REVIEW ARTICLE

Dynamic regulation and dysregulation of the water channelaquaporin-2: a common cause of and promising therapeutic targetfor water balance disorders

Yumi Noda

Received: 22 August 2013 / Accepted: 24 September 2013

� Japanese Society of Nephrology 2013

Abstract The human body is two-thirds water. The

ability of ensuring the proper amount of water inside the

body is essential for the survival of mammals. The key

event for maintenance of body water balance is water

reabsorption in the kidney collecting ducts, which is reg-

ulated by aquaporin-2 (AQP2). AQP2 is a channel that is

exclusively selective for water molecules and never allows

permeation of ions or other small molecules. Under normal

conditions, AQP2 is restricted within the cytoplasm of the

collecting duct cells. However, when the body is dehy-

drated and needs to retain water, AQP2 relocates to the

apical membrane, allowing water reabsorption from the

urinary tubule into the cell. Its impairments result in vari-

ous water balance disorders including diabetes insipidus,

which is a disease characterized by a massive loss of water

through the kidney, leading to severe dehydration in the

body. Dysregulation of AQP2 is also a common cause of

water retention and hyponatremia that exacerbate the

prognosis of congestive heart failure and hepatic cirrhosis.

Many studies have uncovered the regulation mechanisms

of AQP2 at the single-molecule level, the whole-body

level, and the clinical level. In clinical practice, urinary

AQP2 is a useful marker for body water balance (hydration

status). Moreover, AQP2 is now attracting considerable

attention as a potential therapeutic target for water balance

disorders which commonly occur in many diseases.

Keywords Trafficking � Diabetes insipidus �SIADH � Congestive heart failure � Hepatic cirrhosis �Solute-free water diuretics

Introduction

Water is the largest component of the body and accounts for

approximately 60 % of body weight. Maintaining body fluid

homeostasis including fluid volume and concentration is

essential for cell function and whole-organism survival. The

osmolality of body fluid, a concentration of all of the solute

in water, is kept within a narrow range (280–295 mOsm/kg),

in spite of large fluctuations of solute and water intake and

losses. Although this constancy is made by various kinds of

regulatory mechanisms in the body, the most critical regu-

latory capacities are provided by the kidney’s urine con-

centration and dilution mechanisms [1].

Body water volume is maintained by the balance

between the input and output of water, with each side

having regulated and unregulated components. The regu-

lated component of water input is oral intake of fluids in

response to a perceived sensation of thirst. The unregulated

components of water input are metabolic water of oxida-

tion and oral intake of liquids and water in foods that varies

due to psychological factors in excess of homeostatic need.

Water excretion by the kidney is the only route of regulated

water output. The unregulated components of water

excretion are sweat, evaporative loss, gastrointestinal loss,

and the obligate amount of water that is required to excrete

the solutes in the urine. Both input and output of water

This article was presented as the Oshima Award memorial lecture at

the 53rd annual meeting of the Japanese Society of Nephrology, held

at Kobe, Japan, in 2010.

Y. Noda (&)

Department of Nephrology, Nakano General Hospital,

4-59-16 Chuo, Nakano-ku, Tokyo 164-8607, Japan

e-mail: ynodmed2@tmd.ac.jp

Y. Noda

Department of Nephrology, Tokyo Medical and Dental

University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519,

Japan

123

Clin Exp Nephrol

DOI 10.1007/s10157-013-0878-5

have unregulated components that vary tremendously as a

result of factors that are unrelated to body water balance.

Therefore, the regulated components that are urine excre-

tion and water intake caused by thirst must compensate for

the perturbations by the unregulated components. Daily

urine excretion range is as low as 0.5 L to as high as 25 L

depending on the requirements for water balance. When the

kidney’s capacity to conserve water is taxed to the limit

due to dehydration, a sensation of thirst is activated,

causing oral intake to be increased.

Because the solute concentrations in water in the body

must be kept nearly constant, water loss must be regulated by

a mechanism that decouples water and the solutes. The kid-

ney can excrete the appropriate amount of water without

marked perturbations in solute excretion. When water intake

is too large to dilute blood plasma, urine more dilute than

plasma is excreted to concentrate the plasma. When water

intake is too small to concentrate plasma, urine more con-

centrated than plasma is excreted to dilute the plasma. In both

cases, the solute excretion varies little. Renal free water

excretion is mainly regulated by the antidiuretic hormone

vasopressin. Vasopressin is secreted from the posterior pitu-

itary gland into systemic circulation in response to increases

in the effective osmotic pressure, decreases in the effective

circulating volume or pressure, or several other stimuli. In

response to the levels of vasopressin in the plasma, the kidney

is capable of wide variations in free water excretion. The

molecular entity of a major effector of vasopressin in the

kidney is aquaporin-2 (AQP2), which is a member of the

aquaporin (AQP) water channel family [2–6]. The AQP2

channel is exclusively selective for water molecules and

never allows permeation of ions or other small molecules.

AQP2 is abundant in the collecting duct, which is the chief

site of regulation of water reabsorption. Acute stimulation of

vasopressin promotes AQP2 translocation from an intracel-

lular reservoir to the luminal cell surface and its chronic

stimulation increases the cellular abundance of AQP2, both

of which elevate the water permeability of the collecting duct

cells, resulting in the promotion of water reabsorption from

the urinary tubule. AQP2 impairments result in various water

balance disorders including diabetes insipidus, which is a

disease characterized by a massive loss of water through the

kidney, leading to severe dehydration in the body. On the

other hand, dysregulation of AQP2 is also a common cause of

water retention and hyponatremia that exacerbate the prog-

nosis of congestive heart failure and hepatic cirrhosis.

AQP2 in the collecting duct maintains body water

balance by defining final water excretion volume

The collecting duct is the final structure in the nephron.

The collecting duct expresses vasopressin V2 receptor and

AQP2 [7]. Water reabsorption in the collecting ducts is the

key event for maintenance of body water balance. This

process is regulated by AQP2. Under conditions of normal

hydration, AQP is confined to the cytoplasm of the col-

lecting duct cells. When the body is dehydrated and needs

to retain water, AQP2 relocates to the apical membrane,

thus enabling water reabsorption from the urinary tubule

into the cell.

Vasopressin is an antidiuretic hormone secreted from

the posterior pituitary gland. An increase in the tonicity

of body fluid and reduction in effective circulating blood

volume stimulate the secretion of vasopressin [1]. When

circulating vasopressin reaches the kidney, it binds to

vasopressin V2 receptor expressed on the basolateral

plasma membrane of the collecting duct principal cells

and initiates the signal transduction in the cell. V2

receptor is a 371 amino acid protein with 7 membrane-

spanning domains and couples to heterotrimeric G-pro-

teins [8, 9]. Binding of V2 receptor to vasopressin pro-

motes the disassembly of the bound hetertrimeric

G-protein, Gs, into Ga and Gbc subunits. Guanosine

diphosphate/guanosine triphosphate (GDP/GTP) exchange

occurs in the Ga subunit and this activated Gsa then

stimulates adenylate cyclase, resulting in an increase in

intracellular cyclic adenosine monophosphate (cAMP)

levels. Increased cAMP activates protein kinase A that

phosphorylates AQP2 at serine 256. As a consequence of

AQP2 phosphorylation, subapical storage vesicles that

contain AQP2 translocate from the cytoplasm of principal

cells to the luminal cell surface membrane (also called

the apical membrane) and fuse with it. Relocation of

phosphorylated AQP2 to the cell membrane renders the

cell water permeable, resulting in water reabsorption [5,

10]. How this AQP2 phosphorylation event induces the

AQP2 movement is described below.

Upon removal of the vasopressin stimulus, AQP2 is

shuttled back to the cell cytoplasm, a process that restores

the water impermeability of the cell. This internalization

process consists of AQP2 retrieval into early endosomes

that express early endosome antigen 1, and subsequent

transferral of this water channel to storage vesicles that

express Rab-11 [11].

In response to vasopressin, AQP2 recycles between the

luminal cell surface membrane and the intracellular sub-

apical storage vesicles of the collecting duct principal cells.

In the absence of vasopressin, the water permeability of the

cell surface membrane without AQP2 is low. In the pre-

sence of vasopressin, AQP2-containing vesicles fuse with

the luminal membrane, inducing exceedingly high water

permeability of this luminal membrane. Vasopressin

increases the water permeability by a factor of 10–100 in

the collecting duct, inducing a steep and drastic increase in

water reabsorption [12].

Clin Exp Nephrol

123

The phosphorylation process of AQP2

AQP2 phosphorylation event is a requisite for AQP2

translocation to the apical membrane as described above.

This section describes the process of AQP2

phosphorylation.

AQP2 forms homotetramers, and at least three of four

monomers in AQP2 tetramers must be phosphorylated for

successful apical membrane localization [13, 14]. Protein

kinase A and its substrates are present throughout the cell,

therefore, localization of protein kinase A in specific sites

is necessary to effectively phosphorylate its target. This

process is assisted by protein kinase A anchoring proteins

(AKAPs). Tethering of protein kinase A to AKAPs is

required for AQP2 trafficking [15]. A splice variant of

AKAP-18, AKAP-18d, is specifically involved in AQP2

trafficking [16] although the involvement of AKAP-220

has also been reported [17].

AQP2 phosphorylation by kinases other than protein

kinase A is also involved in AQP2 trafficking. Serine 256

in AQP2 is also a substrate for Golgi casein kinase. AQP2

transition through the Golgi apparatus is associated with a

protein kinase A-independent increase in AQP2 phos-

phorylation at serine 256, suggesting that phosphorylation

by Golgi casein kinase may be required for Golgi transition

[18]. van Balkom et al. [19] showed that activation of

protein kinase C mediates AQP2 endocytosis, which is

independent of the phosphorylation state of serine 256. In

addition, a cyclic guanosine monophosphate (cGMP)-

dependent pathway is shown to be involved in AQP2

exocytosis [20], and an inhibitor of cGMP phosphodies-

terase is able to induce AQP2 translocation to the cell

surface [21].

In addition to serine 256, there are three additional

phosphorylation sites nearby the AQP2 C-terminus. These

modifiable residues are serine 261, serine 264 and serine

269. Vasopressin also induces phosphorylation of AQP2 at

serine 264, and serine 264-phosphorylated AQP2 is trans-

located to the plasma membrane similarly to serine

256-phosphorylated AQP2 [22]. On the other hand, vaso-

pressin decreases the phosphorylation levels at serine 261,

and the localization of serine 261-phosphorylated AQP2 is

different from that of serine 256-phosphorylated AQP2,

which suggests distinct roles for these residues in AQP2

trafficking [23]. Lu et al. [24] reported that the phosphor-

ylation state of serine 261 does not affect AQP2 trafficking.

Serine 269 is involved in plasma membrane retention of

AQP2 [25]. Moeller et al. [26] showed that phosphoryla-

tion of serine 264 and serine 269 depends on prior phos-

phorylation of serine 256 and that phosphorylation of

serine 261 partially depends on the phosphorylation of

serine 264 and serine 269. In contrast, serine 256 phos-

phorylation is not dependent on the state of any of the other

phosphorylation sites, suggesting that serine 256 is the

most important phorphorylation site of AQP2.

The role of calcium in the regulation of AQP2

Intracellular calmodulin-dependent protein kinase II (Ca2?)

mobilization is also involved in vasopressin-mediated AQP2

trafficking [27]. In addition to increasing cAMP levels in the

cytoplasm of the principal cells of the collecting duct,

vasopressin binding to V2 receptor triggers a rapid increase

of intracellular Ca2?, which is followed by sustained tem-

poral oscillations of the level of this ion. This process seems

to be involved in AQP2 exocytosis. Balasubramanian et al.

[27] suggest several plausible candidates as downstream

effectors of this signaling cascade, such as calmodulin and

myosin light-chain kinase (MLCK). MLCK is a calmodulin-

dependent kinase that regulates actin filament organization

by phosphorylating the regulatory light chain of myosin II,

and thus also activates myosin motor activity. Myosin II and

its regulatory light chain are found in the AQP2-binding

protein complex [28], supporting their involvement in AQP2

trafficking. Nevertheless, Lorenz et al. [29] demonstrated

that cAMP alone is sufficient to induce AQP2 translocation,

without the need for an increase in cytosolic Ca2? levels in

the inner medullary collecting duct cells.

Involvement of extracellular Ca2? in AQP2 regulation

has also been indicated by several findings [20–33]. Uri-

nary AQP2 correlates with the severity of enuresis, a dis-

ease characterized by nocturnal polyuria and hypercalciuria

[30]. Clinical amelioration demonstrated by a low calcium

diet is accompanied by regulation of urine output through

remodulation of AQP2 expression/trafficking [31]. Drug-

induced hypercalcemia/hypercalciuria causes polyuria and

reduces AQP2 expression in rats [32]. AQP2 translocation

to the apical membrane prompted by forskolin-induced

increases in cAMP levels is inhibited by increased levels of

extracellular Ca2? [33]. This process is probably mediated

by the endogenous calcium-sensing receptor and is asso-

ciated with an increase in F-actin levels.

Other factors involved in AQP2 trafficking

Several other factors have recently been reported to affect

AQP2 trafficking. Nejsum et al. [34] used Madin–Darby

canine kidney epithelial cells transfected with AQP2 and

showed that prostaglandin E2 and dopamine induce inter-

nalization of AQP2, regardless of AQP2 dephosphoryla-

tion. de Seigneux et al. [35] reported that aldosterone

induces basolateral expression of AQP2, suggesting a role

for aldosterone in water metabolism in conditions of

increased sodium reabsorption in the collecting ducts.

Clin Exp Nephrol

123

The Role of the cytoskeleton in AQP2 trafficking

The actin cytoskeleton is reported to function as a barrier for

AQP2 exocytosis [36, 37]. Actin depolymerization is nec-

essary for the cAMP-dependent translocation of AQP2 [38].

In fact, stimulation of prostaglandin E3 receptors has been

shown to inhibit vasopressin-induced inactivation of Rho

GTPase, vasopressin-induced F-actin depolymerization and

AQP2 translocation induced by vasopressin, cAMP or for-

skolin [38]. Rho GTPase activation by bradykinin stabilizes

cortical F-actin and inhibits AQP2 trafficking [39].

GTPase-activating protein Spa-1 (SPA-1) binds to the

C-terminus of AQP2, and this binding is required for AQP2

trafficking [40, 41]. SPA-1 may inhibit Rap1 GTPase-

activating protein, which triggers F-actin disassembly and

may maintain the basal mobility of AQP2 [10, 42]. SPA-1-

deficient mice show impaired AQP2 trafficking and

hydronephrosis [40, 43]. In humans, mutations in the

C-terminus of AQP2, which is the binding region of SPA-

1, cause nephrogenic diabetes insipidus (NDI), a disease

characterized by a massive loss of water through the kidney

[10, 44, 45].

F-actin assembly might have both inhibitory and facil-

itatory effects on AQP2 transport to the cell surface [36,

37]. Drug-induced actin depolymerization inhibits AQP2

translocation from the early endosomes that express early

endosome antigen 1 to the subapical storage vesicles that

express Rab-11 [46]. Myosin II and its regulatory light

chain are found in an AQP2-binding protein complex [28],

and vasopressin induces myosin light-chain phosphoryla-

tion, which enhances myosin–actin filament interaction and

the formation of actin fibers [47]. Myosin has also been

shown to be critical for AQP2 recycling [48].

In addition to acting as a barrier to prevent AQP2 traf-

ficking, actin fibers may function as ‘cables’ that promote

and direct AQP2 transport. Dynamic actin reorganization

may be responsible for the transformation of the actin

barrier into actin cables. Myosin and tropomyosin may be

involved in this cable formation. Measurement of molec-

ular dynamics with high spatiotemporal resolution may be

useful for clarifying the mechanism of this process.

Fusion of AQP2 vesicles with the apical membrane

The docking and fusion of AQP2-containing vesicles with

the apical membrane involves the action of SNARE pro-

teins including VAMP-2, SNAP-23, syntaxin-3 and syn-

taxin-4 [10, 49]. Syntaxin-binding protein 2 (also called

Munc18b) is reported to function as a negative regulator of

SNARE complex formation and AQP2-vesicle fusion to

the apical membrane [50].

AQP2 recycling and endocytosis

AQP2 is a recycling membrane protein. Upon vasopressin

stimulation, AQP2 is transported to the apical membrane,

rendering the cell water permeable as described above.

After vasopressin stimulation is terminated, AQP2 is

shuttled back to the cell cytoplasm, a process that

restores the water impermeability of the cell. This inter-

nalization process consists of AQP2 retrieval into early

endosomes that express early endosome antigen 1, and

subsequent transferral of this water channel to storage

vesicles that express Rab-11 [11]. From Rab-11-positive

vesicles, AQP2 is able to go again to the apical mem-

brane. Actually, this recycling process occurs constitu-

tively, and many signaling pathway are involved for the

regulation of each part of this recycling itinerary. Vaso-

pressin signaling is the most potent and most important

factor that enhances the exocytotic process among the

recycling itinerary.

During the endocytotic process of AQP2 recycling

pathway, AQP2 accumulates in clathrin-coated pits and is

internalized via a clathrin-mediated process [51, 52].

Dynamin is a GTPase that is involved in the formation and

pinching off of clathrin-coated pits to form clathrin-coated

vesicles, and its dominant-negative mutant K44A renders

the protein GTPase deficient and arrests clathrin-mediated

endocytosis. This GTPase-deficient dynamin mutant K44A

is shown to accumulate AQP2 in the plasma membrane

even without vasopressin stimulation [52]. Furthermore,

this dynamin mutant K44A or methyl-b-cyclodextrin is

able to accumulate phosphorylation-deficient mutants in

the cell surface [53] despite AQP2-S256A accumulation in

the cell surface is not induced by vasopressin. Methyl-b-

cyclodextrin depletes membrane cholesterol, resulting in a

rapid inhibition of endocytosis. These data also support the

constitutive recycling of AQP2 and the presence of the

processes that are not dependent on phosphorylation of

AQP2 at serine 256.

A heat shock protein, hsc70, which is important for

uncoating clathrin-coated vesicles, binds to the C-terminus

of nonphosphorylated AQP2 and is required for AQP2

endocytosis [54]. Kamsteeg et al. [55] reported that the

myelin and lymphocyte protein (also known as MAL),

which is involved in the organization of glycosphingolipid-

enriched membrane, interacts with AQP2 and enhances

accumulation of AQP2 in the apical membrane by

decreasing the level of internalization of the protein.

Ubiquitination at lysine 270 of AQP2 is important for

AQP2 endocytosis and degradation [56]. Furthermore,

LIP5, which is involved in multivesicular body formation,

interacts with AQP2 and facilitates its lysosomal degra-

dation [57].

Clin Exp Nephrol

123

The entity of the molecular process of driving AQP2

movement

AQP2 phosphorylation is a requisite for AQP2 transloca-

tion to the apical membrane as described above. But how

this phosphorylation event induces the AQP2 movement

was unknown until recently. In other words, the direct

mechanism which generates motion in AQP2 trafficking

was unknown. To clarify this mechanism, we tried to

identify AQP2-binding proteins and discovered that AQP2

forms a multiprotein motor complex [10, 28, 58, 59].

This intra-complex transaction which may be crucial in

AQP2 regulation was then examined. For this purpose, we

applied fluorescence correlation spectroscopy (FCS) and

fluorescence cross-correlation spectroscopy (FCCS) for the

first time to channel research. As a result, we succeeded in

measuring the spatial and temporal dynamics of compo-

nents of the AQP2 motor complex at the single-molecule

level, and discovered the direct mechanism that drives

channel movement to the targeted site [5, 36, 37, 60].

Under basal conditions, AQP2 binds to G-actin, while

F-actin is stabilized by tropomyosin-5b (TM5b) to form a

barrier that inhibits translocation of AQP2 to the apical

membrane. Vasopressin-triggered AQP2 phosphorylation

releases AQP2 from G-actin and promotes AQP2 associa-

tion with TM5b, which sequesters TM5b from F-actin and

destabilizes the F-actin network, allowing efficient move-

ment of AQP2 to the apical membrane. This molecular

mechanism was confirmed using purified recombinant

proteins reconstituted in proteoliposomes.

Fluorescence correlation spectroscopy (FCS) and fluo-

rescence cross-correlation spectroscopy (FCCS) measure-

ments are particularly powerful for clarifying the dynamics

of multiprotein complexes at the single-molecule level and

at the various locations in the cell. Recent studies have

uncovered that many channels and transporters form mul-

tiprotein complexes and this intra-complex transaction is

crucial in their regulation. Our methods, including FCS,

FCCS, and the reconstituted system of purified proteins are

powerful for investigating the membrane proteins that form

multiprotein complexes, clarifying the pathophysiology

and identifying the therapeutic target for diseases that are

caused by membrane proteins.

Regulation of individual water channel activity

of AQP2

As described above, AQP2 phosphorylation induces its

apical membrane insertion, rendering the collecting duct

cells water permeable. However, whether this phosphory-

lation regulates the water transport activity of individual

AQP2 water channels remained unclear until recently.

Several groups had examined the role of phosphorylation on

osmotic water permeability (Pf) of individual AQP2. Ku-

wahara et al. [61] examined the phosphorylation and Pf of

AQP2 expressed in Xenopus oocytes. They showed that

protein kinase A (PKA) phosphorylated AQP2 at serine 256.

cAMP stimulation increased the Pf of oocytes expressing

AQP2, although this stimulation did not increase the amount

of AQP2 on the oocyte surface, which was examined by

immunoblotting AQP2 using oocyte membranes. This

finding suggested that the Pf of individual AQP2 proteins

was increased by cAMP-mediated phosphorylation of

AQP2. However, the possibility of phosphorylation-induced

translocation of AQP2 could not be excluded in this

experimental system. Moeller et al. [62] also examined the

role of phosphorylation using AQP2 expressed in Xenopus

oocytes. To evaluate the Pf of a single channel, Pf relative to

the plasma membrane abundance was compared among

wild-type (WT) and mutants of AQP2. Both the Pf and the

plasma membrane abundance of the nonphosphorylation-

mimicking mutant S256A-AQP2 were decreased compared

with WT-AQP2, resulting in the Pf relative to the plasma

membrane abundance being similar. This finding suggested

that a lack of phosphorylation at this site had no effect on

individual AQP2 proteins. However, the methods deter-

mining the plasma membrane abundance were semiquanti-

tative, and this study could not exclude the possibility that

the Pf of individual AQP2 proteins was altered by this

mutation. On the other hand, Lande et al. [63] purified en-

dosomes derived from the apical membrane of rat inner

medullary collecting duct cells that were highly enriched for

AQP2. These endosomes contained endogenous PKA and

phosphatase activities that could phosphorylate and

dephosphorylate AQP2. Therefore, these authors prepared

two kinds of samples to detect the effect of phosphorylation

on Pf. For phosphorylated AQP2, AQP2 endosomes were

incubated with exogenous PKA catalytic subunit and aden-

osine triphosphate (ATP) to maximize the phosphorylation

levels. For nonphosphorylated AQP2, the above phosphor-

ylated sample was then incubated with exogenous alkaline

phosphatase, which was shown to remove 95 % of the

phosphate from AQP2. There was no significant difference

in Pf between these two samples, suggesting that the Pf of

AQP2 is not changed by its phosphorylation. However, the

phosphorylation levels of AQP2 endosomes incubated with

exogenous PKA and ATP progressively decreased over

20 min, which might have been caused by endogenous

phosphatase activities. It is possible that the differences in Pf

may have been underestimated due to the existence of

endogenous PKA, phosphatase and other regulatory proteins

in AQP2 endosomes. To clarify whether the Pf of AQP2 is

regulated by its phosphorylation event alone, an experi-

mental system that does not contain any other regulatory

proteins is required.

Clin Exp Nephrol

123

To answer this question without the effects of other

regulatory proteins, we performed large-scale expression of

full-length recombinant human AQP2, purification and

reconstitution in proteoliposomes, and examined the pro-

tein function [64]. This study provides direct evidence that

the water transport activity of AQP2 is enhanced approx-

imately 2-fold by phosphorylation at serine 256. In addition

to AQP2 translocation to the luminal membrane, this study

indicates that the water transport activity of individual

AQP2 proteins is involved in the regulation of water

reabsorption in kidney collecting ducts.

Actually, vasopressin increases the water permeability

by a factor of 10–100 in the collecting duct, inducing a

steep and drastic increase in water reabsorption [12]. Thus,

vasopressin-induced short-term regulation of Pf of the

collecting ducts still seems to be mainly due to AQP2

translocation and the altered water transport activity of

individual AQP2 may have a role of doubling the effect.

Long-term regulation of water permeability

of the collecting duct by altered AQP2 abundance

In addition to short-term regulation of collecting duct water

permeability, long-term regulation also plays an important

role in body water balance. Long-term regulation of col-

lecting duct water permeability is seen when water intake is

restricted for 24 h or more. This response is mainly

induced by an increase in AQP2 abundance due to

increased transcription of the AQP2 gene [65, 66].

Increased AQP2 expression levels during water restriction

is a downstream of vasopressin signaling [67, 68]. How-

ever, there are other factors influencing AQP2 expression

that is indicated by the phenomenon escape from the

antidiuretic effects of vasopressin [69]. ‘Vasopressin

escape is a phenomenon that long-term vasopressin stim-

ulation reduces its antidiuretic efficacy. Despite an increase

in plasma vasopressin, a moderate decrease in AQP2

expression is observed in this condition, contributing to this

phenomenon.

The role of AQP2 in fluctuatling osmolality

Collecting duct cells are the site of AQP2-regulated water

reabsorption, and they are exposed to great fluctuations in

osmotic pressure during transition between antidiuresis and

diuresis. The activity of the promoter of the gene that

encodes the murine AQP2 is enhanced by hypertonicity

and reduced by hypotonicity [70, 71]. Acute hypertonicity

induces AQP2 accumulation at the cell surface, and chronic

hypertonicity induces AQP2 insertion into the basolateral

membrane instead of the apical membrane [72, 73]. Cell

volume regulation in response to changes in the external

osmolality is a fundamental property of cells. When cells

are exposed to hypotonic extracellular fluid, they swell

because of the osmotic water influx. After swelling, the

cells start to recover their original volume. This cellular

defensive process against hypotonic shock is called regu-

latory volume decrease [74]. Hypotonicity induces AQP2

internalization, which may contribute to regulatory volume

decrease by limiting water entry into collecting duct cells

[75]. Furthermore, we discovered that AQP2 regulates

volume decrease by controlling the cytoskeleton [76]. As

well as being a water channel, AQP2 also has the function

of regulating the cytoskeleton, which contributes to volume

regulation properties of collecting duct cells.

Nephrogenic diabetes insipidus

Impairment of AQP2 by any cause results in NDI, which is

characterized by an inability of patients to concentrate

urine in response to vasopressin stimulation. This condition

results in a massive loss of water through the kidney,

leading to severe dehydration in the body. Unlike central

diabetes insipidus, vasopressin administration is ineffective

for patients with NDI. There are congenital and acquired

forms of NDI.

Congenital nephrogenic diabetes insipidus

In [90 % of cases of congenital NDI, the condition results

from loss-of-function mutations in the AVPR2 gene encod-

ing the vasopressin V2 receptor (X-linked NDI). The

remaining cases result from mutations in AQP2 (autosomal

NDI). AQP2, the gene that encodes AQP2, is located in

12q12-q13 and is comprised of four exons. To date, 43

mutations in the AQP2 gene have been reported (The

Human Gene Mutation Database; http://www.hgmd.cf.ac.

uk/ac/index.php) (Fig. 1; Ref. [6]). Two inheritance types

are possible for the disease—autosomal-recessive NDI is

associated with 35 mutations, and autosomal-dominant NDI

is associated with eight mutations. Almost all of the muta-

tions in recessive NDI are located in the core region of the

protein, and they lead to misfolded proteins that become

trapped in the endoplasmic reticulum and targeted for rapid

degradation by the proteasome. On the other hand, AQP2

homotetramers composed only of wild-type proteins are

properly translocated to the apical membrane. This effect

explains the healthy phenotype of the patients’ parents [77].

All mutations in autosomal-dominant NDI are located in

the cytosolic C-terminus of AQP2. This region is important

for AQP2 trafficking and these mutations impair trafficking

to the apical membrane, although the water channel

Clin Exp Nephrol

123

function of these mutants is preserved. Arg254Leu and

Arg254Gly mutations destroy the site for PKA phosphor-

ylation, so that forskolin-induced trafficking to the plasma

membrane is impaired [78, 79]. The Glu258Lys mutant of

AQP2 is missorted to multivesicular bodies and/or lyso-

somes [80]. AQP2 mutants resulting from three gene

deletions, 721delG, 763–772del and 812–818del, have

similar extended C-terminal tails, which contain the baso-

lateral-membrane-sorting dileucine (LeuLeu) motif, so

these mutated proteins are wrongly translocated to the

basolateral membrane [44, 81]. In contrast to the AQP2

mutants associated with the recessive form of the disease,

AQP2 mutants associated with the dominant form of the

disease are not misfolded, so they are able to form het-

erotetramers with WT-AQP2. Because of the dominancy of

the missorting motif in the mutant proteins, tetramers

composed of mutant and WT are missorted, which leads to

severely decreased amounts of AQP2 on the apical mem-

brane. This effect explains the dominant mode of NDI

inheritance in patients with these mutations. Sohara et al.

[82] generated gene knockin mice with the heterozygous

mutant AQP2 resulting from a gene deletion (763–772del)

that produces a mouse model of dominant NDI. The mutant

AQP2 is wrongly translocated to the basolateral mem-

brane; it forms a heterotetramer with WT-AQP2 and shows

a dominant-negative effect on the normal apical sorting of

WT-AQP2. The urine concentrating ability of these gene

knockin mice is severely reduced.

Several other mouse models of NDI caused by AQP2

mutations have also been generated. Yang et al. [83] cre-

ated mice with T126 M knockin mutation in the AQP2

gene. These homozygous mutant mice died within 6 days

after birth, suggesting that the mice may be a highly sen-

sitive organism with regard to water homeostasis, and are

unable to survive with polyuria. Lloyd et al. generated mice

with F204V mutation in the AQP2 gene that survived

beyond the neonatal period and had a much milder form of

NDI [84].

Acquired nephrogenic diabetes insipidus

Acquired NDI is more common than congenital NDI and is

cuased by various conditions including drug treatments,

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Fig. 1 AQP2 mutations causing nephrogenic diabetes insipidus

(NDI). Missense/nonsense mutations, frame shift deletions and splice

mutations that cause the autosomal-recessive form of NDI are shown

in red. Mutations that cause the autosomal-dominant form of NDI are

shown in blue. Phosphrylation sites in the C terminus are shown in

yellow

Clin Exp Nephrol

123

electrolyte disturbances, and urinary tract obstruction.

Dysregulation of AQP2 plays a fundamental role in many

acquired NDI.

Lithium is widely used for treating bipolar disorder and

20–30 % of the patients treated with lithium develop NDI

[85]. In lithium-induced NDI, both AQP2 expression and

its trafficking to the apical membrane are inhibited. Lith-

ium enters cells expressing AQP2 via the epithelial sodium

channel in the apical membrane and accumulates intra-

cellularly. This accumulation leads to the inhibition of

signaling pathways that involve glycogen synthase kinase-

3b (GSK3b). Although the mechanism by which AQP2 is

dysregulated in this context is not established, the

involvement of GSK3b is speculated. Inhibition of GSK3bby lithium increases expression of cyclooxygenase 2 and

the local excretion of prostaglandin E2 [86]. Prostaglandin

E2 is suggested to counteract vasopressin activity by

causing endocytic retrieval of AQP2 from the plasma

membrane, thus impairing the urinary concentrating ability

of the cell. Furthermore, lithium increases the intracellular

accumulation of b-catenin [87], which can serve as an

activator of T-cell factor-dependent transcription. AQP2

downregulation may be achieved via this transcription

mechanism. In addition, AQP3 expression is also

decreased. Moreover, lithium treatment caused a marked

reduction in the fraction of the principal cells in the col-

lecting duct with a parallel increase in the population of

intercalated cells [88]. This restructuring of the collecting

duct, together with down-regulation of collecting duct

AQPs, may be important in lithium-induced NDI.

Hypokalemia and hypercalcemia cause downregulation

of AQP2 expression, which results in a vasopressin-resis-

tant urinary concentrating defect. With regard to hyper-

calcemia, in addition to AQP2, the expression levels of

AQP1 and AQP3 are also decreased [89]. In addition,

hypercalcemia reduces bumetanide-sensitive sodium–

potassium–chloride cotransporter (BSC-1/NKCC2) and an

ATP-sensitive inwardly rectifier potassium channel ROMK

[90], resulting in sodium absorption defects in the thick

ascending limb, which would affect the countercurrent

multiplication.

Ureteral obstruction decreases AQP2 expression and

impairs urine concentrating capacity [91].

A urinary concentrating defect is also observed in

patients with the nephrotic syndrome [92, 93]. AQP2

expression is decreased in nephrotic rats [92, 94]; however,

changes in AQP2 expression levels have not yet been

confirmed in patients with the nephrotic syndrome.

Mouri et al. [95] reported that AQP2 translocation to the

apical membrane is inhibited by metabolic acidosis, a

mechanism that might be responsible for diuresis in

patients with chronic renal failure.

Water retention by urine diluting defects

AQP2 is also plays an important role in the pathophysiol-

ogy of water retention disorders. The best-known example

is congestive heart failure (CHF). Water retention and

hyponatremia are common and clinically important com-

plications of CHF. Plasma vasopressin levels are sup-

pressed by hyponatremia in healthy individuals; however,

these levels are not suppressed in patients with hypona-

tremia who have CHF [96, 97]. In CHF, a decrease in

effective blood volume and arterial filling is sensed by left

atrial baroreceptors, resulting in stimulation of vasopressin

secretion. Upregulation of AQP2 expression and increased

AQP2 trafficking to the apical membrane of principal cells

of the collecting duct have been shown in rat models of

cardiac failure [98, 99]. Futhermore, water retention and

hyponatremia in these rats are reversed by a V2 receptor

antagonist [99]. These findings indicate that hyponatremia

is caused by nonosmotic stimulation of vasopressin, which

promotes the expression and trafficking of AQP2. In

patients with heart failure, V2 receptor antagonists promote

electrolyte-free water excretion and elevate serum sodium

concentration [100–102]. Tolvaptan, a vasopressin antag-

onist, has been shown to improve several symptoms of

heart failure, such as dyspnea, in these patients [103].

Water retention and hyponatremia are observed in

patients with hepatic cirrhosis. In these patients, nonos-

motic secretion of vasopressin occurs secondary to

splanchnic arterial vasodilation and relative arterial un-

derfilling [97]. In cirrhotic rats, AQP2 expression was

increased and correlated with the volume of ascites [104].

In patients with hyponatremic cirrhosis, V2 receptor

antagonists are effective at inducing free water diuresis and

raising plasma sodium levels [101, 105].

During pregnancy, arterial underfilling secondary to

systemic arterial vasodilation with nonosmotic vasopressin

secretion and upregulation of AQP2 is observed [106, 107].

Administration of a V2 receptor antagonist increases

electrolyte-free water excretion in pregnant rats [106].

Syndrome of inappropriate antidiuretic hormone secre-

tion (SIADH) is a condition in which plasma vasopressin

levels are not appropriately suppressed despite hypo-

osmolality. Vasopressin-dependent antidiuresis leads to

impaired water excretion and hyponatremia. SIADH is the

predominant cause of euvolemic hyponatremia and a

commonly encountered disorder [108]. In the chronic

vasopressin excess condition, however, antidiuresis is

attenuated, resulting in water diuresis to some extent. This

has been called the ‘vasopressin escape’ phenomenon

[109]. Saito et al. [71, 109] showed that the impaired uri-

nary concentrating ability in SIADH is caused by dimin-

ished expression of AQP2 in rats.

Clin Exp Nephrol

123

SIADH occurs frequently in association with vascular,

infectious or neoplastic abnormalities in the lung or central

nervous system. In patients with SIADH, the V2 receptor

antagonist OPC-31260 was shown to be effective in

increasing urine volume and plasma sodium levels [110].

However, the long-term effects of its administration are

limited in rats with SIADH [111]. Although AQP2 protein

expression is reduced shortly after administration of the V2

receptor antagonist to rats with SIADH, it increases again

in parallel with the decline of the therapeutic effects.

Urinary AQP2 excretion level is associated with vaso-

pressin activity in the kidney and is, therefore, a clinically

useful biomarker [112, 113]. AQP2 is excreted into the

urine through the secretion of exosomes originating from

internal vesicles of multivesicular bodies [114]. During this

process, the outer membrane multivesicular bodies fuse

with the apical plasma membrane. Urinary AQP2 excretion

is increased by dehydration or vasopressin and decreased

by hydration. Urinary AQP2 excretion is also increased in

patients with CHF and hepatic cirrhosis and in pregnant

women [115–117]. In patients with CHF, administration of

a V2 receptor antagonist produced a significant increase in

urine flow and solute-free water excretion, accompanied

with a dramatic decrease in urinary AQP2 excretion [115].

Augmentation of urinary excretion of AQP2 is also found

in SIADH [118]. Urinary excretion of AQP2 is a sensitive

marker of the antidiuretic activity of vasopressin.

Therapeutic development for water balance disorders

by targeting AQP2 regulation

There is no cure for NDI. Currently, this condition is

managed by salt restriction combined with the adminis-

tration of hydrochlorothiazide diuretics to reduce urine

output [119]. Hydrochlorothiazide reduces sodium reab-

sorption in the distal convoluted tubule, leading to increase

in sodium excretion and extracellular fluid volume con-

traction. As a result, glomerular filtration rate decreases

and the proximal tubular sodium and water reabsorption

increases. Consequently, less water and sodium are deliv-

ered to the collecting ducts, resulting in decrease in urine

volume. This antidiuretic effect is enhanced by a low

sodium intake. Moreoever, hydrochlorothiazide increases

expression of AQP2 and distal renal sodium transporters,

which may also contribute to this antidiuretic action [120].

Additional administration of prostaglandin synthesis

inhibitors or the potassium-sparing diuretic amiloride

enhances the effectiveness of NDI management, although

long-term use of prostaglandin inhibitors is often compli-

cated by gastrointestinal and hematopoietic adverse effects

and renal dysfunction. In any event, current treatment does

not completely obviate the excessive water excretion, as

adult patients undergoing treatment still void 8–10 L per

day. Therefore, extensive efforts to develop therapies are

continuing. Chemical chaperones that facilitate folding of

the mutant protein have been reported to correct the sorting

of NDI-causing AQP2 mutants in cell cultures. However,

chemical chaperones are not suitable for use in vivo

because of the high concentrations that would be required

to achieve clinically meaningful results.

AQP2 phosphorylation by cGMP kinase is involved in

its exocytosis, and the cGMP phosphodiesterase inhibitor

sildenafil citrate induces AQP2 membrane insertion [20,

21]. Therefore, cGMP phosphodiesterase inhibitors are

expected to be effective in the treatment of NDI due to

V2R impairment by bypassing the need for cAMP signal-

ing for AQP2 membrane insertion. However, in healthy

individuals, use of sildenafil citrate has not been associated

with either water retention or hyponatremia, which might

mean that their effect on AQP2 translocation is, at least in

healthy people, negligible.

Li et al. [121] generated an animal model for X-linked

NDI, in which AVPR2, the gene that encodes V2R, was

conditionally deleted. These mice showed symptoms of

NDI, such as urinary concentrating defects and dilatation of

the renal pelvis. An agonist of the EP4 subtype of the

prostaglandin E receptor proved highly effective in ame-

liorating these manifestations of NDI.

Sohara et al. [82] investigated in mutant AQP2 knockin

mice whether phosphodiesterase inhibitors affect the uri-

nary concentration ability of these mice. Among the

inhibitors tested, rolipram increased urine osmolality,

cAMP content in the papillae, AQP2 phosphorylation, and

apical membrane translocation of the mutated AQP2.

Interestingly, rolipram also induced the apical translocation

of WT-AQP2 as a consequence of dehydration.

We showed the interaction of phosphorylated AQP2

with TM5b is essential for AQP2 trafficking to the apical

membrane, suggesting that TM5b is a potential therapeutic

target for NDI [5, 36, 37, 60]. Knockdown of the gene

encoding TM5b corrects the trafficking defect of the

Ser256Ala AQP2 mutant. Specific inhibition of TM5b may

be useful for both congenital and acquired NDI because the

interaction between TM5b and phosphorylated AQP2 is

critical for the final step of AQP2 trafficking.

Suga et al. [122] succeeded in developing a method for

the targeted expression of AQP2 in collecting ducts using

the sendai virus vector system that did not contain the

fusion protein gene (SeV/DF). This vector system directs

high-level transgene expression and is nontransmissible.

Furthermore, in an animal model of NDI, this vector was

administered to the animal retrogradely via ureter to renal

pelvis, which enabled its efficient expression in the col-

lecting duct in a limited area of renal medulla. Viral

delivery of AQP2 in a lithium-induced rat model of NDI

Clin Exp Nephrol

123

led to a reduction in urine output and an increase in urine

osmolality for several days. The authors suggest that this

strategy may be beneficial in patients with NDI, especially

those who are unconscious or in a perioperative situation

because this treatment approach can temporarily reverse

the polyuria phenotype.

AQP2 also plays a critical role in water retention disor-

ders such as CHF, hepatic cirrhosis, and SIADH. Vaso-

pressin V2 antagonists (vaptans) are effective for water

retention and hyponatremia by inducing free water diuresis

as described above. They bind to the V2 receptor, preventing

the hormone’s downstream signaling pathway, resulting in a

decrease in the amount of AQP2 on the luminal membrane.

Tovaptan is shown to improve several symptoms of CHF in

the short-term; however, it has no effect on the long-term

prognosis [103, 123]. There are several studies evaluating

long-term V2 receptor antagonist therapy in chronic hypo-

natremia [124, 125]. These studies showed that serum

sodium increased. However, there is no study showing

improvement in ‘hard’ outcomes such as hospitalization,

morbidity, and mortality. In addition, V2 receptor has

downstream effects other than AQP2 function. It is expected

to develop drugs directly targeting AQP2 that may be more

specific and effective for water retention disorders as a pure

aquaretic. We showed that phosphorylation-induced AQP2

interaction with TM5b is essential for AQP2 targeting to the

luminal membrane [5, 36, 37, 60]. A drug to inhibit the

interaction between AQP2 and TM5b without escape or

side-effects may be a more effective aquaretic.

Water balance disorders commonly occur in many dis-

eases. For NDI, there is currently no cure. Water retention

and hyponatremia are often difficult to manage and worsen

the prognosis of patients with CHF, hepatic cirrhosis and

neurological diseases. Drug development targeting AQP2

is a promising research field for appropriate therapy for

water balance disorders.

Conflict of interest The author declares no competing interest.

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