Brenner and Rector's The Kidney, 8th ed.

CHAPTER 8. Cell Biology of Vasopressin Action

Dennis Brown   Søren Nielsen

  

 

Vasopressin—The Antidiuretic Hormone, 280

  

 

The Vasopressin V2 Receptor (V2R)—A G-Protein Coupled Receptor, 281

  

 

The Aquaporins—A Family of Water Channel Proteins, 286

  

 

Intracellular Pathways of AQP2 Trafficking, 291

  

 

Regulation of AQP2 Trafficking, 293

  

 

Long-Term Regulation of Water Balance, 297

The antidiuretic hormone, vasopressin (VP) plays a multifaceted role in urinary concentration in mammals via activation of a G-protein coupled receptor (GPCR), the vasopressin receptor (V2R). VP in-creases the water permeability of renal collecting ducts by stimulating the plasma membrane accumulation of a water channel, aquaporin 2 (AQP2); it stimulates NaCl reabsorption by thick ascending limbs of Henle to increase the osmolality of the medullary interstitium; it facilitates the transepithelial movement of urea along its concentration gradient in terminal portions of the collecting duct, an impor-tant facet of the renal concentrating me-chanism that allows high levels of urea to be excreted without reducing urinary concentrating ability. Many of the proteins that are involved in fluid and electrolyte transport in the kidney have now been identified and in several cases their function has been verified in animal models, providing a critical link between molecular function and animal physiology. This chapter will focus on two of the major protein elements that constitute the vasopressin-activated renal concentrating mechanism, the V2R and AQP2. Other critical channels and transporters that contribute to urinary concentration are dealt with elsewhere in this volume.

We will address functionally relevant properties of the V2R and AQP2 proteins that have emerged over the past few years, and we will update our understanding of signaling cascades, protein-protein interactions, membrane transport, intracellular trafficking, and synthesis and degradation pathways—areas that continue to evolve rapidly as the powerful new tools of genomics and proteomics are applied to renal physiology. However, in the face of an explosion of information related to the genetic and protein components that interact in these pathways, it becomes even more critical to integrate this information into whole organ and whole animal physiol-ogy in order to fulfill the promise of the emerging “Systems Biology” revolution as it applies to the renal concentrating mechanism.

VASOPRESSIN—THE ANTIDIURETIC HORMONE

Arginine vasopressin, a nine amino acid peptide, is the antidiuretic hormone of most mammals, although members of the pig family have a slightly different peptide known as lysine vasopressin (LVP) in which a lysine replaces the arginine in position 8 of the molecule. VP is synthesized in cells of the supraoptic and paraventricular nuclei of the hypothalamus, and the hormone is transported to nerve terminals in the posterior pituitary where it is stored in secretory granules. Secretion of VP is stimulated by a variety of factors, most notably an increase in plasma osmolality, but also by plasma volume.[1] A recent study indicates that vasopressin gene transcription is activated by decreased plasma volume, but not by increased plasma osmolality,[2] whereas another report shows increased VP heteronuclear RNA (hnRNA) levels in the hypothalamus after acute salt loading of rats.[3] The secretion of vasopressin in response to plasma osmolality is very sensitive, and a change in osmolality as small as 1% can cause a significant rise in plasma VP levels, which then activates regulatory systems necessary to retain water and restore osmolality to normal. Although the physiological response of VP to volume is less sensitive, with a 5% to 10% decrease in volume required to stimulate VP secretion, VP has important clinical applications in the control of vasodilatory shock. [4] [5] Finally, the usual mammalian form of VP, arginine VP (AVP), is an effective agonist for all vasopressin receptor isoforms—the V1a and V1b/V3 forms that are located mainly in blood vessels and hepatocytes,[6] and the pituitary, [7] [8] [9] respectively—as well as the V2R that is expressed in the kidney[10] and in some other tissues, including the inner ear. [11] [12] A modified form of AVP, known as desamino d-arginine[8]vasopressin (dDAVP), is specific for the V2R and has little or no V1-related pressor effect. It is, therefore, commonly used in studies (or in the clinical situation) when V2R activation is required in the absence of the V1 effect.

THE VASOPRESSIN V2 RECEPTOR (V2R)—A G-PROTEIN COUPLED RECEPTOR

The V2R is a 371 amino acid protein [10] [13]—a member of the family of seven membrane-spanning domain receptors that couple to heterotrimeric G-proteins.[14] In the kidney, it is expressed on the plasma membrane of collecting duct principal cells and epithelial cells of the thick ascending limb of Henle. The V2R is also expressed in the endolymphatic sac in the inner ear, [11] [12] [15] [16] and on endothelial cells in a variety of tissues, where it may be involved in a vasodilatory response that includes NO generation and Von Willebrand factor secretion. [17] [18] [19] The presence of vasopressin receptors on these cell types has been demonstrated by a variety of techniques, including functional and morphological assays of vasopressin action both in situ and in isolated tubule and cellular preparations from the kidney. [20] [21] [22] [23] [24] [25] Attempts to localize the V2R using specific antibodies have met with variable success, and some studies have even reported a significant apical staining for the V2R in renal tubules, in addition to the expected basolateral staining and staining inside the cell. [26] [27] Considerable use has also been made of cell culture systems, especially LLC-PK1 cells from porcine kidney, to evaluate ligand receptor interactions and signal transduction mechanisms via stimulatory and inhibitory heterotrimeric GTP-binding proteins.[28] A variety of transfected cell systems, both epithelial and non-epithelial, have proven valuable in elucidating several aspects of the V2R signaling cascade following ligand binding, as well as intracellular pathways of V2R recycling, down-regulation, and desensitization.

In target cells, the V2R is activated by the binding of its ligand, AVP, which stimulates adenylyl cyclase activity and increases cytosolic cAMP levels.[29] The increase in cAMP activates protein kinase A (PKA) and results in the PKA-mediated phosphorylation of several proteins. As will be discussed in more detail later, the vasopressin-sensitive water channel AQP2 is itself phosphorylated under these conditions and accumulates in the apical plasma membrane of collecting duct principal cells, thus increasing transepithelial water permeability and facilitating osmotically driven water reabsorption from the tubule lumen into the renal interstitium ( Fig. 8-1 ). In addition, intracellular calcium is also increased by VP via a mechanism involving interaction with calmodulin,[30] a phenomenon that is also involved in the regulated trafficking of AQP2. [31] [32]

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FIGURE 8-1  Overview of vasopressin-controlled short-term regulation of AQP2 trafficking in AQP2-containing collecting duct cell. Signaling cascades and molecular apparatus involved in vasopressin regulation of AQP2 trafficking are shown. A, Vasopressin binding to the G-protein-linked V2-receptor stimulates adenylyl cyclase leading to elevated cAMP levels and activation of protein kinase A. AQP2 is subsequently translocated to the apical plasma membrane. B, Role of AQP2-phosphorylation in AQP2 recruitment to the plasma-membrane. Protein kinase A phosphorylates AQP2-monomers and phosphorylation of at least three of four AQP2 monomers in an AQP2-tetramer is associated with translocation to the plasma membrane. It is currently unknown if dephosphorylation of AQP2 is necessary for endocytosis of AQP2. C, Overview of cytoskeletal elements, which may be involved in AQP2-trafficking. AQP2 containing vesicles may be transported along microtubules by dynein/dynactin. The cortical actin web may act as a barrier to fusion with the plasma-membrane. D, Changes in the actin cytoskeleton associated with AQP2-trafficking to the plasma membrane. Inactivation of RhoA by phosphorylation and increased formation of RhoARhoGDI complexes seem to control the dissociation of actin fibers seen after vasopressin stimulation. E, Intracellular calcium signaling and AQP2-trafficking. Increases in intracellular Ca2+concentration may arise from stimulation of the V2 receptor. The existence and potential role of other receptors and pathways affecting Ca2+ mobilization is still uncertain but may potentially include AT1 receptors. The downstream targets of the calcium signal are unknown. F, Vesicle targeting receptors and AQP2-trafficking. A number of vesicle targeting receptors, for example, SNARE-proteins have been localized to the AQP2-containing collecting duct cells and cultured cells. The exact role of these remains to be established. V2R, vasopressin-V2-receptors; AC, adenylyl cyclase, PKA, cAMP and protein kinase A (PKA).

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Structure of the V2R

Homologs of the V2R have been cloned from several mammalian species including human, pig, and rat, and the receptor sequences are more than 90% identical. The membrane topology of the receptor and several functionally important features are illustrated in Figure 8-2 . These include (1) an extracellular N-terminus with a consensus site for N-linked glycosylation (N22), (2) a cytoplasmic carboxy terminus and large intracellular loop that contain multiple sites for serine and threonine phosphorylation, and probably play a role in receptor desensitization, internalization, sequestration, and recycling, [33] [34] [36] [37] (3) conserved sites for fatty acylation (palmitoylation), which may serve as an additional membrane anchor in the C-terminal tail, and be involved in membrane accumulation or in endocytosis and MAPK signaling, [38] [39] (4) two highly conserved cysteine residues in the second and third extracellular loops, which may form a disulfide bridge that is important for correct folding of the molecule and stabilization of the ligand binding site, (5) hydrophobic residues at the C-terminus, including a dileucine motif, which are involved in ER to Golgi transfer, and in receptor folding that is required for receptor transport from the ER.[40]

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FIGURE 8-2  Membrane topology of the vasopressin receptor (V2R). The 371 amino acid protein has seven membrane spanning domains, an extracellular N-terminus, and a cytoplasmic C-terminus. Several features of the molecule are illustrated. Residue N22 is a putative N-glycosylation site; a functionally important disulfide bridge occurs between cysteines 112 and 192; the dileucine motif LL339–340 is an endocytotic signaling motif; C341/342 are sites of palmitoylation; phosphorylation sites (serine and threonine residues) between T347 and S364 play a critical role in V2R internalization and recycling. Potential sites of phosphorylation by GRK are indicated with asterisks. (Figure slightly modified from an original kindly provided by Dr. Daniel Bichet, University of Montreal.)

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Interaction of V2R with Heterotrimeric G-Proteins

Upon binding of VP, the V2R assumes an active configuration and promotes the disassembly of the bound heterotrimeric G-protein, Gs, into Ga and Gbg subunits.[29] GDP-GTP exchange occurs on the alpha subunit. This G-protein is located on the basolateral plasma membrane of TAL and principal cells. [41] [42] The activated Gsa then stimulates adenylate cyclase (AC), resulting in an increase in cAMP levels in the cell. In the rat kidney, several AC isoforms are expressed, but AC-6 is the predominant isoform in the adult rat kidney, and AC-4, -5, and -9 have lower expression levels.[43] The calmodulin-sensitive AC-3 is also expressed in the collecting duct, however, and the vasopressin-induced increase in cAMP and AQP2 trafficking in principal cells has been reported to be calmodulin-dependent. [31] [32] The liganded V2R interacts with Gs via its cytosolic domain, and the third intracellular loop of the V2R is involved in this interaction. [44] [45] A peptide corresponding to this loop inhibits V2R signaling though Gs when introduced into cells expressing the V2R.[46] Interestingly, this same peptide also reduces VP binding to the V2R by converting the receptor from a high to a low affinity state.

A complex cross-talk mechanism also results in activation of an inhibitory GTP-binding protein, which down-regulates the vasopressin response. [28] [29] Other factors involved in a blunting of the vasopressin response are receptor down-regulation and desensitization. This results at least in part from a decreased number of receptors at the cell surface as receptors are internalized via clathrin-coated pits. [47] [48] The level of V2R mRNA also decreases rapidly after an elevation of plasma AVP.[49] Many additional mechanisms that down-regulate the vasopressin response have been described. These include destruction of cAMP by cytosolic phosphodiesterases[50] and inhibition of the vasopressin response by prostaglandins,[51] dopamine, [52] [53] adenosine receptor stimulation,[54] adrenergic agonists, [55] [56] endothelin-1,[57] and bradykinin.[58]

The V2R Enters a Lysosomal Degradative Pathway after Internalization

G-protein coupled receptors (GPCR) are constitutively expressed on the plasma membrane and are down-regulated following ligand binding. Ligand-induced changes in receptor conformation are followed by receptor phosphorylation, desensitization, internalization, and sequestration. Phosphorylation triggers the binding of β-arrestin to the V2R. [33] [59] Arrestins uncouple GPCRs from heterotrimeric G-proteins, effectively producing a desensitized receptor.[60] Arrestin-receptor complexes are also capable of recruiting the clathrin adaptor protein AP-2, an important component of the endocytotic mechanism,[61] and the complex is then internalized via clathrin-mediated endocytosis. [47] [48] In most cases, hormone ligands dissociate from their receptors in acidic endosomes, and the receptors subsequently reappear at the cell surface in a process known as receptor recycling. However, different GPCRs recycle back to the cell surface at different rates. The β2-adrenergic receptor (b2AR) is a so-called “rapid recycler”, and pre-stimulation levels of the b2AR are restored on the cell surface within an hour of ligand-induced internalization.[33] In contrast, the same process requires several hours in the case of the V2R. [33] [34] [62]

An earlier study[35] showed that the vasopressin ligand is delivered to lysosomes after binding to the V2R, as are many other ligands that are internalized by receptor-mediated endocytosis, but the fate of the actual vasopressin receptor was not followed in this report. It is known that the V2R forms a stable complex with β-arrestin throughout the internalization pathway [33] [63] and this prolonged association of β-arrestin with the V2R could be responsible for the intracellular retention, but not the final destination of the receptor.[37]

Recent immunofluorescence, biochemical, and ligand binding data have now clearly shown that much of the V2R that is internalized after vasopressin addition to cells enters a lysosomal degradation compartment, and that re-establishment of baseline levels of vasopressin binding sites (V2R) at the cell surface requires de novo protein synthesis. [64] [65] Furthermore, VP stimulation leads to rapid, β-arrestin-dependent ubiquitination of the V2R and increased degradation.[66]

The process of internalization and delivery to lysosomes can be followed using transfected cells expressing the V2R coupled to green fluorescent protein (GFP). Real-time spinning disk confocal microscopy shows that after ligand binding, the V2R-GFP moves from a predominant plasma membrane location to a perinuclear vesicular compartment ( Fig. 8-3 ).[67] Colocalization of the V2R-GFP construct with Lysotracker, a marker of acidic late endosomes and lysosomes, after VP-induced internalization of V2R-GFP is shown in Figure 8-4 , indicating that the perinuclear vesicles are predominantly lysosomes.[64] Western blotting also reveals a time-dependent degradation of the V2R after internalization that is completely inhibited by chloroquine (a lysosome inhibitor) but not by lactacystin (a proteasome inhibitor). Furthermore, re-establishment of pre-stimulation levels of the V2R at the cell surface is significantly inhibited by cycloheximide, illustrating the requirement for new protein synthesis in this process.[64]

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FIGURE 8-3  Spinning disk confocal microscopy (live cell imaging) of LLC-PK1 cells stably expressing V2R-GFP seen at various times (0–90 min) after addition of the ligand, vasopressin (VP). Initially, most of the V2R-GFP is located on the plasma membrane (A). After VP treatment, the V2R-GFP is down-regulated from the cell surface and is progressively internalized (B, C) into a perinuclear compartment that is seen as a bright fluorescent patch (indicated with an arrow in each panel). The degree of internalization can be easily followed in the same cells using this technique. After VP treatment for 90 minutes, virtually no plasma membrane V2R-GFP is detectable—it is all concentrated in an area close to the nucleus (D).  (Figure adapted from a review by Brown D: Imaging protein trafficking. Nephron Exp Nephrol 103:e55–61, 2006.)

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FIGURE 8-4  In non-stimulated LLC-PK1 cells expressing V2R-GFP, the patterns of staining for Lysotracker (A)—a marker of acidic lysosomes and late endosomes—and the V2R-GFP (B) are distinct, with most of the V2R-GFP at the plasma membrane (although some intracellular V2R is also present). After VP-induced down-regulation, the V2R-GFP is internalized as shown in Fig. 8-3 , and accumulates in vesicles, many of which are also stained with Lysotracker (C, D). These data indicate that internalized V2R is mainly trafficked to lysosomes for degradation.  (Figure adapted from Bouley R, Lin HY, Raychowdhury MK, et al: Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: Involvement of a lysosomal degradation pathway. Am J Physiol Cell Physiol 288:C1390–1401, 2005.)

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In summary, the V2R—classified as a “slow-recycling” GPCR—appears to be mainly degraded in lysosomes after ligand-induced internalization. This pathway may have evolved to allow the V2R to function in the harsh environment of the renal medulla, which can be acidic and of high osmolality.[68] Normally, receptors and ligands dissociate in the acidic endosomal environment, but the V2R must actually associate with VP in the acidic renal medulla. Thus, the V2R-VP pair should be resistant to pH-induced dissociation, and the delivery of both the ligand and receptor to lysosomes may be required in order to terminate the physiological response to VP.

Diabetes Insipidus (Central and Nephrogenic)

Diabetes insipidus is the generic name for conditions affecting the VP, V2R, AQP2 axis that result in a failure to maximally concentrate the urine. Patients with this disease produce large amounts of dilute urine—up to 20 L per day in extreme cases. [69] [70] Clinically, this condition is recognizable soon after birth, and if not corrected can result in severe dehydration, hypernatremia, and damage to the central nervous system. The molecular basis for many of these related disorders has been examined and elucidated thanks to the cloning and sequencing of the key proteins involved, the V2R[13] and the vasopressin-sensitive collecting duct water channel, AQP2,[71] as well as the gene coding for the vasopressin/neurophysin/glycopeptide precursor protein from which active vasopressin is derived by further processing. [72] [73]

Congenital Central Diabetes Insipidus

The autosomal dominant form of familial neurohypophyseal (central) diabetes insipidus (adFNDI) has been linked to over 40 different mutations of the gene encoding the vasopressin-neurophysin II (AVP-NPII) precursor. Most of these mutations have been located in either the signal peptide or the neurophysin II moiety, but a mutation in the portion of the gene coding for the VP protein has also been identified.[74] A three-generation kindred with severe adFNDI was found to cosegregate with a novel missense mutation in the part of the AVP-NPII gene encoding the AVP moiety.[75] Normally, newly synthesized pre-pro-AVP-NPII is translocated into the endoplasmic reticulum, where the signal peptide is removed, enabling the prohormone to fold, form the appropriate intrachain disulfide bonds, and dimerize. This conformation permits the prohormone to move to the Golgi where it is packaged into neurosecretory granules, cleaved into its individual moieties (AVP, NPII, and copeptin), and transported along the axon to be stored in nerve terminals until release. Many studies over the past several years have documented that mutations leading to adFNDI result in impaired folding or dimerization of the mutant precursor (or both). This interferes with normal intracellular trafficking and process-ing of the prohormone through the regulated secretory pathway. [75] [76]

The congenital (or acquired) absence of a functional vasopressin hormone can be generally treated by administration of vasopressin or dDAVP, usually via nasal aerosol. In this form of the disease, the V2R and the AQP2 genes and proteins are unaffected, and VP administration leads to the re-establishment of urine concentration. [77] [78] [79] [80] An animal model of CDI, the Brattleboro rat, has proven to be an invaluable system in which many of the consequences of defective urinary concentration resulting from an absence of functional VP have been elucidated. [81] [82] A single base pair deletion in the neurophysin domain of the vasopressin gene was identified in these animals.[83] This frameshift mutation results in the loss of a stop codon and abnormal processing of the neurophysin/vasopressin/glycopeptide precursor, leading to a failure in the production, storage, and secretion of vasopressin.[72] The ability to convert these rats from non-concentrators to concentrators simply by administering exogenous VP has resulted in many important discoveries on the vasopressin signaling cascade that will be alluded to later.

Nephrogenic Diabetes Insipidus

Nephrogenic diabetes insipidus (NDI) on the other hand, results from a loss of an appropriate response of the kidneys to circulating vasopressin, and in most cases cannot be treated simply by administering vasopressin. As for central DI, NDI can be congenital/hereditary or acquired. [69] [77] [84] [85] CNDI was first described over 50 years ago[86] and genetic linkage studies in several families established that the predominant form is an X-linked trait. [87] [88]Acquired disease is more frequent than hereditary NDI, and of all causes, lithium-induced NDI is the most common. [84] [89] This will be addressed in more detail in a later section of this chapter, along with other acquired forms of the disease. Distinct forms of nephrogenic diabetes insipidus are produced by a variety of gene mutations that result in defective targeting and/or function of the V2R or the AQP2 water channel. [69] [85] [90] [91] [92] [93]

Type I CNDI (congenital nephrogenic DI), the more frequent X-linked form, is a recessive disease caused by mutations in the vasopressin receptor.[93] Type II CNDI is an autosomal recessive disease resulting from mutations in the AQP2 water channel [91] [92] [93] [94] although an autosomal dominant form of CNDI has also been described. [95] [96]

Mutations in the V2R

Close to 200 disease-causing mutations have been identified in the V2R[97] (see Fig. 8-2 ), many of which result in the production of a non-functional receptor by target cells in the kidney.[98] While this is predominantly an X-linked disease (the V2R gene is located on the X-chromosome), some very rare female cases have been described, which are believed to be associated with aberrant X-chromosome inactivation.[99] Some of the mutations result in the appearance of premature stop codons, others result in frame shifts that result in nonsense protein sequences, whereas others are single point mutations that cause an amino acid replacement at critical locations in the receptor. Mutations in the V2R sequence that allow the production of a full-length or near full-length protein could result in CNDI by interfering with different aspects of the receptor-ligand signal transduction cascade. For example (1) the receptor could be expressed normally at the cell surface, but not bind vasopressin, (2) the receptor could bind its ligand normally at the cell surface, but fail to couple to its stimulatory GTP-binding protein, so that adenylyl cyclase is not activated, (3) the mutated receptor may be incorrectly folded and might be retained for degradation in the rough endoplasmic reticulum (RER), and may never reach the cell surface—this is an example of a targeting mutation, (4) changes in the ability of the receptor to be phosphorylated may affect several aspects of function, including trafficking and desensitization. [91] [100] The R137H mutation produces NDI because it is constitutively desensitized via an arrestin-mediated mechanism—the mutant V2R is phosphorylated and sequestered in arrestin-containing vesicles even in the absence of agonist.[101] Interestingly, this mutation can result in either severe or mild NDI, indicating that genetic or environmental modifiers (or both) may affect the final phenotype within affected members of the same family.[102]

Some mutations in the V2R have been associated with specific functional defects that are believed to explain the loss of receptor function. For example, many of the missense mutations that have been described (R181C, G185C, and Y205C) result in the addition of cysteine residues in the extracellular loops, and this is believe to interfere with the disulfide bond formation that connects the first and second loops in the wild-type receptor. However, a newly discovered Y205H mutation also abolishes receptor function and leads to NDI, suggesting that loss of the tyrosine is the cause of dysfunction, rather than the addition of a cysteine at least at residue 205.[103]

Many mutations cause folding defects that are recognized by cellular quality control mechanisms, leading to retention and degradation in intracellular compartments. However, different mutations are handled in different ways, and some temporarily escape from the ER before being rerouted back to this compartment for degradation. Cell transfection experiments have shown that whereas the L62P, DeltaL62-R64, and S167L mutants are trapped in the ER, the R143P, Y205C, InsQ292, V226E, and R337X mutant receptors actually reach the ER/Golgi intermediate compartment (ERGIC) before being rerouted to the ER. Differences in the folding characteristics of these receptors that allow interactions with different sets of accessory proteins are thought to explain these differences.[94]

Correcting the Defect: Approaches to Nephrogenic Diabetes Insipidus Therapy Involving the V2R

Several approaches have been considered as potential therapeutic strategies in X-linked forms of NDI that involve V2R mutations. These have developed from our increased understanding of the cell biology and signaling pathways that are involved in the response to VP. If the mutated V2R is mistrafficked in cells but is otherwise functional, then persuading the mutant receptor to move to the cell surface would be therapeutically beneficial. This strategy is also being explored for other diseases of protein trafficking, including cystic fibrosis, in which a single point mutation prevents efficient delivery of the CFTR protein to the cell surface. Instead, it is retained intracellularly and degraded. A variety of approaches including the use of chemical-induced or drug-induced rescue of cell surface expression have been attempted. Among the first chemical chaperones to be tested for the V2R were substances such as glycerol and dimethylsulfoxide. Additional reagents (thapsigargin/curcumin and ionomycin) that modify calcium levels in cellular compartments were also tested.[105] However, their reported efficacy in partially restoring transport activity to cells and tissues expressing the DF508 CFTR mutation that leads to cystic fibrosis [106] [107] has subsequently been contested in an independent study.[108] Furthermore, of 9 V2R mutants tested, the surface expression of only one of them—V2R-V206D—was increased using these reagents.[105]

However, the use of V2R antagonists to increase cell surface expression and functionality of mutant V2R protein seems more promising.[109] Small, cell permeant non-peptidic antagonists were shown to rescue the cell surface appearance of 8 mutant receptors that were tested, whereas a non-permeable antagonist had no positive effect. [110] [111] Importantly, the antagonist SR49059, which was shown to be effective in three patients harboring the R137H V2R mutation, acts by improving the maturation and cell surface targeting of the mutant receptor.[112] Furthermore, the pharmacological V2R antagonist SR121463B resulted in greater maturation and surface expression of the V2R mutations V206D and S167T than chemical chaperones.[105]

Finally, aminoglycoside antibiotics are known to suppress premature stop codons in some cases. In the case of the V2R, an E242X mutation produces a premature stop codon in humans, and when introduced into mice, this mutation causes NDI. However, urine concentrating ability can be restored by administering the antibiotic Geneticin (G418) to mice, and the AVP-mediated cAMP response is increased by G418 in cultured cells expressing this V2R mutation.[113] This provides a potential means of suppressing NDI that is caused by a premature stop codon in the V2R.

THE AQUAPORINS—A FAMILY OF WATER CHANNEL PROTEINS

The first water channel (aquaporin) was identified and characterized as a Nobel Prize winning discovery by Peter Agre and his associates in 1988.[114] This protein—originally known as CHIP28—is now known as aquaporin 1 (AQP1). [115] [116] [117] Functional studies in Xenopus oocytes (injected with AQP1 mRNA) and liposomes (reconstituted with purified AQP1 protein) [117] [118] [119] confirmed its role as the long-sought erythrocyte transmembrane water channel protein whose existence had been proposed for many years prior to its ultimate identification. The AQP1 protein is homologous to the lens fiber channel-forming protein MIP26 (Major Intrinsic Protein of 26 kDa, now renamed AQP0), which was cloned several years earlier[120] but whose physiological function was a matter of speculation. AQP1 is expressed in many cells and tissues with high constitutive water permeability, in addition to erythrocytes, including proximal tubules and thin descending limbs of Henle in the kidney, [121] [122] the choroid plexus,[123] reabsorptive portions of the male reproductive tract that are embryologically related to renal tubules,[124]parts of the inner ear,[125] and many others. [126] [127]

AQP2, the collecting duct vasopressin-sensitive water channel was then discovered by homology cloning from the renal medulla,[71] and a variety of studies that are described in more detail later confirmed that it is the principal cell water channel that is involved in distal urinary concentration ion the kidney. [128] [129] [130] [131] [132] Other aquaporins were subsequently discovered in rapid succession, and at the time of writing,12 mammalian homologs are known. Although aquaporins show considerable homology among different mammalian species,[133] homology among the different aquaporins from the same species may be as little as 35%. Aquaporins have also been found in virtually all species examined, including bacteria and plants. The membrane topography and some key features (e.g., phosphorylation sites) of the aquaporins (AQP2) are illustrated in Figure 8-5 .

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FIGURE 8-5  Membrane topology of the aquaporin 2 (AQP2) water channel. This 271 amino acid protein spans the lipid bilayer six times. Both N- and C-termini are in the cytoplasm. Phosphorylation sites for PKC, PKA, and casein kinase II (CKII) are shown.

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Other Permeability Properties of Aquaporins

Aquaporins were so named because they function as transmembrane water channels. However, the single channel water permeability of different aquaporins varies greatly. AQP1, AQP2, and AQP4 have high permeabilities whereas and AQP0 and AQP3 have much lower permeabilities.[134] Furthermore, some aquaporins including AQP3, AQP8, and AQP9 also allow the passage of other molecules, including urea, glycerol, ammonia, and other small solutes.[134] [135] [136] [137] [138] [139] [140] [141] Based on such properties and phylogenetic considerations, aquaporins were divided into one of two groups, the “orthodox set” (aquaporins) and the “cocktail set” (aquaglyceroporins).[142]Distinct physiological functions for aquaporins in the transport of non-water molecules, including glycerol, are beginning to emerge.[143]

Remarkably, water channels do not allow the passage of protons, a property that was first shown using isolated apical endosomes from rat kidney papilla. [144] [145] Crystallographic evidence has provided a structural explanation for their ability to prevent proton conductance.[146] This and other structural features of the aquaporins that contribute to their remarkable specificity will be described in more detail later. However, some results in oocytes and liposomes indicate that AQP1 serves as a CO2 channel, [147] [148] [149] [150] (reviewed in Ref 151) whereas whole animal studies using AQP1-deficient mice have refuted this claim.[152] Other groups examining the potential role of erythrocyte AQP1 in CO2 transport have produced data in favor[147] or against [153] [154] a role of AQP1 in this process. Recent developments have not reached a final consensus, and the role of AQP1 in transmembrane CO2 permeability in mammalian cells remains controversial.[155] Support of this idea, however, comes from plant systems in which aquaporin-mediated CO2 permeability is reported to be an important step in the photosynthetic process. [156] [157] [158]

Although some aquaporins including AQP1[159] and AQP8 have been reported to allow passage of ammonia in some expression systems, including yeast and oocytes, [135] [138] the physiological relevance of this has been questioned in AQP8 knockout mice.[160] AQPs 7 and 9 have both been implicated in arsenite transport in mammalian cells.[161]

Aquaporin 2: The Vasopressin-Sensitive, Collecting Duct Water Channel

Many studies have localized aquaporin mRNA or protein (or both) in a wide range of cell types, and aquaporins have been attributed a wide range of functions in the normal physiology of many tissues and organ systems. This section will focus on aquaporin 2 (AQP2), which was identified as the VP-regulated water channel in kidney collecting duct principal cells.[71] VP stimulation of the kidney collecting duct results in the accumulation of AQP2 on the plasma membrane of principal cells via a membrane trafficking mechanism that involves the recycling of AQP2 between intracellular vesicles and the cell surface ( Fig. 8-6 ). [94] [128] [129] [130] [134] [162] [163] However, it should be mentioned that AQP3, present in the basolateral membrane of principal cells,[137] is also regulated at the expression level by vasopressin or dehydration (or both),[164] although no evidence for an acute regulation of this basolateral channel has been forthcoming. Hormonal (VP) stimulation of the collecting duct epithelium increases its plasma membrane water permeability, which in turn allows the luminal fluid to equilibrate osmotically with the surrounding interstitium. The osmolality in the renal inner medulla reaches about 1200 mOsm/kg in humans, and thus the urine can reach the same concentration in the presence of vasopressin. The mechanism by which the apical plasma membrane of collecting duct principal cells shifts from a low-to-high permeability state upon vasopressin action is the subject of much of the remainder of this chapter, and involves the redistribution of AQP2 from cytoplasmic vesicles to the plasma membrane under the influence of a signaling cascade that is triggered by the binding of VP to the V2R in these cells.

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FIGURE 8-6  Increased plasma membrane expression of AQP2 in principal cells of Sprague Dawley kidney perfused in the presence of 5 μM mbCD (an endocytosis inhibitor) or 4 nm dDAVP for 60 minutes. Kidneys were then fixed, sectioned, and immunostained using anti-AQP2 antibodies. Examples of tubules sectioned transversely from the inner stripe of the outer medulla (A, C, E) and longitudinally in the inner medulla/papilla (B, D, F) are illustrated. Under control conditions (A, B), AQP2 has a cytosolic distribution in principal cells. After perfusion with 5 μM mbCD (C, D), AQP2 shows an increased apical localization in principal cells of the inner stripe and inner medulla. After perfusion with 4 nm dDAVP, a similar and expected increased apical localization of AQP2 in medullary collecting ducts is seen in the isolated perfused kidney preparation. dDAVP also induced a basolateral localization of AQP2 in the inner medulla (F and arrows, inset) but not in the inner stripe (E) principal cells. Bar = 40 mm.

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Although AQP2 was discovered in the kidney, it is also expressed in a limited number of non-renal epithelia. These are the vas deferens of the male reproductive tract, [165] [166] the inner ear in which its expression is regulated by vasopressin, [15] [16] and the colon.[167] Interestingly, AQP2 in the vas deferens is inserted into the apical plasma membrane in a non-regulated, constitutive pathway, implying that the same aquaporin can be regulated in different ways depending on the cell type in which it is expressed.[166]

Intramembranous Particle Aggregates—An Early Morphological Hallmark of Membrane Water Permeability

The “cell biological” era of vasopressin action can be considered to have begun in 1974, when Chevalier and colleagues described an alteration in the appearance of frog urinary bladder plasma membranes in parallel with an increase in epithelial water permeability induced by another neurohypophyseal hormone, oxytocin.[168] Freeze-fracture electron microscopy revealed numerous small aggregates of intramembranous particles (IMPs), which represent integral membrane proteins, on the apical plasma membranes of frog bladder epithelial cells under these conditions. The correlation between this membrane structural change and hormonally induced transepithelial water flow was strengthened by a large body of subsequent work from several groups. [169] [170] [171] [172] [173] The data suggested that the IMP aggregates were water-permeable patches, and that the individual IMPs in each aggregate were the morphological correlate of a putative (but not yet identified at that time) water channel protein that spanned the lipid bilayer.

In support of this idea, apical plasma membrane IMP aggregates or IMP clusters were induced by VP administration in both amphibian epidermis and the mammalian collecting duct. [174] [175] IMP clusters were not present in mice with hereditary diabetes insipidus that cannot concentrate their urine.[176] In the amphibian epidermis, both ADH and isoproterenol stimulate water flow, and both caused IMP aggregates to appear.[177] In all three target epithelia (amphibian urinary bladder and skin, and mammalian collecting duct), there was a dose response relationship between the number of IMP aggregates in the membrane, and the magnitude of the water permeability response.

AQP4 Splice Variants and Orthogonal Arrays of Intramembranous Particles

The IMP aggregates seen by freeze-fracture EM in toad epidermis are virtually identical to the characteristic orthogonal arrays of IMPs (OAPs) that have now been identified as AQP4. [178] [179] [180] These AQP4 arrays are present on the basolateral plasma membrane of collecting duct principal cells where AQP4 is located, [181] [182] [183] as well as on other cell types that express AQP4 including gastric parietal cells [184] [185] and astroglial cells.[178] AQP4 is an unusual water channel in that two splice variants are expressed—known as M1 and M23—due to the presence of alternative transcription initiation sites in the AQP4 gene. [186] [187] Two recent reports have shown that M23 is more abundant in most cells, and that this variant arranges into typical orthogonal arrays when expressed in cultured cells. [188] [189] M1 expression does not result in orthogonal arrays but interestingly, M1 co-expression in cells along with M23 actually is disruptive to OAP formation. Furthermore, the single channel water permeability of M23 when arranged into OAPs is significantly greater than that of M1, which does not form OAPs. These data raise the intriguing possibility that OAP formation enhances membrane water permeability due to AQP4, and that M1 expression and incorporation into the membrane disrupts OAPs and may decrease membrane water permeability. Interestingly, the water permeability of AQP4 expressed in LLC-PK1 cells and oocytes was reported to be gated (decreased) by PKC phosphorylation of residue S180, without any apparent change in membrane distribution of this protein, [190] [191] but the expression of OAPs in these cells was not examined. Thus, it is possible that the basolateral membrane permeability of collecting duct principal cells may be modulated by AQP4 phosphorylation. Indeed there is one report showing that the basolateral membrane permeability of collecting ducts from the outer and inner medulla increases after dehydration and/or vasopressin (desmopressin) action, but the aquaporin responsible for this was not clearly identified.[192] However, it is unlikely to be AQP4 because the cell swelling effect was abolished by mercuric chloride, and AQP4 is known to be insensitive to this inhibitor.[193] Another basolateral aquaporin, AQP3, is up-regulated at the transcriptional level by VP[164] and could be responsible for the increased basolateral permeability. Furthermore, AQP2 can also be inserted into basolateral membranes of principal cells under some conditions, and thereby increase the permeability of this membrane domain. [194] [195] [196]

Aquaporin 2 Recycling: The “Shuttle Hypothesis” of Vasopressin Action

Based on many studies using vasopressin-sensitive amphibian epithelia, it was proposed that “water channels” are located on intracellular vesicles that fuse with the apical plasma membrane upon vasopressin stimulation, and then are retrieved back into the cell by endocytosis after vasopressin washout. The internalized water channels can then be re-inserted back into the plasma membrane upon subsequent re-stimulation by vasopressin. This so-called “shuttle hypothesis” of vasopressin action, proposed by Wade and associates in 1981,[173] was an elegant idea that has guided studies on the cell biology of vasopressin action for the past two decades. Developments in aquaporin cloning and expression in various cell systems, as well as in vivo studies, over the past decade have allowed a direct examination of many of the subcellular mechanisms underlying vasopressin regulation of collecting duct water permeability. The basic principles of the shuttle hypothesis (i.e., that water channels [AQP2] recycle between an intracellular vesicle pool and the plasma membrane [ Fig. 8-1 ]) have withstood the test of time, but the details of this process remain to be fully elucidated at the cellular and mechanistic level. The following sections will provide an update on specific parts of the recycling itinerary of AQP2, and will outline the current state of our understanding of the regulation of the complex pathways that lead to a VP-induced increase in collecting duct water permeability.

Vasopressin-Regulated Trafficking of AQP2 in Collecting Duct Principal Cells

AQP2 was first identified by Fushimi and colleagues and sequencing of this protein from rat allowed several groups to develop antibodies against AQP2. [71] [195] [196] These reagents were used to show that AQP2 is abundantly expressed in the apical plasma membrane of collecting duct principal cells, as well as in numerous intracellular vesicles.[195] This distribution was consistent with the shuttle hypothesis of VP action (see Fig. 8-1 ). The onset and offset phase of VP action were then examined in vitro and in vivo, and the immunocytochemical data showed clearly that VP induced a striking and reversible redistribution of AQP2 from intracellular vesicles to the apical plasma membrane of principal cells (see Fig. 8-6 ). [197] [198] [199] [200] Rapid internalization of AQP2 was induced by VP washout in isolated perfused collecting ducts, and in whole animals infused with a V2R antagonist or water loading.[201] [202] [203] These studies, taken together, provided strong evidence that vasopressin acutely regulates the osmotic water permeability of collecting duct principal cells by inducing exocytosis of AQP2 from intracellular vesicles to the apical plasma membrane, and that AQP2 removal from the membrane by endocytosis restores the low baseline water permeability of the apical plasma membrane of principal cells.

The internalized AQP2 that accumulates in endosomes after VP withdrawal follows a complex intracellular pathway prior to re-insertion into the plasma membrane. Studies in cells stably transfected with AQP2 have shown that recycling of AQP2 can occur in conditions in which protein synthesis is inhibited (see later), indicating that de novo protein synthesis is not required for this process to occur.[204] However, not all AQP2 is recycled. There is significant accumulation of AQP2 in larger cellular structures including multivesicular endosomes (MVEs) in response to treatment of rats with a VP antagonist.[201] Late endosomes often have the appearance of MVBs, and proteins in this compartment could be moved to lysosomes for degradation, be transferred to a recycling compartment via vesicular carriers that bud from the MVB, or be directly transported to the cell surface via other distinct transport vesicles that derive from the MVBs. It has been shown that AQP2 is “secreted” into the tubule lumen, where it can be found partially associated with small vesicles called exosomes in the urine. [205] [206] The amount of AQP2 in the urine increases in conditions of antidiuresis when more AQP2 is present in the apical membrane of principal cells. The physiological relevance of this urinary excretion of AQP2 is unknown but interestingly, urinary AQP2 correlates with the severity of nocturnal enuresis in children, and lowering urinary calcium levels (by low calcium diet) has a beneficial effect in reducing the severity of the enuresis and reducing AQP2 secretion in hypercalcemic children treated with dDAVP.[207]

Reconstitution of Aquaporin Expression in Non-Polarized Cells

Expression of Aquaporins in Xenopus Oocytes

Xenopus oocytes were the first expression system that was used to demonstrate that aquaporin 1 (CHIP28) was a functional transmembrane water channel.[117] This system has been used in many subsequent studies to assess the water (and solute) permeability of virtually all mammalian aquaporins. Membrane permeability resulting from injection of an appropriate mRNA is measured by computer-assisted analysis of oocyte swelling in response to a hypotonic buffer, as initially described by Verkman and colleagues.[208] This system has also proven useful in assessing the function of mutated aquaporins including those that are known to cause NDI in humans, posttranslational modifications (phosphorylation, glycosylation), as well as potential modifiers of aquaporin permeability by co-expressing proteins such as CFTR.[209] Many of the mutant AQP2 proteins are not expressed at the cell surface of oocytes, probably due to folding defects that cause retention and ultimate degradation in the rough endoplasmic reticulum or retention (or both) in the Golgi apparatus. [96] [210] [211] Oocytes have also been useful for dissection of the role of aquaporin oligomerization in cell surface expression.[212]

Expression of Aquaporins in Non-Epithelial Cells

Expression systems such as CHO cells have been extremely useful for morphological and functional studies on the different aquaporins. However, by definition, they cannot be used for studies on factors that regulate the polarized expression of aquaporins in renal epithelia. Freeze-fracture studies on transfected CHO cells revealed that the AQP1 protein assembles as a tetramer in the lipid bilayer, [213] [214] a result in agreement with biochemical cross-linking data[215] and with data from cryo- and atomic force microscopy of 2D crystals of AQP1. [216] [217] [218] Transfection of CHO cells with AQP4 cDNA showed that this protein forms a characteristic pattern of orthogonal IMP arrays (OAPs) that are found in several cell types, including collecting duct principal cells (on the basolateral plasma membrane). [180] [182] A comparison of membrane IMP organization in CHO cells expressing AQPs 1-5 showed that only AQP4 forms OAPs, that AQP2 does not spontaneously form IMP aggregates, and that AQP3 has a limited tendency to form small, densely packed clusters of IMPs.[219]

When various AQP2 mutations were expressed in CHO cells, important information was gathered concerning the abnormal intracellular location and the defective functional activity of these proteins.[210] CHO cells were also used to demonstrate that chemical chaperones could increase the delivery of misfolded AQP2 protein to the cell surface, a potentially important observation in terms of managing autosomal CNDI. [220] [221]

Expression of AQP2 in Polarized Epithelial Cells

Transfected Cells Expressing Exogenous AQP2

Early observations revealed that renal epithelial cell lines that are commonly used for cell biological studies (LLC-PK1, MDCK, OMCD, IMCD, OK) showed little or no endogenous expression of AQP2. Cultures of cells from the inner medullary collecting duct (IMCD) showed a progressive loss of AQP2 mRNA expression over the first 4 days of culture.[222] Transcription of the AQP2 gene appears to be rapidly inactivated in these cells cultures and was shown, at least in part, to be mediated by repressors present in its 5′-flanking region.[222] Several laboratories, therefore, developed stably transfected cells and used them to dissect intracellular processes related to AQP2 trafficking and V2R signaling. cAMP-dependent translocation of AQP2 was first reconstituted in LLC-PK1 cells ( Fig. 8-7 ),[223] and subsequently in transfected rabbit collecting duct epithelial cells,[224] MDCK cells,[225] and primary cultures of inner medullary collecting duct cells.[226] Two lines of stably transfected LLC-PK1 and MDCK renal epithelial cells were produced that retained constitutive (AQP1) and vasopressin-regulated (AQP2) membrane localization of these aquaporins. [204] [223] [225] [227] AQP1-transfected LLC-PK1 and MDCK cells showed constitutive plasma membrane expression of the protein, whereas AQP2-transfected LLC-PK1 cells had a baseline intracellular vesicular labeling that relocated to the plasma membrane only after increasing cytosolic cAMP levels with forskolin or vasopressin stimulation (see Fig. 8-7 ). Interestingly, transfected MDCK cells also show constitutive membrane AQP2 expression under baseline conditions unless they are pre-treated with indomethacin, which is presumed to reduce cAMP levels and induce AQP2 internalization in these cells.[225] After indomethacin treatment, AQP2 can then be returned to the cell surface by vasopressin/forskolin exposure of the cells. Functional studies showed that AQP1-transfected cells had a high constitutive water permeability, whereas AQP2- transfected cells acquired the same degree of permeability only after stimulation.[223] Similar data were obtained using transformed rabbit collecting duct epithelial cells.[224]

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FIGURE 8-7  Immunofluorescence staining for AQP2 in LLC-PK1 cells expressing wild-type AQP2 (A-C) or a mutant in which the S256 residue has been replaced by alanine (S256A) (D). Under baseline conditions, both wild-type (A) and the S256A mutation (not shown) are mainly located on intracellular vesicles, with very little plasma membrane staining. After vasopressin (VP) treatment, the wild-type AQP2 relocates to the plasma membrane (B), whereas the S256A mutation remains on intracellular vesicles (not shown). However, when endocytosis is inhibited in these cells by application of the cholesterol-depleting drug methyl-b-cyclodextrin (MBCD), both wild-type and S256A AQP2 accumulate at the cell surface (C, D). This result shows that both wt AQP2 and S256A AQP2 are constitutively recycling between intracellular vesicles and the plasma membrane, and that inhibiting endocytosis (using MBCD) is sufficient to cause membrane accumulation, even in the absence of S256 phosphorylation of AQP2.

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Tissue slices [228] [229] and isolated papillary collecting ducts[230] have also been used to dissect vasopressin and forskolin-stimulated AQP2 trafficking events. These systems more closely mimic the in vivo situation than pure cell culture models, and may be very useful to elucidate many of the signaling cascades that are involved in regulating AQP2 trafficking or that are involved in modulating the vasopressin response.

Cells Expressing Endogenous AQP2

Endogenous expression of AQP2 has been reported in a collecting duct cell line known as mpkCCD(cl4). [231] [232] These cells have the advantage that factors regulating AQP2 expression at the transcriptional levels can be addressed because endogenous flanking regions that contain promoter, repressor, and enhancer elements are presumably present. The involvement of the tonicity-responsive enhancer binding protein (TonEBP) in regulating AQP2 gene transcription in response to hypertonicity was demonstrated using mpkCCD(cl4) cells.[233] These cells have not yet been used extensively to address questions related to VP-induced AQP2 trafficking, however.

Expression of Multiple Basolateral Aquaporins (AQP2, AQP3, and/or AQP4) in Principal Cells

Although VP regulates collecting duct water permeability by modulating the amount of AQP2 in the apical plasma membrane of principal cells, AQP2 is also localized in the basolateral plasma membrane of these cells in some regions of the collecting duct. The bipolar expression of AQP2 is most evident in the inner medulla (see Fig. 8-6F ) and the cortical connecting segment. [195] [198] [234] [235] In the inner medulla, basolateral expression of AQP2 is increased by VP and oxytocin. [194] [196] Intriguingly, the basolateral membrane of principal cells contains two other aquaporins—AQP3 and AQP4—although their relative abundance varies in different regions of the collecting duct, with AQP3 expression being predominant in the cortex and decreasing toward the inner medulla, with the reverse pattern for AQP4, which is most abundant in the inner medulla. [164] [183] No AQP4 expression could be detected in the connecting segment.[236] In view of this apparent redundancy of basolateral aquaporin expression in some principal cells, the physiological role of basolateral AQP2 is unclear. Whether all three of these aquaporins are ever co-expressed in the same basolateral membrane has not been definitively examined, but clearly the following AQP pairs can be present in the basolateral membrane of the same principal cell: AQP2/AQP3 (connecting segments), AQP2/AQP4 (inner medulla), and AQP3/AQP4 (outer medulla). Although AQP2 and AQP3 have similar water permeabilities, AQP3 may also function as a solute channel under normal circumstances. However, AQP3 knockout mice have a severe concentrating defect, indicating an important role in the urinary concentrating mechanism.[237] In addition, AQP3 message and protein are both up-regulated by VP,[164] although trafficking to the membrane appears not to be acutely regulated by VP.

AQP4 on the other hand has a much greater single channel water permeability than AQP2 and AQP3; this may be due to the arrangement of the M23 AQP4 variant into tightly packed OAPs within the plasma membrane.[189]Interestingly, the involvement of AQP4 in urine concentration is less clear cut than that of AQP2 and AQP3. First, AQP4 knockout mice have only a minor concentrating defect that becomes detectable only after water deprivation,[238] although the water permeability of isolated collecting ducts from these animals is reduced to about 25% of that measured in wild-type tubules.[239] Second, kangaroo rats that can concentrate their urine to more than 5000 mOsm/kg do not express AQP4 in any cell type in their kidneys, indicating that AQP4 is not necessary for the extreme concentrating ability of these rodents.[240] Indeed, the complete absence of AQP4 from kangaroo rat kidneys suggests that expression of this channel might even be detrimental in some way to maximal urinary concentration.

As mentioned earlier, the amount of AQP2 at the basolateral membrane of principal cells in some collecting duct regions appears to be regulated. Both VP and oxytocin have been reported to cause basolateral AQP2 insertion in the inner medullary collecting duct, [194] [195] and one report has shown that basolateral membrane water permeability in this region is increased after VP treatment in a mercurial-sensitive manner, ruling out the contribution of the mercurial-insensitive AQP4 to this process.[192] Recent data indicate that interstitial osmolality may be at least partially responsible for the basolateral targeting of AQP2 in the inner medulla and in MDCK cells.[196] However, hypertonicity cannot be the only factor involved in this change in polarity of AQP2 inser-tion because cortical connecting segments in an isotonic environment also show an abundant basolateral insertion of AQP2. [234] [235]Furthermore, a recent study found basolateral AQP2 in IMCD cells in vasopressin-deficient Brattleboro rats in vivo.[234] The physiological role of basolateral AQP2 and the signaling events that lead to basolateral delivery are the subject of ongoing research in several laboratories.

The apical to basolateral distribution of AQP2 in connecting segments and cortical collecting ducts can also be modified by aldosterone in rats with two types of diabetes insipidus. In lithium-treated rats, aldosterone treatment increases urine output even more than lithium alone, and causes a significant redistribution of AQP2 to the basolateral plasma membrane in these cortical segments.[241] A similar effect is seen in aldosterone treated Brattleboro rats that lack endogenous vasopressin. Although the mechanism for this profound effect on AQP2 polarity is unknown, it is clearly independent of VP action. However, in humans, a frameshift mutation in AQP2 that results in basolateral targeting when expressed in polarized MDCK cells causes NDI.[242] This shows that, as expected, increased basolateral expression of AQP2 is not sufficient to increase transepithelial water permeability in the collecting duct. Whether basolateral AQP2 represents a mechanism to further increase the water permeability of the basolateral membrane under some conditions (despite the presence of other water channels in the same membrane), or whether it represents a transient step in an indirect apical targeting pathway for the AQP2 protein remains uncertain.

Apical and Basolateral Expression of AQP2 in Cell Cultures

In the original study describing trafficking of AQP2 expressed exogenously in transfected cells,[223] AQP2 was inserted into the basolateral plasma membrane of LLC-PK1 cells after vasopressin stimulation (see Fig. 8-7 ). In MDCK and rabbit collecting duct cells, a predominant apical insertion of exogenous AQP2 was described. [224] [225] However, in primary cultures of IMCD cells, AQP2 is inserted both apically and basolaterally, but the predominant pattern in vitro reflects basolateral insertion. [31] [226] This pattern is reminiscent of the basolateral expression of AQP2 that is seen in IMCD cells in the renal inner medulla in situ.[195] Thus, regulated trafficking of AQP2 occurs in a variety of transfected cell lines, and these unique targeting properties can be used to examine how polarity signals on proteins are interpreted by different cell types, and how they are translated by the intracellular transport machinery.

Studies on transfected epithelial cells have also shown that motifs in the sixth transmembrane domain of AQP2, including a dileucine motif, are involved in regulated trafficking of this water channel.[243] Domain-swap experiments however, show that while the cytoplasmic C-terminus of AQP2 is necessary for regulated insertion of AQP2, it is not sufficient, implying that other domains of the protein play a role in this process.[244] One study has identified an AQP2 mutation causing NDI in humans that adds a C-terminal extension containing both a tyrosine- and a leucine-based basolateral targeting motif to the AQP2 protein.[242] Presumably, if active in humans, this would lead to basolateral insertion of AQP2 in the collecting duct and would prevent the VP-induced increase in epithelial permeability.

INTRACELLULAR PATHWAYS OF AQP2 TRAFFICKING

As discussed earlier, early studies using model amphibian epithelia led to the shuttle hypothesis of water channel trafficking, according to which water channels were stored in intracellular vesicles before insertion into the apical plasma membrane following VP stimulation of target cells. Thus, the water channels were said to be part of a “regulated” membrane recycling pathway. More recent data have shown that in fact, AQP2 is recycled continually between intracellular vesicles and the cell surface. This section will describe the known pathways that AQP2 passes through during its recycling itinerary. The potential mechanisms by which this pathway is regulated will be discussed in a later section.

Role of Clathrin-Coated Pits in Water Channel Recycling

Clathrin-coated pits concentrate and internalize selected populations of many plasma membrane proteins, including receptors (with or without their cognate ligands), transporters, and channels. The role of clathrin-coated pits in V2R internalization has been shown previously. [47] [63] Based on morphological studies on collecting duct principal cells in situ, it was proposed that coated pits were also involved in the endocytotic step of water channel recycling long before aquaporins were identified.[245] IMP clusters believed to represent water channels were shown to correspond to sites of clathrin-coated pit formation at the cell surface.[245] Studies using horseradish peroxidase to follow apical membrane endocytosis during vasopressin stimulation of principal cells supported a role of clathrin-mediated endocytosis in water channel retrieval from the plasma membrane, and the rate of endocytosis was increased by VP washout to terminate the permeability response. [246] [247]

These early studies were confirmed by direct visualization of AQP2 in clathrin-coated pits by immunogold electron microscopy ( Fig. 8-8 ).[250] A relationship between IMP clusters first described more than two decades earlier [174] [175] and AQP2 endocytosis was shown using a technique known as fracture-labeling. In a transfected cell culture system (LLC-PK1 cells), AQP2 is concentrated in these coated-pit related IMP clusters after stimulation of the cells with forskolin followed by a 10-minute washout period (see Fig. 8-8 ).[250] Thus, IMP clusters in principal cells are markers of endocytotic, but probably not exocytotic events, and may result from a concentration of AQP2 protein into clathrin-coated membrane domains during the internalization phase of the vasopressin-induced recycling process. Whether exocytosis of AQP2 from intracellular stores results in the immediate formation of detectable membrane IMP clusters (representing patches of AQP2) at the sites of vesicle fusion with the plasma membrane remains uncertain.

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FIGURE 8-8  Aquaporin 2 is internalized by clathrin-coated pits. A, immunogold labeling of AQP2 in clathrin-coated pit (arrow) at the apical plasma membrane of collecting duct principal cells. An antibody against an external epitope of AQP2 was used. Panels B and C show label-fracture images of LLC-PK1 cells expressing AQP2. Immunogold label for AQP2 is located in IMP clusters on the membrane (B, arrows) and is associated with membrane invaginations that resemble clathrin-coated pits (C, arrows). Bars = 0.25 mm.

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Finally, when clathrin-mediated endocytosis was inhibited by the expression of a dominant negative form of the protein dynamin in LLC-PK1 cells, AQP2 accumulated on the plasma membrane and was depleted from cytoplasmic vesicles.[250] Dynamin is a GTPase that is involved in the formation and pinching off of clathrin-coated pits to form clathrin-coated vesicles. [248] [249] The dominant negative form has a single point mutation K44A that renders the protein GTPase-deficient, and arrests clathrin-mediated endocytosis.

AQP2 Localization in Intracellular Compartments during Recycling

Observations on the complex recycling pathways followed by AQP2 have been greatly facilitated by studies on transfected cells. Recycling of the AQP2 protein was directly demonstrated in cycloheximide-treated, AQP2-transfected LLC-PK1 cells, in which several rounds of exo- and endocytosis of AQP2 could be followed despite the complete inhibition of de novo AQP2 synthesis.[204] Several studies have been carried out to identify the intracellular compartments in which AQP2 resides during this recycling process. After internalization from the plasma membrane via clathrin-coated pits, AQP2 enters an early endosomal compartment that can be identified using antibodies against EEA1 (early endosomal antigen 1).[252] However, in collecting duct principal cells in situ, the endosomes that are formed during water channel recycling are highly specialized because they are non-acidic, and lack important functional subunits of the vacuolar H+ATPase. [144] [145] It has been reported that after forskolin washout from transfected MDCK cells, AQP2 enters an apical storage compartment that is sensitive to wortmannin and LY294002, which are phosphatidylinositol 3-kinase inhibitors. In the same cells, AQP2 is localized in a subapical recycling compartment that is distinct from organelles such as the Golgi, the TGN (trans-Golgi network), and lysosomes.[252]Furthermore, this AQP2 compartment does not contain transferrin receptor, and it is distinct from vesicles that contain Glut4 (another recycling protein) in adipocytes that co-express AQP2 and Glut4.[253] Stimulation of these coexpressing cells with forskolin results in the membrane accumulation of AQP2, but not of Glut4. Similarly, stimulation of cells with insulin causes membrane accumulation of Glut4 but not AQP2.[253] Together, these data suggest that prior to insertion into the cell surface, AQP2—like Glut4 in smooth muscle cells and adipocytes—is located in specialized vesicles that are not easily identified using markers of known intracellular compartments, although in the adipocyte system AQP2 showed significant overlap with the distribution of vesicle associated membrane protein (VAMP) 2. Whether these vesicles represent a novel organelle that appears in cells transfected with AQP2, or whether AQP2 usurps an already existing pathway and modifies it based on intrinsic signals within the AQP2 sequence, remains unclear. It is likely that as newly synthesized AQP2 is loaded into transporting vesicles as it exits the TGN, the fate of the vesicles is indeed determined by signals on the AQP2 protein itself that will be discussed in more detail later.

However, experiments in which the recycling of AQP2 has been artificially interrupted show that in transfected LLC-PK1 cells, AQP2 can be concentrated in a clathrin-positive, Golgi-associated compartment by lowering the incubation temperature of the cells to 20°C, or by incubating cells with bafilomycin, an inhibitor of the vacuolar H+ATPase.[254] This accumulation occurs even in the presence of cycloheximide, an inhibitor of protein synthesis, indicating that recycling AQP2 is also accumulating in this juxta-nuclear compartment. It is known that the 20°C block prevents exit of proteins from the trans Golgi,[255] and that clathrin-coated vesicles are enriched in this cellular compartment.[256] However, some portions of the so-called “recycling endosome”, which is located in a similar juxtanuclear region of the cells, also have clathrin-coated domains.[257] Therefore, the AQP2 could be recycling either via the trans Golgi, via a specialized clathrin-coated recycling endosome, or both. Indeed recycling AQP2 is at least partially colocalized with internalized transferrin in recycling endosomes in LLC-PK1 cells[258] and is partially colocalized with rab11, a marker of the recycling endosomal compartment, in subapical vesicles.[259]

AQP2 is a Constitutively Recycling Membrane Protein

Whereas AQP2 was originally believed to be present in subapical vesicles awaiting a signal (VP stimulation) to move to the cell surface, it is now clear that AQP2 in fact recycles continually between intracellular vesicles and the cell surface, both in transfected cells in culture and in principal cells in situ. In this respect, AQP2 resembles the glucose transporter, Glut4, which also recycles constitutively, but whose plasma membrane accumulation is increased by insulin. [260] [261] [262] This provides the opportunity to modulate the plasma membrane content of AQP2 by increasing the rate of exocytosis, decreasing endocytosis, or both. Indeed such a dual action of VP was predicted by Knepper and Nielsen[263] by comparing mathematical models of VP-induced permeability changes to actual experimental data from isolated perfused collecting ducts.

Data showing that AQP2 recycles constitutively have been obtained by blocking the AQP2 recycling pathway either in an intracellular perinuclear compartment identified as the TGN as discussed earlier,[254] or at the cell surface.[250] [264] When cells are infected with a dynamin K44A virus, clathrin-mediated endocytosis is arrested, and in parallel, AQP2 accumulates at the plasma membrane in a VP-independent manner.[250] This process takes several hours as the mutant dynamin is expressed in cultured cells and the endo-genous wild-type dynamin is overwhelmed. A more rapid means of preventing clathrin-mediated endocytosis is to treat cells with the cholesterol-depleting drug, methyl-b-cyclodextrin. [265] [266] When this was done in LLC-PK1 cells expressing AQP2, the water channel accumulated at the plasma membrane in a matter of minutes, indicating that it is recycling rapidly through the plasma membrane and that inhibition of endocytosis is sufficient to cause membrane accumulation of AQP2 ( Fig. 8-7C ).[264] Importantly, this drug also causes a significant accumulation of AQP2 in the apical membrane of collecting duct principal cells in situ ( Fig. 8-6C ),[267] confirming the relevance of the cell culture studies to the intact organ. This observation raises the exciting possibility that inhibition of endocytosis is a potential pathway by which AQP2 can be accumulated at the cell surface of collecting duct principal cells in patients with X-linked NDI. How recent insights into AQP2 trafficking and signaling might provide novel strategies to alleviate the symptoms of NDI will be discussed in more detail later.

REGULATION OF AQP2 TRAFFICKING

Our understanding of AQP2 recycling continues to evolve in parallel with new discoveries related to the targeting and trafficking of membrane proteins in general. These include the discovery of alternative signaling pathways for AQP2 trafficking in addition to the “conventional” cAMP pathway, the role of phosphorylation by various kinases, the involvement of the actin cytoskeleton, and the gradual discovery of accessory interacting proteins. However, as will be pointed out, several fundamental questions related to the cell biology of VP action remain unanswered, the most im-portant of which is precisely how phosphorylation of AQP2 on residue S256 induces membrane accumulation of this water channel.

Role of Phosphorylation in AQP2 Trafficking

The AQP2 sequence contains several putative phosphorylation sites for kinases including protein kinase A (PKA) and protein kinase G (PKG), protein kinase C (PKC), Golgi casein kinase, and casein kinase II (see Fig. 8-5 ). A recent study using phosphoproteomics on inner medullary protein samples identified an additional site at S261 on AQP2 that may be a MAP kinase site.[268] Most work has focused on the role of PKA-induced phosphorylation of S256 in the vasopressin-induced signaling cascade because this site appears to be critical to the vasopressin-induced membrane accumulation of AQP2. [269] [270] Upon VP binding to the V2R, activation of protein kinase A by increased levels of cytosolic cAMP leads to phosphorylation of S256 on the cytoplasmic C-terminus (see Fig. 8-1 ). This S256 residue is required for a cAMP-induced increase in water permeability of oocytes expressing AQP2.[271]

Phosphorylation could in theory modulate the water permeability of AQP2 already in the plasma membrane, or it could be involved in the regulated trafficking of vesicles containing AQP2 and insertion of AQP2 into the plasma membrane. The permeability of AQP4 [190] [191] and of several plant water channels is regulated by phosphorylation, [272] [273] and some structural features of the phosphorylation-dependent gating mechanism were recently elucidated for SoPIP2, a spinach plasma membrane aquaporin.[274] In addition, the ion channel properties of AQP0 (MIP26) are modulated by a calcium/calmodulin-mediated event.[275] Some reports have suggested that PKA-induced phosphorylation may increase the permeability of AQP1 to cations, [276] [277] [278] but this result is controversial and has not been repeated in other laboratories.[279] However, evidence against a role of phosphorylation in gating AQP2 was obtained by Lande and colleagues[280] who showed that the water permeability of isolated kidney papillary vesicles containing AQP2 was not modified significantly by PKA or phosphatase treatment of the AQP2-containing vesicles. However, a more direct assessment of the effect of phosphorylation on AQP2 permeability in systems overexpressing AQP2 or using purified protein in liposomes has not been performed to date.

In contrast, regulation of membrane permeability by AQP2 trafficking has been established in a variety of experimental systems. Using a point mutation of AQP2, serine 256 to alanine (S256A) expressed in LLC-PK1 cells, it was clearly shown that phosphorylation of the S256 residue by PKA is required for the VP-induced accumulation of AQP2 in the plasma membrane. [269] [270] VP also stimulates S256 phosphorylation of native AQP2 in collecting duct principal cells in situ. [281] [282] The in vivo importance of S256 phosphorylation was shown by the identification of a mutant AQP2 in a patient with NDI that destroys the consensus PKA phosphorylation site. This S254L mutation, when expressed in epithelial cells, is retained in intracellular vesicles and is not phosphorylated upon forskolin addition.[283] Furthermore, PKA and several protein kinase A anchoring proteins (AKAPs) are enriched in AQP2-immunopurified vesicles from IMCD cells. Inhibition of forskolin-induced AQP2 translocation with a peptide that prevents PKA-AKAP interaction demonstrated that, besides its enzymatic activity, tethering of PKA to subcellular compartments is essential for AQP2 translocation. [284] [285] Of particular interest and importance is the finding that the rat AKAP Ht31 directly interacts with the actin modifying GTPase RhoA, which plays a crucial role in modulating AQP2 trafficking (see later). Most recently, an AKAP18 splice variant—AKAP18d—was shown to colocalize with AQP2 in IMCD cells. Elevation of cAMP caused the dissociation of AKAP18d and PKA suggesting a role for this novel AKAP in the VP response.[286]

However, more recent data have shown that phosphorylation of AQP2 at S256 is not necessary for its exocytotic insertion into the plasma membrane. As indicated earlier, AQP2 follows a constitutive recycling pathway and the S256A mutant, from which the PKA phosphorylation site is absent, also accumulates on the plasma membrane upon inhibition of endocytosis with either K44A dynamin or methyl-b-cyclodextrin ( Fig. 8-7D ).[264] Thus, while VP-induced accumulation of AQP2 at the cell surface requires S256 phosphorylation, exocytotic insertion of AQP2 into the plasma membrane is independent of this phosphorylation event.

However, dephosphorylation of AQP2 at S256 is not necessary for its internalization. Prostaglandin E2 stimulates removal of AQP2 from the surface of principal cells when added after AVP treatment, but does not alter the phosphorylated state of AQP2.[282] In support of this, it was shown in cell cultures that PKC-mediated endocytosis of AQP2 is also independent of the phosphorylation state of this water channel and in addition, the AQP2 mutant S256D—which mimics the phosphorylated state of the channel—is constitutively expressed mainly at the cell surface in overexpressing cells.[287] However, internalization of S256D AQP2 can be induced by treating cells with either PGE2 or dopamine, but only after pre-exposing the cells to forskolin.[288] The authors concluded that PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and that preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization. These data imply that phosphorylation of another intracellular target or targets (presumably by forskolin-stimulated elevation of cAMP) is necessary for AQP2 endocytosis to occur, but these proteins remain to be identified.

Preventing dephosphorylation of AQP2 with the phosphatase inhibitor okadaic acid also has the expected effect of increasing cell surface accumulation of AQP2 in cultured cells but surprisingly, the same effect of okadaic acid was observed in the presence of the PKA inhibitor H-89. The authors concluded that okadaic acid stimulates the membrane translocation of AQP2 in a phosphorylation-independent manner.[289] The transduction mechanism responsible for this effect remains to be determined, but these data support the idea that AQP2 can accumulate on the plasma membrane in an S256 phosphorylation-independent manner.

The mechanism by which phosphorylation of AQP2 on residue S256 affects the steady-state redistribution of AQP2 is unknown. No other phosphorylation sites on AQP2 have yet been shown to be involved in its VP-induced membrane accumulation, although one report has suggested that a Golgi casein kinase mediated phosphorylation of S256 is involved in the passage of AQP2 through the Golgi apparatus in its biosynthetic pathway.[290] One possibility is that phosphorylation results in a modified interaction of vesicles with the cytoskeleton, via microtubule or microtubule motors (or both). These proteins are a driving force for intracellular vesicle movement, and must be brought into play in order for vesicles to be transported through the cytosol in the direction of the plasma membrane. Alternatively, phosphoryla-tion could inhibit the endocytotic step of AQP2 recycling, leading to accumulation at the cell surface, although results discussed earlier show that phosphorylated AQP2 can still be internalized.

Actin, Actin-Associated Proteins, and AQP2 Trafficking

The literature on the role of actin in water channel trafficking dates back over three decades, but its role in this process still remains unclear. Actin has been shown to associate directly with AQP2, [291] [292] implying a functional relationship that remains to be clearly demonstrated. VP exposure was reported to depolymerize actin in the toad bladder and collecting duct principal cells, [169] [293] [294] allowing water channel containing vesicles to break through the “actin barrier” and fuse with the plasma membrane. However, actin depolymerization resulting from the inactivation of RhoA, a GTPase that regulates the actin cytoskeleton, increases the membrane accumulation of AQP2 and membrane water permeability in cultured cells even in the absence of VP. [295] [296] Cytochalasins, which disrupt actin filaments, markedly inhibit the vasopressin response in target epithelia, [297] [298] [299] [300] but such treatment increases AQP2 membrane accumulation in cultured renal epithelial cells in some studies. [295] [296] Although actin depolymerization was originally believed to simply remove a physical barrier that prevented vesicles fusion with the plasma membrane, it is now clear that the role of actin in vesicle trafficking and vesicle endo- and exocytosis is much more complex.

Role of Actin Polymerization in AQP2 Trafficking

The potential role of actin in AQP2 membrane insertion was directly examined in transfected CDB cells in culture. Exposure of these cells to Clostridium toxin B, which inhibits RhoGTPases that are involved in regulating the actin cytoskeleton,[301] caused actin depolymerization and an accumulation of AQP2 in the plasma membrane.[296] A similar AQP2 translocation was seen in cells treated with the downstream Rho kinase inhibitor, Y-27632.[295] This occurred in the absence of any detectable elevation of intracellular cAMP. Conversely, expression of constitutively active RhoA in these cells induced stress fiber formation, indicating actin polymerization, and inhibited the normal AQP2 translocation response to forskolin. Although these data provide strong evidence for a major regulatory role of the actin cytoskeleton in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the cell surface, it remains unclear whether the net accumulation of AQP2 under these conditions is due to increased exocytosis or decreased endocytosis. There is a considerable body of evidence showing that actin depolymerization inhibits endocytosis, although whether apical and/or basolateral endocytosis is most affected remains a matter of debate. [302] [303] [304] Interestingly, actin depolymerization was sufficient to provoke membrane accumulation of AQP2 in either the apical or the basolateral plasma membrane, depending on the transfected cell type that was examined. [295] [296] In contrast to the data discussed earlier, showing membrane accumulation of AQP2 after actin depolymerization, a more recent study using transfected MDCK cells reported that AQP2 was concentrated in an EEA1-positive early endosomal compartment upon actin filament disruption by either cytochalasin D or latrunculin.[259] These contrasting effects may reflect the use of different model systems, and the physiological role played by actin on AQP2 trafficking in renal principal cells in situ remains to be determined.

Identification of Actin-Associated Proteins Potentially Involved in AQP2 Trafficking

Many studies in other systems have implicated actin and associated proteins such as the myosins, as well as microtubules (see later), in sequential transport steps of vesicle trafficking. [305] [306] Several actin-associated proteins appear to be involved in AQP2 trafficking, and in some cases have been localized on or close to vesicles that contain AQP2 by immunocytochemistry or immuno-isolation of vesicles coupled to mass spectrometry/proteomic analysis.[307] Immunogold localization showed that myosin I, an actin-associated motor protein is associated with AQP2 containing vesicles.[308] Various myosin isoforms including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB were associated with vesicles prepared for the proteomic analysis by immunoprecipitation with AQP2 antibodies, although the profile of identified proteins indicates that virtually all compartments in the secretory and recycling pathways were represented in the immunoprecipitated material.[307] Myosin light chain kinase, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy were also found in rat IMCD cells and were implicated in a calcium/calmodulin regulated pathway leading to AQP2 membrane accumulation.[309] These data supported previous work from the same group that Ca2+ release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.[31] A role of Epac (exchange protein directly activated by cAMP) in VP-induced calcium mobilization and AQP2 exocytosis in perfused collecting ducts has also been shown.[310] However, the role of calcium in the vasopressin response was questioned by another group who provided capacitance data in support of a cAMP-dependent, but calcium-independent exocytotic process after VP stimulation.[311]

In another study, AQP2-interacting proteins were identified by mass spectrometry in a complex containing ionized calcium binding adapter molecule 2, myosin regulatory light chain smooth muscle isoforms 2-A and 2-B, alpha-tropomyosin 5b, annexin A2 and A6, scinderin, gelsolin, alpha-actinin 4, alpha-II spectrin, and myosin heavy chain nonmuscle type A.[312] The proteins were suggested to comprise a multiprotein motor complex involved in AQP2 trafficking. Interestingly, the gelsolin-like protein adseverin is much more highly expressed in collecting duct principal cells than gelsolin (which is abundant in intercalated cells), indicating that it might be a physiologically important player in calcium-activated actin remodeling in these cells.[313] In addition to myosins, moesin, a member of the ERM (ezrin-radixin-moesin) family of scaffolding proteins, has also been implicated in the apical trafficking process.[314] The GTPase Rap1 and the signal-induced proliferation-associated gene-1 (SPA-1) may also have a role in regulating AQP2 trafficking.[315] Activation of Rap1 was found to inhibit AQP2 plasma membrane targeting, possibly by increasing actin polymerization. This effect is most likely mediated by SPA-1.

Based on these studies, it is clear that actin and its complex array of regulatory proteins play critical roles in the membrane accumulation and recycling of AQP2. However, the precise steps in the pathway, and how these processes are regulated by vasopressin have not been established in any detail, other than to show that disruption of the cytoskeleton has end results compatible with a perturbation of the physiologically regulated process. How AQP2 interaction with the cytoskeleton is affected by phosphorylation, for example, is completely unknown.

Microtubules and AQP2 Trafficking

Many studies have shown that vesicles can move along microtubules and that such transport may be driven by microtubule mechanoenzymes or motors. [316] [317] [318] It is, therefore, not surprising that microtubule-depolymerizing agents such as colchicine and nocodazole have long been known to partially inhibit the VP-induced water permeability increase in target epithelia. [298] [299] [300] [319] [320] [321] [322] Colchicine treatment disrupts the apical localization of AQP2 in rat kidney principal cells, and causes it to be scattered on vesicles throughout the cytoplasm.[197] Furthermore, cold treatment, which depolymerizes microtubules, also inhibits the vasopressin response, indicating that caution must be exercised in the interpretation of data from cell or tissue preparations that involve a cold incubation step as part of the experimental procedure.[229] However, relatively little insight is available concerning the mechanism(s) by which AQP2-containing vesicles interact with the microtubule network in a regulated manner.

As is the case for the actin cytoskeleton, microtubules have an array of accessory proteins that are necessary for their biological activity. Two large protein families are of particular importance for microtubule-based vesicle movement—these are ATPases known as motor proteins, the dyneins,[323] and the kinesins.[324] The ones with minus end-directed motors such as dynein will transport vesicles toward the microtubule-organizing center whereas plus end-directed motors (such as kinesin) will transport vesicles in the opposite direction. [325] [326] [327] Both immunoblotting using immunoisolated vesicles and double-immunogold microscopy revealed that dynein and dynactin, a protein complex thought to link dynein to microtubules and vesicles, are associated with AQP2-bearing vesicles,[328] consistent with the view that microtubule motor proteins are involved in the vasopressin-regulated trafficking of AQP2-bearing vesicles. Furthermore, early studies had shown that an inhibitor of the dynein ATPase, erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA), significantly reduced the effect of VP on water flow in the amphibian urinary bladder model system.[329] However, although treatment of transfected epithelial cells with nocodazole or colchicine to depolymerize microtubules resulted in a dispersion of AQP2 vesicles throughout the cytoplasm, forskolin-induced membrane accumulation was apparently not inhibited in these cells as judged by immunofluorescence staining.[259] Even in earlier work using toad bladder and collecting duct epithelia, the effect of VP on transepithelial water flow was only partially inhibited by microtubule disruption (about 65% in collecting ducts)[320]; this could be accounted for by a reduction in aquaporin delivery that might not be detectable without careful quantification. This also supports the idea that microtubules are involved in the long-range trafficking of vesicles toward the plasma membrane, but that the final step of approach and fusion involves a cooperative interaction between the microtubule and actin-based cytoskeleton. [306] [326] [330] [331] Thus, in cells in which AQP2 containing vesicles are already quite close to the cell surface, AQP2 delivery to the membrane might be less dependent on intact microtubules. Furthermore, recent studies have shown that a protein complex exists at the plus ends of microtubules that is involved in cellular processes involving force generation at the interface between microtubule ends and the actin-rich cortical cytoskeleton.[332] Thus, the delivery of AQP2 vesicles to the sub-plasma membrane region probably involves both microtubules and the actin cytoskeleton, as well as their respective cohorts of accessory and motor proteins that are also involved in membrane trafficking processes in most cell types. Most of the specific protein–protein interactions, as well as their regulation, which render the process VP-sensitive in the case of AQP2 trafficking in the collecting duct remain to be elucidated.

SNARE Proteins and AQP2 Trafficking

As for most if not all membrane fusion events, it has been postulated that the docking step for vasopressin-induced exocytosis of AQP2-bearing vesicles could be mediated by vesicle targeting proteins. [333] [334] [335] [336] The final delivery steps of vesicle tethering, docking, and fusion involve a complex series of protein-protein interactions that are combined under the name “the SNARE hypothesis”. [337] [338] [339] [340] [341] This process requires a complex interaction between integral membrane proteins, the “SNAREs”, present in the vesicle (v-SNAREs) and the target membrane (t-SNAREs) as has been suggested for docking of synaptic vesicles to the presynaptic plasma membrane in the central nervous system. In the synapse, this core com-plex consists of a v-SNARE (VAMP-2) and two t-SNAREs (syntaxin-1 and SNAP-25),[342] which form a 7S core complex that binds the ATPase NSF (“N-ethylmaleimide sensitive factor”) via an intervening soluble NSF attachment protein (a-SNAP) to form a larger 20S complex. The formation of this complex is thought to be vital to the eventual vesicle fusion process. In the collecting duct principal cell, several proteins of the SNARE complex are associated with AQP2-containing vesicles or the apical plasma membrane (or both) of principal cell cells. These include VAMP-2,[336] the t-SNAREs syntaxin-4[334] and SNAP23,[343] and Hrs-2, an ATPase that may regulate exocytosis via interaction with SNAP25.[344] Other proteins that are involved in exocytotic processes in other cell types, such as rab3, rab5a, and a synaptobrevin II-like protein,[345] as well as cellubrevin[346] have also been identified in isolated vesicles containing AQP2. Several additional SNARE proteins, including syntaxin-7, syntaxin-12, syntaxin-13 were identified in a proteomic screen using vesicles immunoisolated with anti AQP2 antibodies.[307] However, as mentioned earlier, such isolated vesicles represent a mixed population that contain AQP2, but are not all in the exocytotic pathway. While a role of some of these proteins in exocytosis is likely by analogy with other secretory pathways, the functions of most of these SNARE proteins in AQP2 trafficking have not been formally examined. One exception is VAMP 2/synaptobrevin. Studies on collecting duct cells in culture have shown that treatment with tetanus toxin, which cleaves VAMP2, abolishes vasopressin-induced AQP2 translocation to the plasma membrane.[347] Interestingly, the related protein cellubrevin, also present in principal cells,[346] is involved in the exocytosis of the vacuolar ATPase in proton secreting cells of the epididymis,[348] which closely resemble intercalated cells of the collecting duct. Thus, SNARE proteins are part of a ubiquitous fusion machinery that is required for vesicle exocytosis. Whether they have any specific features in principal cells that allow them to be specifically regulated upon vasopressin action to modulate AQP2 insertion into the plasma membrane is unknown and requires additional studies.

cAMP-Independent Membrane Insertion of AQP2—Potential Strategies for Treating Nephrogenic Diabetes Insipidus

Important progress has been made in the past 3 or 4 years in our understanding of intracellular signaling or alternative trafficking pathways that bypass the V2R, cAMP, PKA cascade and that allow membrane accumulation of AQP2 even in the absence of a functional V2R. This is especially important for the generation of novel strategies to alleviate the symptoms of X-linked NDI, in which a mutated V2R is defective for a number of reasons, as described earlier. Principal cells in these patients still produce AQP2, but the defective V2R signaling mechanism means that it does not accumulate at the cell surface in order to increase urine concentration upon an increase in circulating VP levels. Recent developments in understanding the cell biology of AQP2 trafficking have provided some hope that AQP2 can in fact accumulate at the cell surface independently of VP signaling in collecting ducts of these patients. Two promising approaches will be discussed below.

Activation of a cGMP Signaling Pathway

In both cell cultures ( Fig. 8-9 ) and principal cells in kidney slices in vitro ( Fig. 8-10 ), several hormones and drugs that increase cGMP levels also induce AQP2 accumulation at the cell surface. These include sodium nitroprusside (a nitric oxide donor), L-arginine (which stimulates nitric oxide synthase), and atrial natriuretic peptide.[228] Similar data showing that ANP increases AQP2 surface expression in vivo after 90 minutes of infusion have also been reported.[349] This effect was paralleled by an increased apical expression of EnaC, and may represent a direct or a compensatory effect to increase sodium and water reabsorption, which would prevent volume depletion in response to prolonged ANP infusion. Importantly, it has also been shown that elevation of intracellular cGMP using the phosphodiesterase (PDE5) inhibitor sildenafil citrate (Viagra) also increases cell surface expression of AQP2 both in vitro and in vivo ( Fig. 8-11 ).[350] Although no significant increase in urinary concentration was detectable in rats treated with Viagra for 90 minutes, possibly due to increased renal blood flow as a result of vasodilatation, this observation nevertheless provides a strategy to induce the VP-independent cell surface accumulation of AQP2. Adaptation of this approach to the human condition will require further dose-response studies, as well as the potential development of PDE5 inhibitors that may be more selective for tubular epithelial cells, or that may be delivered more specifically to these cells at an appropriate concentration to elicit the required response.

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FIGURE 8-9  Effect of activation of the cGMP pathway on AQP2 membrane accumulation in cells and tissues. Immunofluorescence staining of an AQP2-c-myc construct expressed in LLC-PK1 epithelial cells. Under basal conditions, the AQP2 is located on intracellular, perinuclear vesicles (A, CON). After 10 minutes stimulation with the nitric oxide donor sodium nitroprusside (SNP) to increase cGMP (B), forskolin (FK) to increase cAMP (C), or with a permeant cGMP analog (D), AQP2 is relocated to the plasma membrane. Bar = 10 mm.

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FIGURE 8-10  The nitric oxide donor, sodium nitroprusside (SNP), has a vasopressin-like effect on AQP2 distribution in principal cells. Rat kidney slices were incubated in vitro without (A) or with SNP for 15 minutes. A marked redistribution of AQP2 to the apical plasma membrane is induced by SNP treatment (B, arrows) compared to the scattered intracellular location of AQP2 under non-stimulated conditions (A). See Ref 284 for more details. Bar = 20 mm.

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FIGURE 8-11  Viagra stimulates membrane accumulation of AQP2 in rat kidney inner stripe collecting duct principal cells after acute in vivo treatment of Brattleboro rats. Animals were injected with saline, dDAVP, or sildenafil through the jugular vein. Injection of saline was used as a control (A) and compared to 25 mg/kg dDAVP (B) and (C) 4 mg/kg of sildenafil. In controls (A), AQP2 is diffusely located throughout the subapical cytoplasm of principal cells. dDAVP (B) and sildenafil (C) both induce a marked redistribution of AQP2, which appears as a narrow, brightly stained band at the apical pole of principal cells, consistent with plasma membrane staining (arrows). Bar = 10 mm.

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Inhibition of AQP2 Endocytosis

As discussed earlier, it is now evident that AQP2 recycles constitutively between the cell surface and intracellular vesicles. It has been shown using dominant negative dynamin (K44A dynamin) and methyl-b-cyclodextrin treatment that AQP2 accumulation at the cell surface can be achieved simply by blocking endocytosis [250] [264] (Figs. 8-6C, D and 8-7C, D [6] [7]). It has also been shown that endocytosis of AQP2 is stimulated by PGE2 (or dopamine) under some conditions[288] and that activation of PKC by PMA treatment of cells also stimulates AQP2 endocytosis, independently of its S256 phosphorylation state.[287] Thus, an attractive possibility is to modulate the endocytotic pathway in X-linked NDI as a means of increasing its cell surface expression. Potential approaches are to decrease prostaglandin production using specific COX2 inhibitors, or to use a discovery approach to screen small chemical libraries for specific inhibitors of endocytosis that might be applied in vivo. For example, such a strategy was recently used to identify a small chemical named “dynasore” that is a specific inhibitor of the dynein GTPase, a protein critically involved in clathrin and caveolin-mediated endocytosis.[351]

LONG-TERM REGULATION OF WATER BALANCE (see also Chapter 13 )

In addition to the acute regulation of collecting duct water permeability and body water balance, long-term regulation of water balance also plays an important role in the homeostatic response. De Wardener and colleagues showed several decades ago that prolonged dehydration is as potent as acute vasopressin treatment in increasing urinary concentration, while water loading efficiently reduced the urinary concentrating capacity.[352] Although multiple nephron segments are involved in these effects, Lankford and colleagues[353] demonstrated that part of this long-term adaptational regulation occurs in the kidney collecting duct; isolated perfused collecting tubules dissected from thirsted rats displayed much higher osmotic water permeability than tubules from water-loaded rats. It was later shown that AQP2 expression levels markedly increase in response to dehydration with an increased abundance of AQP2 in the apical plasma membrane.[195] Both vasopressin-dependent and -independent signal transduction pathways are involved in this process. [198] [354] [355] [356] [357] Thus, collecting duct water permeability and body water balance are regulated in a concerted fashion by short-term and long-term mechanisms, both critically involving AQP2.

Other studies have also documented that the expression of AQP3, a water channel that is abundantly expressed in the basolateral plasma membranes of collecting duct principal cells, is also regulated by vasopressin[358] suggesting that adaptational regulation of AQP3 may also be involved. DDAVP treatment of vasopressin-deficient Brattleboro rats results in a significant increase in AQP3 expression (in addition to AQP2), whereas AQP1 and AQP4 remain unchanged.[358] The key role of AQP3 in urinary concentration was demonstrated in transgenic mice lacking AQP3, which had a very severe urinary concentrating defect.[237] One complicating factor is that the expression of AQP2 was (surprisingly) markedly down-regulated in the cortex and outer medulla but not in the inner medulla of these knockout mice. The mechanisms responsible for this segment-specific down-regulation of AQP2 remain unknown. There are major differences in the different segments also with regard to the expression and localization of AQP2.[234] Because most water is reabsorbed in the proximal regions (i.e., the connecting tubule and cortical collecting duct) reduced expression of AQP2 in the renal cortex could contribute to the development of severe polyuria in AQP3 knockout mice. Because AQP4 is not present or only present at low levels in the CNT and CCD, [163] [235] [236] it would not be able to provide a compensatory exit route for basolateral water flow, which it may accomplish in the IMCD where AQP4 is abundantly expressed. Moreover AQP2 is normally present in the basolateral plasma membrane in the CNT (at least in the rat) and could therefore also potentially be involved in basolateral exit of water. [234] [235] Thus, the absence of AQP4 and reduced levels of both apical and basolateral AQP2 may participate in the polyuria in AQP3-deficient mice. However, the precise role of basolateral AQP4 in urinary concentration is also unclear, because (1) AQP4 knockout mice have only a slight impairment of concentrating ability,[238] and (2) AQP4 is completely absent from kidneys of the desert rodent, which can concentrate urine up to 5000 to 6000 mOsm/kg.[240] Clearly, further studies are needed to provide a better understanding of the functional interactions among the different collecting duct aquaporins.

AQP2 and Nephrogenic Diabetes Insipidus

Hereditary nephrogenic diabetes insipidus, resulting in the inability to produce a concentrated urine, can be a result of mutations in the V2R, as described earlier, or can be a result of mutations in the AQP2 water channel. The latter form is characterized mainly as an autosomal recessive disorder,[359] although some mutations causing a dominant phenotype have been described.[95] In addition, there are several known forms of acquired NDI that also lead to a concentrating defect, and these are much more common than the hereditary forms of the disease. Many of these disorders have now been associated with alterations in the expression of AQP2 in principal cells. This section will summarize both hereditary and acquired NDI with emphasis on the role of AQP2 in these pathological conditions.

Autosomal Recessive Nephrogenic Diabetes Insipidus and Mutations in AQP2

Important data on the role of AQP2 in non X-linked NDI was obtained by examining the AQP2 molecule in human disease.[360] In the first patient studied[211] two distinct point mutations were found that resulted in a substitution of Cys for Arg[187], and Pro for Ser[216]. The R187C mutation occurs in a region of the third extracellular loop that is strongly conserved in members of the aquaporin family. The S216P mutation is located in the last transmembrane domain of AQP2. When mRNAs coding for these mutated proteins (which had the predicted size of 29 kD) were expressed in Xenopus oocytes, no increase in membrane osmotic water permea-bility was detected above that seen in water-injected controls.[211]

Mutations in the AQP2 gene that result in production of a full-length protein could have at least two consequences that would result in a loss of principal cell vasopressin-sensitivity; (1) the production of an AQP2 channel that is still vasopressin-sensitive, and is inserted into the plasma membrane after vasopressin action, but has lost the capacity to function as a water channel; (2) the production of a water channel that is still functional, but that is no longer targeted to the plasma membrane after vasopressin action. Oocyte expression studies have shown that the missense mutations R187C and S216P are impaired in their delivery to the cell surface.[361] The trapped form of these AQP2 mutants is a 32 kD high mannose form, indicating that the protein is blocked in the RER[361] in much the same way as the DF508 CFTR mutation is trapped inside the RER. Recent data indicate that some point mutations, including mutations in asparagine residue 123, significantly affect AQP2 water permeability.[362] The plasma membrane expression of this mutation was only slightly decreased from that shown by the wild-type protein in oocytes. Most recently, an additional AQP2 mutation has been found that causes the protein to be blocked at the level of the Golgi apparatus.[96]

An unusual dominant form of autosomal NDI due to a single nucleotide deletion (727DG) was ascribed to the ability of the mutated AQP2 monomers to form tetramers with normal protein in heterozygotes, and to block the delivery of the entire tetramer to the plasma membrane. The AQP2 tetramers were retained in late endosomes and lysosomes.[95] In addition, a frameshift mutation in the AQP2 molecule has been identified in some patients that causes basolateral targeting of AQP2 in epithelial cells in culture.[242] This mutation results in the addition of both a leucine- and a tyrosine-based basolateral targeting motif to the AQP2 COOH terminus.

Acquired Water Balance Disorders

Acquired forms of NDI are much more common than the hereditary forms described earlier, and they arise as a consequence of drug treatments, electrolyte disturbances, following urinary tract obstruction, as well as a variety of other causes that are listed in Table 8-1 . There is considerable experimental support for the view that dysregulation of AQP2 plays a fundamental role in the development of polyuria associated with multiple acquired forms of nephrogenic diabetes insipidus,[357] and some quantitative data on AQP2 levels in various experimental conditions is summarized in Figure 8-12 . Dysregulation of AQP3 often accompanies AQP2 down-regulation and may also participate in the development of polyuria, but this has not been examined in as great a depth as the AQP2 contribution. Moreover, in some forms of acquired NDI, defects in the expression of important renal sodium transporters that participate in the urinary concentrating ability have also been encountered.


TABLE 8-1   -- Physiologic or Pathophysiologic Conditions Associated with Altered Abundance or Targeting (or Both) of Aquaporin-2

Reduced Abundance of AQP2

Increased Abundance of AQP2

  

 

With Polyuria

  

 

Genetic defects

  

 

Brattleboro rats (central DI)

  

 

DI +/+ Severe mice (low cAMP)

  

 

AQP2 mutants (human)

  

 

V2 receptor variants (human)[*]

  

 

With Expansion of Extracellular Fluid

  

 

Volume

  

 

Vasopressin infusion (SIADH)

  

 

Congestive heart failure

  

 

Hepatic cirrhosis (CCI4-induced noncompensated)?

  

 

Pregnancy

  

 

Acquired NDI (rat models)

  

 

Lithium treatment

  

 

Hypokalemia

  

 

Hypercalcemia

  

 

Postobstructive NDI

  

 

Bilateral

  

 

Unilateral

  

 

Low-protein diet (urinary concentrating defect without polyuria)

  

 

Water loading (compulsive water drinking)

  

 

Chronic renal failure (5/6 nephrectomy model)

  

 

Ischemia-induced acute renal failure (polyuric phase in rat model)

  

 

Cisplatin-induced acute renal failure

  

 

Calcium channel blocker (nifedipine) treatment (rat model)

  

 

Age-induced NDI

  

 

With Altered Urinary Concentration without

  

 

Polyuria

  

 

Nephrotic syndrome models (rat models)

  

 

PAN-induced

  

 

Adriamycin-induced

  

 

Hepatic cirrhosis (CBL, compensated)

  

 

Ischemia-induced acute renal failure (oliguric phase in rat model)

  

 

With Polyuria

  

 

Osmotic diuresis (DM model in rat)

 

cAMP, cyclic adenosine monophosphate; CBL, common bile duct ligation; CCI4, carbon tetrachloride; DI, diabetes insipidus; DM, diabetes mellitus; NDI, nephrogenic diabetes insipidus; PAN, puromycin aminonucleoside; SIADH, syndrome of inappropriate secretion of antidiuretic hormone.

 

*

Reduced V2-receptor density has a profound effect on AQP2 targeting and expression.

 

000078

000519

FIGURE 8-12  Quantitation of AQP2 levels in various conditions of fluid and electrolyte imbalance, including acquired NDI.

000519

 

Lithium Treatment

One per thousand of the population is on lithium treatment in the Western world, and of these 20% to 30% develop clinically significant polyuria. Rats treated with lithium for 1 month showed a dramatic decrease in AQP2 expression both in the inner medulla as well as in cortical and outer medullary parts of the collecting duct. [363] [364] This down-regulation was paralleled by the progressive development of severe polyuria with a daily urinary output matching their own weight. The reduction in AQP2 expression was originally believed to result from an impairment in the production of cAMP in collecting duct principal cells, consistent with the presence of a cAMP-responsive element in the 5′ untranslated region of the AQP2 gene [365] [366] and with the recent demonstration that mice with inherently low cAMP levels also have low expression of AQP2.[367] However, recent data have shown that the reduction in AQP2 levels is independent of adenylyl cyclase activity and cytosolic cAMP concentration.[368] Blood dDAVP levels were clamped in Brattleboro lacks lacking endogenous VP, and these animals showed no difference in dDAVP-induced cAMP generation between kidneys of rats with lithium-induced NDI and control rats. These data were supported by in vitro experiments using the collecting duct cell line mpkCCD(c14)—lithium did not alter VP-stimulated cAMP elevation in these cells, but it did decrease AQP2 mRNA levels.[368] Thus, the mechanism by which lithium reduces AQP2 expression remains unknown.

There was a very slow recovery in AQP2 expression and restoration of urinary concentration after cessation of lithium treatment[364] consistent with clinical findings. Kwon and colleagues[363] used two different treatment protocols—one leading to moderate lithium-induced NDI and one leading to severe lithium-induced NDI. In both protocols there was a dramatic down-regulation of AQP2 expression and a comparable down-regulation also of the basolateral AQP3 to less than 10% of control levels. Thus, down-regulation of AQP3 may also play a significant role. Interestingly, lithium treatment also caused a marked decrease in the fraction of principal cells in collecting duct in cortex and inner medulla[369] with a parallel increase in the population of intercalated cells. This architectural restructuring of the collecting duct, together with down-regulation of collecting duct aquaporins, is likely to be important in lithium-induced NDI. After a 4-week recovery period (cessation of lithium treatment), urine production, AQP2 protein levels, as well as the fraction of principal cells returned completely to control levels.[369] The mechanism underlying the change in principal/intercalated cell ratio after lithium treatment remains unclear, but other procedures such as chronic carbonic anhydrase inhibition by acetazolamide also increase the intercalated cell population in the medulla, implying a phenotypic plasticity of collecting duct epithelial cells.[370]

In addition to the large increase in urinary water excretion, lithium-induced NDI is also associated with significant sodium wasting.[371] The molecular explanation for this has been investigated by functional and biochemical analysis. Kwon and colleagues did not find changes in the expression levels of proximal tubule and thick ascending limb sodium transporters that could be ascribed any role in the sodium wasting or in the reduced urinary concentrating ability.[363] However, amiloride had a lower effect on sodium excretion in rats with lithium-induced NDI than in controls, suggesting that potential changes in the epithelial sodium channel ENaC function could be involved in the sodium wasting.[372] Immunoblotting and immunocytochemical analysis demonstrated a marked reduction in protein expression levels of alpha, beta, and gamma subunits in the cortical and outer medullary collecting duct principal cells in rats with lithium-induced NDI,[373] but there were no changes in proximal tubule and thick ascending limb sodium transporters, consistent with the data of Kwon and colleagues.[363] Thus reduction in ENaC subunits appears to represent the molecular background for the sodium wasting observed in lithium treatment.

Interestingly, treatment of rats with amiloride attenuates the severity of lithium-induced NDI in rats,[374] and in humans, [375] [376] presumably due to an inhibition of lithium uptake into principal cells via the ENaC channel.

Hypokalemia and Hypercalcemia

Hypokalemia and hypercalcemia are both associated with significant vasopressin-resistant polyuria, and both were associated with a reduced expression of AQP2 levels and polyuria in rats. [377] [378] The polyuria associated with these conditions is less severe than that seen in lithium-induced NDI and consistent with this, a less marked reduction in AQP2 was observed. In addition, expression of AQP2 is reduced in hypercalcemic rats contributing to the development of polyuria.[379] With regard to hypercalcemia, it was found that in addition to down-regulation of AQP2, the expression levels of AQP1 and AQP3 protein were also reduced.[379] Hypercalcemia is known to be associated with sodium absorption defects in the thick ascending limb, which would affect the counter current multiplication system. Wang and associates examined the protein expression levels of several key sodium transporters, including the vasopressin-regulated bumetanide-sensitive sodium potassium 2 chloride co-transporter BSC-1 or NKCC-2.[380] BSC-1 expression was markedly reduced both in cortex and in the inner stripe of the outer medulla (ISOM). Also ROMK protein expression was reduced. In contrast there was no reduction in NHE3 and Na, K-ATPase (alpha subunit) in ISOM. Thus reduced TAL BSC-1 and ROMK in hypercalcemia is likely to participate in the known defect in TAL sodium reabsorption and hence in the impaired urinary concentration together with reduced AQP2 and AQP3 expression in the collecting duct.

Ureteral Obstruction

Chronic urinary outflow obstruction with impaired ability of the kidney to concentrate urine is a common condition amongst elderly men, whereas acute obstruction can also cause a similar concentrating defect at all ages. Rat models in which one or both ureters were reversibly obstructed [381] [382] showed that after 24 hours, AQP2 expression was markedly reduced, even before release of the obstruction. Following release of the obstruction, there was a vasopressin-resistant and persistent polyuria, and an increase in solute-free water clearance. Although urine output was almost normalized after a week, the animals still had a concentrating defect. Consistent with this, AQP2 expression levels were significantly reduced to about 25% of control levels 2 days after release of obstruction and remained at about 50% of controls at 7 days. Thus, the persisting concentrating defect is likely to be related to the continued depression in AQP2 levels.[381] Interestingly, unilateral ureteral obstruction also showed significantly reduced renal AQP2 levels indicating that local intrarenal factors are involved in the signaling pathways resulting in reduced expression. Thus there is increasing evidence that down-regulation of AQP2 expression plays a major role for the development of polyuria associated with acquired forms of NDI. This has been further supported by additional studies showing a marked down-regulation of collecting duct aquaporins (AQP2–4) as well as proximal tubule AQP1.[383] Moreover ureteral obstruction and release of ureteral obstruction is also associated with a marked down-regulation of key renal sodium transporters including the TAL transporters involved in countercurrent multiplication.[384] Thus there is growing evidence that many of the energy-consuming processes involved in urinary concentration are dramatically down-regulated in conditions that result in obstruction of the urinary tract. However, there is very little information regarding the signaling processes that are responsible for this down-regulation. In support of a role of inflammatory processes, alpha-MSH treatment of rats with ureteral obstruction or release of obstruction markedly prevented the down-regulation of several key aquaporins and sodium transporters. In particular, the down-regulation of AQP1 and Na,K-ATPase was significantly inhibited.[385] Importantly, it has been found that acute ureteral obstruction leads to marked upregulation of COX-2 in the inner medulla and that selective COX-2 inhibition prevents the observed dysregulation of AQP2, BSC-1, and NHE3.[386] These data indicate that COX-2 may be an important factor contributing to the impaired renal water and sodium handling in response to bilateral ureteral obstruction.

Cirrhosis and Congestive Heart Failure

In some conditions with extracellular fluid expansion such as hepatic cirrhosis and congestive heart failure, which are known to be associated with hyponatremia and a defect in urinary dilution, an increase in AQP2 expression has been found, for example in rats with severe CCl4-induced hepatic cirrhosis. [387] [388] In contrast, conditions with compensated biliary hepatic cirrhosis showed a reduction in AQP2 expression.[389] In experimental congestive heart failure, a marked increase in AQP2 expression and apical targeting was observed, [251] [390] but no change in AQP2 expression was noted in rats with compensatory heart failure.[251] Vasopressin-V2-receptor antagonist treatment of rats with severe congestive heart failure normalized AQP2 expression and eliminated sodium and water retention.[390] Both observations support the view that dysregulation of AQP2 may play a significant role in the development of water retention and hyponatremia. A baroreceptor-mediated increase in circulating vasopressin is likely to play a key role in the increase in AQP2 expression and targeting. It remains to be established why vasopressin-escape does not take place in rats with congestive heart failure in contrast to what is seen in normal experimental SIADH rats.[391]

It should be emphasized that other mechanisms including changes in NaCl transporter expression are also likely to play a major role in the development of sodium and water retention. Recently, it was demonstrated that the expression of BSC-1 (or NKCC2) in the thick ascending limb of the loop of Henle was increased in rats with mild congestive heart failure.[392] Moreover, vasopressin-mediated renal water reabsorption (evaluated by the aquaretic response to selective V(2)-receptor blockade) was significantly increased. Losartan treatment normalized expression of BSC-1 and decreased the protein expression levels of AQP2. This was associated with normalization of daily sodium excretion and normalization of the aquaretic response to V(2)-receptor blockade. Together, these results indicate that, in rats with congestive heart failure, losartan treatment inhibits increased sodium reabsorption through BSC-1 in the thick ascending limb of the loop of Henle and water reabsorption through AQP2 in the collecting ducts, which in part may result in an improved renal function.

Dysregulation of AQP2 (as well as AQP3 and AQP1) is also associated with multiple other water balance disorders including experimental nephrotic syndrome, [393] [394] low-protein feeding,[395] experimentally induced chronic renal failure,[396] and experimental ischemic renal failure. [363] [397] There is substantial evidence that the down-regulation of AQP2 in many pathological conditions is a primary event in acquired NDI. The changes in AQP2 expression in kidney cortex are identical with those seen in the inner medulla[377] indicating that a local effect of interstitial tonicity is not a major factor. Moreover, treatment with the loop diuretic furosemide causes the washout of the medullary osmotic gradient by blocking salt reabsorption in the loop of Henle, but resulted in no significant change in AQP2 expression after either 1 or 5 days treatment. [357] [377] This also indicates that the high urine flow itself is not responsible for the decrease in AQP2 expression in experimental NDI. Indeed homozygous Brattleboro rats express substantial levels of intracellular AQP2 in principal cells (although less than in normal rats), despite high urine volume. This is further supported by the observation that AQP2 expression was increased in polyuric, glycosuric streptozotocin-diabetic rats that had a raised AQP2 level,[398] probably as a consequence of increased circulating vasopressin. These results strengthen the view that the decrease in AQP2 is, at least in part, a cause rather than a consequence of the polyuria.

Thus, dysregulation of aquaporins is likely to play a significant role in a variety of water balance disorders associated with common and often severe kidney, liver, and heart diseases. Studies are underway in many laboratories to fully examine the signal transduction pathways, and to define the exact role of dysregulated expression and targeting in the development of these conditions. Further analysis of aquaporins and aquaporin cell biology, in combination with our increasing understanding of ion channel and transporter expression and function in the kidney, is expected to provide further insights into the molecular understanding of water balance and its disorders.

Acknowledgment

Work from the laboratories of the authors was carried out with the support of National Institutes of Health (NIH) Grant DK38452 (DB) and a grant from the National Danish Research Foundation (Grundforskningsfonden; SN) and the Danish Medical Research Council (SN). We thank our many excellent colleagues for their invaluable contributions to our research endeavors over the past several years.

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