulated AQP2 Translocation
Studies of a wide variety of vesicular-trafficking processes have pointed to a key role for local increases in intracellular calcium concentration ([Ca2+]i) in triggering the fusion of vesicles to their target membranes (62, 63, 64). Whether vasopressin stimulated exocytosis of AQP2-containing vesicles in collecting duct cells requires calcium as an intracellular mediator is still unclear. Vasopressin, acting via V2 receptors, causes a transient increase in [Ca2+]i and calcium oscillations in IMCD cells (65, 66, 67, 68, 69, 70). Buffering of intracellular calcium with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid(BAPTA) completely blocked the Pf response to vasopressin in IMCD (65, 68), suggesting that intracellular calcium mobilization is required for exocytotic insertion of AQP2. Removal of extracellular Ca2+ in perfused IMCD did not prevent the initial rise [Ca2+]i induced by vasopressin but inhibited the sustained oscillations (65). These observations indicate that vasopressin induces calcium release from intracellular stores, but extracellular calcium is required to sustain the [Ca2+]i oscillations likely needed to prolong the exocytotic activity induced by vasopressin. Little is known about the mechanism of the vasopressin-induced increase in intracellular Ca2+, although previous studies established that it occurs in the absence of activation of the phosphoinositide signaling pathway (71). In primary cultures of IMCD cells, ryanodine, a ryanodine receptor antagonist, inhibits vasopressin-dependent redistribution of AQP2 to the plasma membrane, suggesting a role for ryanodine-sensitive stores in the calcium-dependent AQP2 trafficking (65, 68).
However, reports from other authors do not support a regulatory function for calcium in AQP2 insertion into the plasma membrane (72). By analysis of endocytic capacitance in primary cultured epithelial cells from renal inner medulla, these authors showed that cAMP is sufficient for triggering the exocytic recruitment of AQP2, which is not evoked by vasopressin-induced intracellular calcium increases.
SNAREs for Directing Vesicular Trafficking of AQP2
Fusion of AQP2 vesicles with the plasma membrane is a key terminal step in vasopressin-regulated water transport. This fusion event is possibly mediated by SNARE proteins through a process regulated by accessory proteins whose roles are still unclear. SNARE proteins have been detected in the collecting duct principal cells and colocalize with AQP2-bearing vesicles (73, 74). In particular, syntaxin 4 is present in the apical plasma membrane of collecting duct principal cells (75, 76), and soluble N-ethylmaleimide-associated protein-23 (SNAP23) has also been found in collecting duct principal cells both in the apical plasma membrane and AQP2-bearing vesicles (77). SNAP23 has been demonstrated to interact with syntaxin-4 and vesicle-associated membrane protein 2 (VAMP2) (78). However, another study, using different antibodies, reported an opposite polarity for sintaxin-4 in principal cells (79).
The functional involvement of VAMP2 in AQP2 targeting came from the demonstration that tetanus neurotoxin was efficient in cleaving the synaptobrevin-like protein in intact collecting duct renal cells, and cAMP-stimulated AQP2 targeting to the plasma membrane was completely abolished in treated cells (80). This represents the first evidence for the functional requirement of VAMP2 in cAMP-induced AQP2 exocytosis in renal cells. The identification and functional involvement of the counterpart VAMP2 proteins is still lacking.
Other Accessory Proteins Involved in the AQP2 Shuttle
Heterotrimeric GTP binding proteins of the Gi family has also been proposed to be essential for AQP2 targeting (81). Treatment of cultured rabbit cortical collecting duct cells with pertussis toxin or incubation of permeabilized cells with synthetic peptides reproducing the COOH terminus of the Gi-3 subunit abolished cAMP-induced AQP2 trafficking to the cell apical plasma membrane. This leads to the conclusion that Gi-3 plays a facilitatory role in AQP2 targeting. On the other hand, it has also been proposed that proteins of the Gi family impair AQP2 translocation to the plasma membrane via activation of the calcium-sensing receptor (82). The precise functional role of this G protein in AQP2 trafficking remains to be clarified.
Very recently Noda et al. (58, 83) identified two AQP2 binding proteins, actin and signal-induced proliferation-associated protein 1 (SPA-1). SPA-1 is a specific GTPase-activating protein for Rap1, whose activity was shown to be required for AQP2 trafficking to the apical membrane.
Attenuation of Vasopressin 2 Receptor Signaling
Luminal calcium
Recent data support the hypothesis that high calcium levels (>2 mM) may act as a negative regulator of V2 receptor signaling in the kidney. Calcium plays a role in AQP2-bearing vesicles trafficking not only at second messenger level but also as a first messenger acting through a specific G protein-coupled receptor. Purified AQP2 endosomes contain a CaR (82) similar to that identified in both the parathyroid gland and renal thick ascending limb (TAL) (84, 85). CaRs have been shown to sense the extracellular concentration of divalent cations, including Ca2+ or Mg2+ (84, 86, 87) and activate various signal transduction pathways in multiple cell types (88, 89). Although little significant transepithelial transport of Ca2+ or Mg2+ occurs in IMCD, the luminal concentrations of these divalent cations undergo significant alterations as a result of vasopressin elicited water reabsorption. Studies in isolated perfused rat IMCD show that apical membrane exposure to CaR agonists rapidly reduces AVP-elicited Pf without an alteration in P urea (82). In addition, during sustained hypercalcemia in chronically hypercalcemic rats, the Pf in inner medullary collecting duct did not increase significantly after vasopressin and was accompanied by an 87% reduction in AQP2 protein (90), which may contribute to the lack of vasopressin responsiveness. Moreover it has been observed that extracellular calcium acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in renal AQP2 expressing CD8 cells (52).
The signal transduction pathways linking urinary calcium levels to AQP2 expression/trafficking in the collecting duct is poorly understood. A Gi-coupled receptor activation signaling may mediate the extracellular calcium effects trough CaR (91, 92, 93). In some cells expressing CaR, CaR agonists can inhibit cAMP accumulation through a mechanism involving a CaR-induced increase in [Ca2+]i, which then inhibits a calcium-sensitive form of adenylate cyclase (94). Alternatively, a Ca2+-dependent increase in [Ca2+]i can impair cAMP accumulation by an increase in cAMP hydrolysis through a Ca2+-mediated process (95, 96). Another intriguing possibility is that CaR-mediated Rho activation (97) induces stabilization of cortical F-actin cytoskeleton that impairs AQP2 targeting, as previously suggested (49, 50). Thus, the apical membrane CaR may provide a mechanism for the modulation of vasopressin-elicited water transport in IMCD by altering AQP2 vesicle apical membrane retrieval. The presence of this apical CaR permits the IMCD cell to continuously integrate AVP water transport with alterations in luminal Ca2+ preventing further calcium concentration and thus protecting against a potential risk of calcium oxalate- or calcium phosphate-containing kidney stone formation.
Prostaglandin
In the kidney, prostaglandin E2 (PGE2) is involved in the regulation of epithelial solute and water reabsorption (98). PGE2 and its synthetic analog sulprostone are known to antagonize vasopressin-mediated water reabsorption. It has been reported that PGE2 stimulates internalization of AQP2 from the plasma membrane of principal cells when added after vasopressin treatment without affecting its phosphorylated state (99). In IMCD cells, preincubation of the cells with sulprostone, a synthetic analog of PGE2, inhibited the effect of vasopressin on AQP2 translocation independently of inositol-1,4,5-trisphosphate or [Ca2+]i (51) (Fig. 1). AQP2 down-regulation is also present in long-standing urinary obstruction and persists even after the obstruction is resolved. Quite interestingly in humans, AQP2 urinary excretion in postobstructed kidney was associated with a local increase in PGE2 synthesis, which may be, in part, responsible for selective down-regulation of AQP2 trafficking/expression (100).
Bradykinin (BK)
The nonapeptide BK is a known stimulator of prostaglandin production in the kidney through activation of phospholipase A2, which stimulates AA release. In a rabbit collecting duct cell line expressing endogenous BK2R receptors, BK inhibited deoxy-1-desamino-8-D-arginine vasopressin-dependent cAMP production. This event was mimicked by PGE2 and suppressed with indomethacin, suggesting that the signal transduction initiated by BK included PGE2 synthesis (101). However, in the same cells stably transfected with AQP2, recent data indicate that BK causes an increase of Rho activity associated with stabilization of cortical F-actin network, thus impairing AQP2 targeting (102). These effects counteract the physiological vasopressin stimulation. This indicates that BK may directly contribute to a decrease in the kidney concentrating ability impairing cell surface expression of the vasopressin sensitive AQP2 water channel (Fig. 1).
Body Fluid Regulation in Microgravity, Involvement of AQP2
Continuous sodium retention not being followed by water has been observed in astronauts (103). This is in agreement with the persistent plasma volume deficit occurring in microgravity despite the continuously activated counterregulatory endocrine and autonomous nervous systems. As discussed above, extracellular CaRs might sense alterations of calcium concentration in the collecting duct lumen and in turn activate a signal transduction cascade influencing AQP2 trafficking/expression. Because astronauts, in addition to their water-handling disorder, experience hypercalciuria during their presence aboard space vehicles, it has been hypothesized that the impaired water handling occurring in astronauts during space flight, is due to an attenuated AQP2 response to vasopressin as a consequence of persistent hypercalciuria (104, 105). The link between hypovolemia-induced activation of fluid retaining systems and reduction in bone mineral density, promoting hypercalciuria during space flight, with its postulated modulating effect on renal water and sodium handling, shall stimulate space physiology research during real space flight and on the ground.
Concluding Remarks
After the discovery of the vasopressin-sensitive AQP2 water channel, significant progress has been made in understanding the molecular basis of AQP2 trafficking between intracellular vesicles and the cell surface. These events are regulated by hormone-induced protein phosphorylation and involve the interaction and/or functional involvement of several regulatory proteins as GTP binding proteins, cytoskeletal elements, ERM, and SNAREs. Additional features of AQP2 shuttle have emerged recently related to intracellular pathways activated by physiological molecules such as prostaglandins, bradykinin, and divalent cations like calcium that counteract vasopressin response (Fig. 1). These analyses provide insight into the physiology and pathophysiology of water balance leading to possible therapeutic intervention for water balance disorders including NDI, heart failure, and hypertension.
Acknowledgments
We thank all colleagues who contributed to these studies.
Footnotes
This work was supported by the "Ministero della Ricerca Scientifica e Tecnologica," and the CEGBA (Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario), and Laboratorio Analisi del Gene.
First Published Online September 8, 2005
Abbreviations: AKAP, PKA anchoring protein; AQP2, aquaporin-2; AVP, arginine vasopressin; BK, bradykinin; [Ca2+]i, intracellular calcium concentration; CaR, calcium receptor; ERM, ezrin-radixin-moesin; NDI, nephrogenic diabetes insipidus; p-AQP2, phosphorylated AQP2; Pf, osmotic water permeability coefficient; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; SNARE, soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor; VAMP2, vesicle-associated membrane protein.
Accepted for publication August 5, 2005.
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Abstract
In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.
Introduction
A MAJOR BREAKTHROUGH in understanding how the kidney regulates water transport was the discovery that vasopressin stimulates the translocation of a specific water channel protein, aquaporin-2 (AQP2), from intracellular vesicles to the cell surface. Elucidating how this process is regulated has remained a challenge because it represents a convergence of two different and complex fields of research, namely vesicle transport and signal transduction. AQP2 protein possesses the typical aquaporin fold observed in the aquaporin water channel family members, as recently confirmed by crystallization and structural characterization by atomic force microscopy (1).
The trafficking of the vasopressin-sensitive water channel AQP2, reviewed in several excellent publications (2, 3, 4, 5, 6, 7, 8), is the paradigm of how cells control the movement of membrane proteins through complex pathways in response to external stimuli and how, by doing so, they regulate their function. In response to an increase in vasopressin levels, AQP2, intracellularly localized in resting collecting duct principal cells, becomes exposed on the cell surface on which it facilitates water reabsorption and consequently urine concentration. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins by a clathrin-mediated mechanism (3, 9, 10) and their recycling to the original stores, in which they are retained. These complex mechanisms include the spatiotemporal modulation of several regulatory proteins. The role of some accessory proteins such as soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) proteins and small GTPases in AQP2 shuttle has been recently clarified. Moreover, actin cytoskeleton remodeling regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking.
In addition to a vasopressin-regulated pathway, it has been shown that AQP2 recycles constitutively in epithelial cells, and it can be inserted in different membrane domains. In epithelial cell lines, AQP2 has been shown to be targeted to the apical or basolateral surface, although the polarity signals are still to be clarified (3). Osmolality environment can influence the final membrane destination. Madin-Darby canine kidney cell growth in a hypertonic medium showed a basolateral localization of AQP2 on hormonal stimulation (11). Moreover, hypertonicity has been proved to modulate AQP2 expression in immortalized mouse collecting duct principal cells (12).
On the other hand, maintaining AQP2 membrane compartment identity and fusion specificity is of critical importance because mislocalization or promiscuous fusion of AQP2 vesicles could compromise vasopressin responsivity.
Defective AQP2 trafficking can lead to severe diseases like congestive heart failure, liver cirrhosis, and nephrogenic diabetes insipidus (NDI), a condition characterized by the kidney’s inability to produce concentrated urine due to the insensitivity of the distal nephron to vasopressin (2, 13, 14, 15, 16). NDI can be acquired or congenital. Congenital NDI can be due to either mutation of the V2 receptor gene or mutations in AQP2 gene. To date, 30 mutations in the AQP2 gene have been reported (17, 18, 19).
The acquired forms of NDI may occur at any time in life and are associated with urinary concentrating defect due to decreased expression of AQP2 or impaired targeting of these channels to the apical plasma membrane. Many of these forms are reversible and secondary to drug treatment and metabolic disturbances. For example, studies in rats have shown the reversible down-regulation of AQP2 after treatment with lithium and hypokalemia (20, 21). However, patients treated with lithium with an average exposure of 8 yr recovered only in a very small percentage, whereas most failed to subside after lithium cessation (16).
Whereas many aspects of AQP2 shuttle have been clarified in more than 10 yr of investigation, several features are still unclear.
This brief review discusses recent advances in some selected aspects of this research field.
Role of Phosphorylation in Regulated AQP2 Exocytosis
Binding of vasopressin to V2 receptors on the basolateral surface of principal cells stimulates the G protein Gs, which activates adenyl cyclase. The resulting increase in intracellular cAMP leads to activation of protein kinase A (PKA) that phosphorylates many substrates including the C-terminal Ser-256 of AQP2.
It has to be underlined that the AQP2 primary structure exhibits several potential consensus sequences for at least five kinases, namely PKA, protein kinase C (PKC), protein kinase G, casein kinase II and Golgi casein kinase. To date, however, the only site that has been demonstrated to be involved in AQP2 trafficking is Ser-256. This was first investigated by Kuwahara et al. (22) in 1995 using site-directed mutagenesis. In this study, the author demonstrated that oocytes expressing wild-type, but not Ser-256-mutated, AQP2 responded to cAMP injection by increasing membrane water permeability (Pf). In vitro phosphorylation studies also showed that mutating Ser-256 prevented AQP2 phosphorylation, indirectly implicating this site for PKA-mediated phosphorylation.
Whether AQP2 phosphorylation can regulate channel osmotic Pf was addressed by Lande et al. (23), who showed that Pf of endosomes containing phosphorylated or dephosphorylated AQP2 was not significantly different, suggesting that phosphorylation regulate AQP2 accumulation to the plasma membrane rather than gating the channel. Stopped-flow light-scattering in LLC-PK1 cells transfected with wild-type or S256A AQP2 demonstrated that Ser-256 substitution impaired the surface expression of AQP2 in response to forskolin (24). Moreover, phosphorylation experiments in intact cells demonstrated significant phosphorylation of wild-type AQP2 and only minimal phosphorylation of S256A AQP2 (24).
In a separate study, Katsura et al. (25) showed that vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of AQP2-expressing LLC-PK1 cells with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89).
Studies using intact rat renal tissue confirmed data obtained using cultured renal cells. Physiological serum levels of vasopressin were able to induce a time- and dose-dependent increase in AQP2 phosphorylation, and the same effect was obtained using the vasopressin V2 receptor agonist 1-desamino-8-D-arginine vasopressin or forskolin (26). Moreover, phosphorylation state-specific antiserum revealed an increase in AQP2 phosphorylation at Ser-256 after incubation of renal tissue with arginine vasopressin (AVP). Using the same antibody as a tool to determine the subcellular localization of phosphorylated AQP2 (p-AQP2), it was shown that a consistent amount of the water channel, localized in both the plasma membrane and intracellular vesicles, is constitutively phosphorylated, even in low circulating vasopressin states (27). Because AQP2 is expressed at the plasma membrane as a tetramer, coinjection in oocytes of different ratios of AQP2-S256D and AQP2-S256A mimicking p-AQP2 and non-p-AQP2, respectively, demonstrated that at least three monomers in an AQP2 tetramer should be phosphorylated for steady-state plasma membrane localization (28).
The possible role of the other putative kinase sites in the trafficking and hormonal regulation of AQP2 has been investigated by van Balkom et al. (29). Three putative casein kinase II (Ser-148, Ser-229, Thr-244), one protein kinase C [PKC (Ser-231)], and one PKA (Ser-256) site were mutated to mimic a constitutively AQP2 nonphosphorylated/phosphorylated state and expressed in Madin-Darby canine kidney cells (29). The subcellular localization and the forskolin responsiveness of all the proteins, except for the Ser-256 mutants, were no different from wild-type AQP2. Not surprisingly, AQP2-S256A was retained in intracellular vesicles, and AQP2/S256D was localized to the plasma membrane. Interestingly though, PKC activation after forskolin treatment, increased the endocytosis of all the mutants (including AQP2/S256D), without affecting phosphorylation at Ser-256. The authors concluded that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state (29).
In an apparent contradiction to these findings, Valenti et al. (30) demonstrated that AQP2 fusion with the plasma membrane might occur even in the absence of phosphorylation by PKA. Using the phosphatase inhibitor okadaic acid in an AQP2-transfected renal cell line, the author demonstrated that by increasing the phosphorylation state of intracellular proteins, it is possible to induce translocation of AQP2 and increase Pf independent of AQP2 phosphorylation state, even in the presence of the PKA inhibitor H89 (30). AQP2 phosphorylation, therefore, may not be responsible solely for water channel translocation in renal cells, and other pathways may exist to account for AQP2 membrane translocation. In agreement with this finding, it has been shown that AQP2 recycles constitutively and rapidly between intracellular storage compartments and the cell surface in renal cells using a phosphorylation-independent process (31).
Another signaling pathway that leads to AQP2 membrane insertion was demonstrated by Bouley et al. (32) in both transfected epithelial cells and kidney collecting duct. In this pathway, elevation of intracellular cGMP levels induced AQP2 redistribution, probably through a protein kinase G-dependent AQP2 phosphorylation at Ser-256 (32). This cGMP-mediated process does not involve V2 receptor signaling and intracellular cAMP elevation. More recently the same group showed that the cGMP phosphodiesterase type 5 inhibitor, sildenafil citrate (Viagra), caused plasma membrane accumulation of AQP2 mimicking vasopressin effect in LLC-PK1 cells (33). The results of this study provide the backbone for a promising approach in the treatment of several forms of NDI characterized by V2 receptor dysfunction. In a recent work (34), AQP2 phosphorylation dynamics during maturation from the endoplasmic reticulum to the vasopressin regulated compartment have been reported. Interestingly, AQP2 transition in the Golgi apparatus was associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256, possibly accounted for by the activity of a Golgi-localized serine kinase, the Golgi casein kinase (34). According to this hypothesis, the phosphorylation-defective E258K AQP2 mutant causing a dominant form of NDI would be routed from Golgi to lysosomes for degradation (34).
Despite this plethora of experimental evidence indicating phosphorylation as a key event regulating different steps in AQP2 intracellular trafficking, nobody has so far demonstrated a direct interaction of p-AQP2 with any motor or regulatory proteins. That would be the challenge of investigators in the field in the years to come.
PKA Anchoring Proteins (AKAPs)
Compartmentalization of signaling proteins through association with anchoring proteins ensures specificity in signal transduction. In this respect, the phosphorylation by PKA and also the tethering of PKA by AKAPs to subcellular compartments are prerequisites for AQP2 translocation. In IMCD cells, AQP2 trafficking required anchoring of PKA to AKAPs. Preincubation of cells with the synthetic peptide, S-Ht31, which prevents the binding between AKAPs and the regulatory subunit of PKA, significantly impaired hormonal-dependent AQP-2 trafficking (5, 35, 36). Klussman et al. (36) cloned and sequenced the full length of the rat ortholog Ht31, showing that it not only contains the anchoring domain but also directly interacts with RhoA in vivo, thus integrating PKA and RhoA, both of which are involved in the vasopressin-dependent water reabsorption.
A new splice variant of AKAP18, AKAP18, has been found to be colocalized with AQP2 in IMCD cells, and increases in cAMP produced the dissociation of AKAP18 and PKA, suggesting that AKAP18 plays a role during AQP2 trafficking (37).
Cytoskeletal Dynamics and Rho Signaling
Cytoskeleton remodeling at the cell cortex participates in several cellular processes including endocytosis and exocytosis. In amphibian bladder vasopressin-induced osmotic water flow decreases on colchicines and nocodazole treatment, indicating that microtubule might play a role in the regulation of water permeability (38, 39, 40). Moreover, it has been reported that the driving force to promote intracellular trafficking along microtubules is provided from several motor proteins like dynactin and dynein, which have been found in immunoisolated AQP2-bearing vesicles (41).
Actin cytoskeletal dynamics have also been shown to be essential in the trafficking of AQP2-bearing vesicles. It was already known from previous studies that in mammalian collecting duct and in the amphibian bladder vasopressin increased the apical sorting of water channels was associated with depolymerization of the actin cytoskeleton (42, 43, 44). Consistently, in toad bladder epithelial cells, cytochalasin D, in the presence of vasopressin, significantly increased the fusion rate of water channel-bearing vesicles, compared with vasopressin alone, likely indicating that actin filaments might retard fusion processes (45, 46). On the other hand, in renal collecting duct CD8 cells (47), okadaic acid, a specific phosphatase inhibitor, caused actin depolymerization at the cell cortex and increase in the osmotic water transport (30). Recent studies highlighted the importance of the small GTPase Rho as a key player regulating AQP2 trafficking through regulation of the actin network (48, 49). RhoA inhibition occurring in response to forskolin stimulation represents a physiological step in the signal transduction cascade activated by hormonal stimulation leading to a partial depolymerization of the actin cytoskeleton, which facilitates the apical targeting of AQP2 (50). Consistent with these observations, Rho-dependent actin polymerization observed either on prostaglandin EP3 receptor activation (51) or by selective activation of the sensing calcium receptor (CaR) significantly impaired vasopressin-stimulated AQP2 trafficking (52). These data indicate that RhoA exerts a bidirectional control in the regulation of AQP2 trafficking though the regulation of actin network organization.
Ezrin-Radixin-Moesin (ERM) Protein Involvement in AQP2 Trafficking
ERM proteins cross-link actin filaments with plasma membrane proteins and are important integrators at the cell cortex, where they participate in the reorganization of F-actin-containing cytoskeletal structures.
Rho activity is controlled by several regulators, and ERM proteins function both upstream and downstream of Rho GTPases, implying that there could be a feedback loop for Rho pathway autoregulation (36, 53, 54, 55). Recent data have provided the first evidence that AQP2 translocation to the plasma membrane requires the functional involvement of the ERM protein moesin (56). ERM may represent scaffolding proteins mediating AQP2 interaction with actin. Interestingly, immunoaffinity chromatography experiments in rat kidney papilla revealed that AQP2 interacts with not only several actin binding proteins but also - and -isoforms of actin (57, 58). Proteomic analysis of immunoisolated AQP2-bearing vesicles from renal inner medullary collecting duct suggested the existence of a dynamic complex including myosin IC, nonmuscle myosin IIA and IIB, myosin VI, and myosin IXB involved in the generation of force necessary to promote AQP2 trafficking to the plasma membrane (59). More recently Chou et al. (60) showed that Pf is partially due to the activation of calcium/calmodulin-dependent myosin light chain kinase. Moreover, consistent with these observations, AQP2-containing exosomes isolated from human urine also expressed several cytoskeletal proteins like ERM, many isoforms of actin, cofilin, dynein, and tubulin (61).
Role of Calcium in Vasopressin-Stimulated AQP2 Translocation
Studies of a wide variety of vesicular-trafficking processes have pointed to a key role for local increases in intracellular calcium concentration ([Ca2+]i) in triggering the fusion of vesicles to their target membranes (62, 63, 64). Whether vasopressin stimulated exocytosis of AQP2-containing vesicles in collecting duct cells requires calcium as an intracellular mediator is still unclear. Vasopressin, acting via V2 receptors, causes a transient increase in [Ca2+]i and calcium oscillations in IMCD cells (65, 66, 67, 68, 69, 70). Buffering of intracellular calcium with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid(BAPTA) completely blocked the Pf response to vasopressin in IMCD (65, 68), suggesting that intracellular calcium mobilization is required for exocytotic insertion of AQP2. Removal of extracellular Ca2+ in perfused IMCD did not prevent the initial rise [Ca2+]i induced by vasopressin but inhibited the sustained oscillations (65). These observations indicate that vasopressin induces calcium release from intracellular stores, but extracellular calcium is required to sustain the [Ca2+]i oscillations likely needed to prolong the exocytotic activity induced by vasopressin. Little is known about the mechanism of the vasopressin-induced increase in intracellular Ca2+, although previous studies established that it occurs in the absence of activation of the phosphoinositide signaling pathway (71). In primary cultures of IMCD cells, ryanodine, a ryanodine receptor antagonist, inhibits vasopressin-dependent redistribution of AQP2 to the plasma membrane, suggesting a role for ryanodine-sensitive stores in the calcium-dependent AQP2 trafficking (65, 68).
However, reports from other authors do not support a regulatory function for calcium in AQP2 insertion into the plasma membrane (72). By analysis of endocytic capacitance in primary cultured epithelial cells from renal inner medulla, these authors showed that cAMP is sufficient for triggering the exocytic recruitment of AQP2, which is not evoked by vasopressin-induced intracellular calcium increases.
SNAREs for Directing Vesicular Trafficking of AQP2
Fusion of AQP2 vesicles with the plasma membrane is a key terminal step in vasopressin-regulated water transport. This fusion event is possibly mediated by SNARE proteins through a process regulated by accessory proteins whose roles are still unclear. SNARE proteins have been detected in the collecting duct principal cells and colocalize with AQP2-bearing vesicles (73, 74). In particular, syntaxin 4 is present in the apical plasma membrane of collecting duct principal cells (75, 76), and soluble N-ethylmaleimide-associated protein-23 (SNAP23) has also been found in collecting duct principal cells both in the apical plasma membrane and AQP2-bearing vesicles (77). SNAP23 has been demonstrated to interact with syntaxin-4 and vesicle-associated membrane protein 2 (VAMP2) (78). However, another study, using different antibodies, reported an opposite polarity for sintaxin-4 in principal cells (79).
The functional involvement of VAMP2 in AQP2 targeting came from the demonstration that tetanus neurotoxin was efficient in cleaving the synaptobrevin-like protein in intact collecting duct renal cells, and cAMP-stimulated AQP2 targeting to the plasma membrane was completely abolished in treated cells (80). This represents the first evidence for the functional requirement of VAMP2 in cAMP-induced AQP2 exocytosis in renal cells. The identification and functional involvement of the counterpart VAMP2 proteins is still lacking.
Other Accessory Proteins Involved in the AQP2 Shuttle
Heterotrimeric GTP binding proteins of the Gi family has also been proposed to be essential for AQP2 targeting (81). Treatment of cultured rabbit cortical collecting duct cells with pertussis toxin or incubation of permeabilized cells with synthetic peptides reproducing the COOH terminus of the Gi-3 subunit abolished cAMP-induced AQP2 trafficking to the cell apical plasma membrane. This leads to the conclusion that Gi-3 plays a facilitatory role in AQP2 targeting. On the other hand, it has also been proposed that proteins of the Gi family impair AQP2 translocation to the plasma membrane via activation of the calcium-sensing receptor (82). The precise functional role of this G protein in AQP2 trafficking remains to be clarified.
Very recently Noda et al. (58, 83) identified two AQP2 binding proteins, actin and signal-induced proliferation-associated protein 1 (SPA-1). SPA-1 is a specific GTPase-activating protein for Rap1, whose activity was shown to be required for AQP2 trafficking to the apical membrane.
Attenuation of Vasopressin 2 Receptor Signaling
Luminal calcium
Recent data support the hypothesis that high calcium levels (>2 mM) may act as a negative regulator of V2 receptor signaling in the kidney. Calcium plays a role in AQP2-bearing vesicles trafficking not only at second messenger level but also as a first messenger acting through a specific G protein-coupled receptor. Purified AQP2 endosomes contain a CaR (82) similar to that identified in both the parathyroid gland and renal thick ascending limb (TAL) (84, 85). CaRs have been shown to sense the extracellular concentration of divalent cations, including Ca2+ or Mg2+ (84, 86, 87) and activate various signal transduction pathways in multiple cell types (88, 89). Although little significant transepithelial transport of Ca2+ or Mg2+ occurs in IMCD, the luminal concentrations of these divalent cations undergo significant alterations as a result of vasopressin elicited water reabsorption. Studies in isolated perfused rat IMCD show that apical membrane exposure to CaR agonists rapidly reduces AVP-elicited Pf without an alteration in P urea (82). In addition, during sustained hypercalcemia in chronically hypercalcemic rats, the Pf in inner medullary collecting duct did not increase significantly after vasopressin and was accompanied by an 87% reduction in AQP2 protein (90), which may contribute to the lack of vasopressin responsiveness. Moreover it has been observed that extracellular calcium acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in renal AQP2 expressing CD8 cells (52).
The signal transduction pathways linking urinary calcium levels to AQP2 expression/trafficking in the collecting duct is poorly understood. A Gi-coupled receptor activation signaling may mediate the extracellular calcium effects trough CaR (91, 92, 93). In some cells expressing CaR, CaR agonists can inhibit cAMP accumulation through a mechanism involving a CaR-induced increase in [Ca2+]i, which then inhibits a calcium-sensitive form of adenylate cyclase (94). Alternatively, a Ca2+-dependent increase in [Ca2+]i can impair cAMP accumulation by an increase in cAMP hydrolysis through a Ca2+-mediated process (95, 96). Another intriguing possibility is that CaR-mediated Rho activation (97) induces stabilization of cortical F-actin cytoskeleton that impairs AQP2 targeting, as previously suggested (49, 50). Thus, the apical membrane CaR may provide a mechanism for the modulation of vasopressin-elicited water transport in IMCD by altering AQP2 vesicle apical membrane retrieval. The presence of this apical CaR permits the IMCD cell to continuously integrate AVP water transport with alterations in luminal Ca2+ preventing further calcium concentration and thus protecting against a potential risk of calcium oxalate- or calcium phosphate-containing kidney stone formation.
Prostaglandin
In the kidney, prostaglandin E2 (PGE2) is involved in the regulation of epithelial solute and water reabsorption (98). PGE2 and its synthetic analog sulprostone are known to antagonize vasopressin-mediated water reabsorption. It has been reported that PGE2 stimulates internalization of AQP2 from the plasma membrane of principal cells when added after vasopressin treatment without affecting its phosphorylated state (99). In IMCD cells, preincubation of the cells with sulprostone, a synthetic analog of PGE2, inhibited the effect of vasopressin on AQP2 translocation independently of inositol-1,4,5-trisphosphate or [Ca2+]i (51) (Fig. 1). AQP2 down-regulation is also present in long-standing urinary obstruction and persists even after the obstruction is resolved. Quite interestingly in humans, AQP2 urinary excretion in postobstructed kidney was associated with a local increase in PGE2 synthesis, which may be, in part, responsible for selective down-regulation of AQP2 trafficking/expression (100).
Bradykinin (BK)
The nonapeptide BK is a known stimulator of prostaglandin production in the kidney through activation of phospholipase A2, which stimulates AA release. In a rabbit collecting duct cell line expressing endogenous BK2R receptors, BK inhibited deoxy-1-desamino-8-D-arginine vasopressin-dependent cAMP production. This event was mimicked by PGE2 and suppressed with indomethacin, suggesting that the signal transduction initiated by BK included PGE2 synthesis (101). However, in the same cells stably transfected with AQP2, recent data indicate that BK causes an increase of Rho activity associated with stabilization of cortical F-actin network, thus impairing AQP2 targeting (102). These effects counteract the physiological vasopressin stimulation. This indicates that BK may directly contribute to a decrease in the kidney concentrating ability impairing cell surface expression of the vasopressin sensitive AQP2 water channel (Fig. 1).
Body Fluid Regulation in Microgravity, Involvement of AQP2
Continuous sodium retention not being followed by water has been observed in astronauts (103). This is in agreement with the persistent plasma volume deficit occurring in microgravity despite the continuously activated counterregulatory endocrine and autonomous nervous systems. As discussed above, extracellular CaRs might sense alterations of calcium concentration in the collecting duct lumen and in turn activate a signal transduction cascade influencing AQP2 trafficking/expression. Because astronauts, in addition to their water-handling disorder, experience hypercalciuria during their presence aboard space vehicles, it has been hypothesized that the impaired water handling occurring in astronauts during space flight, is due to an attenuated AQP2 response to vasopressin as a consequence of persistent hypercalciuria (104, 105). The link between hypovolemia-induced activation of fluid retaining systems and reduction in bone mineral density, promoting hypercalciuria during space flight, with its postulated modulating effect on renal water and sodium handling, shall stimulate space physiology research during real space flight and on the ground.
Concluding Remarks
After the discovery of the vasopressin-sensitive AQP2 water channel, significant progress has been made in understanding the molecular basis of AQP2 trafficking between intracellular vesicles and the cell surface. These events are regulated by hormone-induced protein phosphorylation and involve the interaction and/or functional involvement of several regulatory proteins as GTP binding proteins, cytoskeletal elements, ERM, and SNAREs. Additional features of AQP2 shuttle have emerged recently related to intracellular pathways activated by physiological molecules such as prostaglandins, bradykinin, and divalent cations like calcium that counteract vasopressin response (Fig. 1). These analyses provide insight into the physiology and pathophysiology of water balance leading to possible therapeutic intervention for water balance disorders including NDI, heart failure, and hypertension.
Acknowledgments
We thank all colleagues who contributed to these studies.
Footnotes
This work was supported by the "Ministero della Ricerca Scientifica e Tecnologica," and the CEGBA (Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario), and Laboratorio Analisi del Gene.
First Published Online September 8, 2005
Abbreviations: AKAP, PKA anchoring protein; AQP2, aquaporin-2; AVP, arginine vasopressin; BK, bradykinin; [Ca2+]i, intracellular calcium concentration; CaR, calcium receptor; ERM, ezrin-radixin-moesin; NDI, nephrogenic diabetes insipidus; p-AQP2, phosphorylated AQP2; Pf, osmotic water permeability coefficient; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; SNARE, soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor; VAMP2, vesicle-associated membrane protein.
Accepted for publication August 5, 2005.
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