REVIEWS

Aquaporin 2 regulation: implications

for water balance and polycystic
kidney diseases
Emma T. B. Olesen   1,2 ✉ and Robert A. Fenton   3 ✉
Abstract | Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma
membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2
receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels,
has a major role in this targeting process. Although a rise in cAMP levels and activation of protein
kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models
have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated
AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a
known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2
phosphorylation events appears to be rate-limiting in this process, which suggests the
involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several
regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in
the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2
in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that
might be exploited for the treatment of water balance disorders and provide a rationale for
targeting these pathways in autosomal dominant polycystic kidney disease.

Single effect
The difference in osmotic
pressure that is created by
the active transport of sodium
chloride out of the tubular fluid in
the thick ascending limb into the
interstitium; the hyperosmotic
gradient in the interstitium drives
the passive movement of water
from the tubular fluid of the
descending limbs and collecting
ducts into the interstitium.
1
Department of Biomedical
Sciences, Faculty of Health
and Medical Sciences,
University of Copenhagen,
Copenhagen, Denmark.
2
Department of Endocrinology
and Nephrology, North Zealand
Hospital, Hillerød, Denmark.
3
Department of Biomedicine,
Faculty of Health, Aarhus
University, Aarhus, Denmark.
✉e-mail: etbo@sund.ku.dk;
robert.a.fenton@
biomed.au.dk
https://doi.org/10.1038/
s41581-021-00447-x
A cornerstone of internal medicine is the assessment
and treatment of water and electrolyte disorders, and
the mechanisms underlying water preservation are a
major aspect of kidney physiology. Adult human kid-
ney glomeruli filtrate on average 180 l of plasma per day
and 99% of the filtered water is reabsorbed back into the
circulation through the epithelium of the kidney tubule
and the collecting duct. This reabsorption process occurs
mainly through transcellular water transport, which is
crucially dependent on osmosis-driven water movement
through aquaporin (AQP) water channels (Fig. 1). The
kidney proximal tubule and the adjacent initial portion
of the long-looped thin descending limb1 are constitu-
tively water permeable and reabsorb ~90% of filtered
water. AQP1, which is present in the apical and basolat-
eral plasma membrane in these segments, and potentially
AQP7 in the apical plasma membrane of the proximal
straight tubule, are responsible for this water flux1,2. The
remaining portion of the loop of Henle and the distal con-
voluted tubule (DCT) are impermeable to water and the
transport of ions in these segments occurs mainly through
secondary active transport against the transepithelial
gradient. In the thick ascending limb (TAL), Na+ and Cl−
reabsorption through Na–K–Cl cotransporter 2 (NKCC2;
also known as SLC12A1 or BSC1) and K+ recycling via
renal outer medullary potassium channel 1 (ROMK1;
encoded by KCNJ1) provide the single effect for the coun-
tercurrent multiplication mechanism3, which dilutes the
urine and generates a corticomedullary osmotic gradient
in the interstitium. In combination with urea transport
processes, this gradient provides the driving force for
regulated water reabsorption in collecting duct principal
cells, which express AQP2, AQP3 and AQP4. The cellu-
lar abundance of AQPs 2–4 and the presence of AQP2 in
the apical plasma membrane, which is crucial to water
reabsorption, are regulated by the antidiuretic peptide
hormone vasopressin (also known as arginine vasopres-
sin or antidiuretic hormone). Vasopressin is released
from the neurohypophysis in response to increases in
plasma osmolality or activation of baroreceptors through
decreases in effective circulating volume (Fig. 1). In the
collecting duct, vasopressin-mediated stimulation of
the vasopressin V2 receptor (V2R; encoded by AVPR2),
which is a GS protein-coupled receptor (GSPCR), increases
cAMP levels and activates protein kinase A (PKA). In the
TAL, stimulation of V2R by vasopressin also enhances
the countercurrent multiplication and maximizes the
osmotic gradient required for water reabsorption.
Defects in the vasopressin–AQP2 axis result in water
balance disorders (reviewed in refs4,5). The majority of

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Key points
• Targeting of the collecting duct water channel aquaporin 2 (AQP2) to the plasma
membrane is essential for the maintenance of mammalian water homeostasis.
• Although vasopressin signalling via the Gs protein-coupled vasopressin V2 receptor
(V2R) is a major receptor-mediated pathway that modulates trafficking of AQP2 is a major adverse effect of tolvaptan treatment.
to the plasma membrane, this targeting can also occur independently of V2R.
• The canonical V2R signalling pathway — increased cAMP levels, activation of protein
kinase A (PKA) and AQP2 phosphorylation — cannot fully explain AQP2 plasma
membrane targeting, and cAMP–PKA-independent pathways exist.
• Novel regulators of AQP2 plasma membrane targeting that are independent of V2R
and cAMP include A-kinase anchoring protein (AKAP)–PKA disruptors, Src inhibition,
Wnt5a, fluconazole and epidermal growth factor receptor antagonists.
• Research on the signalling networks and regulatory steps that govern AQP2
trafficking has identified promising pharmacological targets for water balance
disorders.
• Investigating plasma membrane targeting of AQP2 using agents that do not increase
cellular proliferation and epithelial secretion is required to delineate the independent
role of AQP2 in cyst progression in autosomal dominant polycystic kidney disease.
these disorders arise from inappropriately high water
reabsorption despite normal extracellular osmolality,
for example, due to vasopressin secretion in response
to decreased effective circulating volume in the absence
of alterations in plasma osmolality. Water is conse-
quently reabsorbed by the kidney despite an isosmotic or
hypo-osmotic extracellular microenvironment. Patients
with these disorders mainly present with oedema, which
occurs frequently in patients with heart failure or liver
cirrhosis, and hyponatraemia, which is a hallmark of
the syndrome of inappropriate antidiuretic hormone.
Conversely, loss of vasopressin-secreting neurons, for
example, due to traumatic brain injury, leads to central
diabetes insipidus, which is characterized by reduced
circulating vasopressin, reduced AQP2 and low urine
osmolality compared with healthy individuals. Central
diabetes insipidus frequently causes dehydration and
hypernatraemia, but responds to exogenous vasopressin
administration6. Reductions in AQP2 levels or plasma
membrane accumulation can also be caused by lithium
treatment or hypercalcaemia, or, in very rare cases, by
genetic disorders that disrupt the function of the V2R
or AQP2. This loss of AQP2 or V2R functionality
causes vasopressin-resistant polyuria, which is termed
nephrogenic diabetes insipidus (NDI) and can lead to
hypernatraemia and dehydration if high water intake
is not maintained. Understanding the regulation of
water balance at the molecular level might therefore not
only improve the therapeutic options for the treatment
of water balance disorders, but might also clarify the
pathophysiology of diseases that originate in the kidney
tubular system.
In this Review, we highlight new developments in
the field of AQP2 regulation, with a particular focus
on the intracellular signalling pathways that link recep-
tor stimulation to the targeting of AQP2 to the plasma
membrane. In addition, we discuss the possible role
of water transport in an important kidney tubular
disease — autosomal dominant polycystic kidney disease
(ADPKD). Specific treatments that can prevent kidney
failure in high-risk patients with ADPKD are not cur-
rently available, although the V2R antagonist tolvaptan
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delayed disease progression in clinical trials7–9. We dis-
cuss how advances in the field of AQP2 regulation might
translate to clinical benefit for patients with ADPKD
and might lead to the development of novel treatment
options to treat ADPKD and/or alleviate polyuria, which
Acute regulation of water balance
The importance of vasopressin and the V2R for
the maintenance of water homeostasis is evident in
Brattleboro rats, which lack vasopressin secretion and
develop central diabetes insipidus10, and in mice with
an Avpr2 deletion11, which triggers NDI (Table 1). The
V2R is expressed in cells of the TAL, DCT and inner
medulla collecting duct (IMCD), where it stimulates
phosphorylation and activity of NKCC2, the NaCl
cotransporter NCC (also known as SLC12A3) and
AQP2, respectively12–15. Of note, V2R expression is lower
in the TAL and DCT than in the collecting duct, and
vasopressin-induced cAMP levels are 15–20-fold lower
in the TAL than in the collecting duct12,16,17. The V2R
selectively activates adenylate cyclase type 6 (AC6) in the
IMCD18 to produce cAMP, which is a major V2R second
messenger and leads to the activation of a range of protein
kinases, including PKA; V2R activation also increases
intracellular calcium19. Although the actions of the V2R
are crucial for maintaining urine concentration at base-
line, rats treated with a V2R antagonist could increase
urine osmolality during water restriction20, which sug-
gests that receptors other than V2R might contribute
to water homeostasis (water restriction experiments
in Avpr2-knockout mice have not been reported).
Alternative receptors shown to be important for
regulation of AQP2 include the angiotensin II receptor
type I (AT1; encoded by AGTR1), the bile acid recep-
tor (also known as farnesoid X-activated receptor), the
oxysterols receptor LXRβ, the oestrogen receptor (also
known as ERα), the peroxisome proliferator-activated
receptor-γ, and the glucocorticoid and mineralocorticoid
receptors21–24. The G protein-coupled bile acid receptor 1
(encoded by GPBAR1; also known as TGR5) can also
inc­rease cAMP levels, AQP2 protein abundance and
AQP2 plasma membrane targeting in IMCD cells,
and Gpbar1-knockout mice had a urine-concentrating
defect both at baseline and throughout a 36-h period
of water restriction23. However, whether GPBAR1 has a
direct role in water homeostasis in the collecting duct
remains unclear because Gpbar1-knockout mice also
have reduced vasopressin levels. AQP2 plasma mem-
brane targeting and/or cellular water permeability can
also be increased by the GPCR ligands calcitonin25,
glucagon26, secretin27, angiotensin II28 or selective ago-
nists for the prostaglandin receptors EP2 (ref.29) and EP4
(ref.11), which suggests that alternative GPCRs might
have a role in water balance, especially during water
restriction.
Novel aspects of AQP2 regulation
Long-term vasopressin-mediated regulation of AQP2
involves protein synthesis and degradation, whereas
acute regulation involves apical plasma membrane inser-
tion and retrieval. At baseline (that is, in non-stimulated
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Stimulation of baroreceptors
or osmoreceptors
Hypothalamus

Pituitary
gland
Vasopressin
release

PCT

TAL CD

Urine Cl– Na K+
+
K+
H2O H2O H2O H2O
ROMK1

NKCC2

Apical
AQP1 AQP7 K+ AQP2
Endocytosis

PCT TAL CD
Translocation
Translocation Activation
AQP3 AQP4
Basolateral
V2R
Vasopressin
H2O H2O H2O
Blood

Recycling endosomes
Organelles in the endocytic
pathway, in which plasma
membrane proteins and lipids
that are internalized by
endocytosis are sorted and
processed for export back to
the cell surface for reuse.
Fig. 1 | Kidney aquaporins and transcellular water transport. Most of the glomerular filtrate is reabsorbed iso-osmotically
through aquaporin 1 (AQP1) in the proximal convoluted tubule (PCT) and, to a lesser extent, through AQP7 in the late
proximal tubule segments; AQP1 is also expressed in the thin descending limbs and descending vasa recta (not shown).
Water reabsorption in these segments of the kidney is not regulated by vasopressin, which in response to a reduction in
circulating blood volume or an increase in plasma osmolality, is released from the pituitary gland following the activation of
baroreceptors or osmoreceptors in the hypothalamus. In the kidney, vasopressin binds to the vasopressin V2 receptor (V2R)
and enhances NaCl reabsorption in the thick ascending limb (TAL) via the concerted actions of Na–K–Cl cotransporter 2
(NKCC2) and renal outer medullary potassium channel 1 (ROMK1), which increases interstitial osmolality. Vasopressin also
enhances AQP2 levels in the apical plasma membrane of collecting duct (CD) principal cells by stimulating AQP2
translocation to the apical plasma membrane from intracellular storage vesicles and inhibiting AQP2 endocytosis. The
sustained expression of AQP2 in the apical plasma membrane, in combination with basolateral expression of AQP3 and
AQP4, provides a pathway for osmotic reabsorption of water.
cells), the trafficking pool of AQP2 is predominantly which renders it permeable to water. Upon removal of
present in recycling endosomes30. Following stimulation vasopressin, AQP2 builds up in clathrin-coated pits in
of the collecting duct principal cell, these vesicles traf- the plasma membrane of AQP2-transfected LLC-PK1
fic, dock and fuse with the apical plasma membrane, cells (AQP2–LLC-PK1; Table 1), where it undergoes

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Table 1 | Advantages and limitations of experimental models used in AQp2 research

Model Description
IMCD cells Primary culture derived from
freshly isolated inner medulla
mpkCCD14 Immortalized cell line derived
cells from mouse cortical collecting
duct
MCD4 cells Cell line derived from mouse
cortical collecting duct and
transfected with AQP2
MDCK–AQP2 Canine kidney epithelial cells
cells transfected with AQP2
(AQP2–) Porcine proximal tubule cell line
LLC-PK1 cells transfected with AQP2
Brattleboro rats Rats with a spontaneous single-
nucleotide mutation in Avp that
results in defective vasopressin
secretion
Pkd1-knockout Mice with inducible Pkd1
mice deletion or Pkd1+/− mice
AQP2, aquaporin 2; dDAVP, D-amino D-arginine vasopressin; IMCD, inner medulla collecting duct; MDCK, Madin–Darby canine kidney; V2R, vasopressin V2 receptor.
Benefits
Retain most features of native IMCD including
V2R expression and (inducible) expression of
AQP2
Retain many features of native tissue
Express V2R, and V2R stimulation induces AQP2
expression
Form polarized monolayers on semi-permeable
supports and AQP2 is targeted to the apical
plasma membrane in response to vasopressin
Collecting duct cell line
No previous stimulation is required for
constitutive AQP2 expression
AQP2 accumulates on the apical membrane
in response to V2R stimulation
Native AQP2 expression is low and cells are
thus suitable for transfection with wild-type
AQP2 or AQP2 harbouring point mutations (for
example, mutations of specific phosphorylation
or ubiquitylation sites)
Cells form polarized monolayers on
semi-permeable supports
Stimulation with vasopressin increases cAMP
levels and AQP2 plasma membrane targeting
No native AQP2 expression, thus cells are
suitable for transfection with wild-type or
mutated AQP2
A rat model for studying vasopressin-
independent mechanisms
Response to vasopressin or dAVP is higher than
in wild-type rats, making it a useful model for
studying vasopressin effects in general
Mice survive until adulthood, whereas Pkd1−/−
mice die perinatally
Inducible Pkd1 deletion leads to cyst
development within weeks of induction
Cautionary issues
Need to be stimulated with cAMP or
vasopressin to sustain AQP2 expression;
cAMP or vasopressin are usually removed
~16 h before experiments
Possible inter-experimental variation in
cell growth or cell purity
AQP2 traffics primarily to the basolateral
membrane following V2R stimulation
V2R is internalized in culture
Immortalized cells might differ from native
tissue
Often cultured in media containing
hormones and/or growth factors
Usually stimulated with dDAVP for several
days to induce AQP2 expression before
experiments; dDAVP is usually removed
1–4 h before experiments
Transfected cell line
Regulation of Aqp2 transcription cannot
be investigated owing to the use of a
non-native promoter
Variable response to V2R stimulation
and hence studies of AQP2 membrane
targeting are frequently performed with
the adenylyl cyclase stimulator forskolin
Tubular origin not completely clarified
AQP2 reportedly traffics to apical and
basolateral sides
Transfected cell line
Limited availability of commercial
antibodies reactive to canine epitopes
Transfected cell line
Not a collecting duct cell line
Does not consistently respond to dDAVP
Studies require the use of vasopressin and
might be confounded by the activity of
V1Rs
AQP2 reportedly traffics to the basolateral
membrane upon stimulation with
vasopressin
Rats might have enhanced vasopressin-
independent V2R signalling owing to
binding of alternative hormones such
as oxytocin
Inducible deletion requires chemical
induction
Pkd1+/− mice have slow cyst progression
(occurs at 7–8 months of age)

dynamin-dependent endocytosis31. In vivo, and in stud-
ies with primary IMCD cells and the mpkCCD14 cell line
(Table 1), AQP2 colocalizes with clathrin, the early endo­
some markers Ras-related protein Rab5A–early
endosome antigen 1 (EEA1) and the lysosomal marker
cathepsin D at baseline; however, whether endocytosis
of AQP2 in these experimental systems is also depend-
ent on dynamin is unclear32–35. Following endocytosis,
Rab7 and vacuolar protein sorting-associated pro­tein 35
(Vps35) are required for sorting AQP2 within Rab5+
early endosomes, either to the lysosome for degradation36
or to the plasma membrane via recycling endosomes

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Transcytosis
The transport of
macromolecular cargo from
one side of a cell to the other
within a membrane-bounded
carrier (or carriers); this
mechanism is used by
multicellular organisms to
selectively move material
between two environments
without altering their unique
compositions.
Multivesicular bodies
(MVBs). Specialized
endosomes that contain
membrane-bound intraluminal
vesicles formed by budding of
the membrane into the lumen
of the MVB; MVBs can fuse
with lysosomes, where their
contents are degraded, or they
can fuse with the plasma
membrane for their contents to
be released into the
extracellular space.
(Rab11+)33,37. In addition to the recycling endosome
pathway, AQP2 can traffic from the basolateral to the
apical plasma membrane via microtubule-dependent
transcytosis38,39. Below, we provide a brief overview of
new findings in the field of AQP2 regulation, includ-
ing novel insights into the role of post-translational
modifications and cytoskeletal remodelling.
AQP2 phosphorylation and ubiquitylation
AQP2 exists functionally as a tetramer (Fig. 2a). Each
monomer consists of six membrane spanning α-helices
and two of the connecting loops contain Asn–Pro–Ala
(NPA) motifs that interact in the membrane to form the
pathway for translocation of water (Fig. 2b,c). Stimulation
with vasopressin triggers a variety of phosphorylation
events in the collecting duct40,41, including changes in
the phosphorylation of AQP2 (ref.42) at the carboxyl
terminus. In mammals, AQP2 can be phosphorylated
at four sites — Ser256, Ser261, Ser264 and Ser269 (in
humans, Ser269 is conserved as Thr269) (Fig. 2d). Ser256
and Ser269 seem to be the most relevant to AQP2 tar-
geting and accumulation in the plasma membrane. The
Ser256 site is essential for vasopressin-induced accu-
mulation of AQP2 in the plasma membrane in the cell
models studied thus far. However, AQP2 seems to be
constitutively phosphorylated at this site in vivo, with
only minor increases following vasopressin stimulation,
which suggests that Ser256 phosphorylation alone is not
the rate-limiting step of AQP2 plasma membrane target-
ing in vivo during V2R stimulation43. By contrast, AQP2
phosphorylation at Ser269 markedly increases in vivo in
the apical plasma membrane42 during V2R stimulation43.
However, although this site seems to be involved in
AQP2 accumulation in the plasma membrane (see
below), studies of cell lines with mutations in this resi-
due suggest that it is not required for cAMP-dependent44
or rapid V2R-mediated targeting of AQP2 to the plasma
membrane45. Furthermore, the precise role of Ser269 in
urine concentration and water homeostasis has not been
addressed.
Emerging evidence indicates that phosphoryl-
ation of AQP2 facilitates its protein–protein bind-
ing properties and consequently affects its cellular
distribution46,47. For example, AQP2 phosphorylation
at Ser256 reduces its binding to the lysosomal traffick-
ing regulator LYST-interacting protein 5 (LIP5; also
known as vacuolar protein sorting-associated protein
VTA1 homologue) and is predicted to reduce the sort-
ing of AQP2 into multivesicular bodies (MVBs), decrease
its lysosomal degradation and increase AQP2 protein
half-life44,48. Ser269 is contained within the class I PDZ
motif of AQP2 (Fig. 2d) and might enhance its interaction
with PDZ domain-containing proteins such as sorting
nexin-27, which directs AQP2 away from lysosomes;

a H2O b
AQP2 N P A
monomer
Extracellular

1 2 3 4 5 6

Intracellular
N P A
N terminus C terminus
H2O

c d
H2O

221 F
P
1 P
2

A K S L A
L S E R V
N N L
3 K
256

P P
4 A
5 RV E R E G
A V S Q R E L
E R W E
D T
L D P
6

H
S PDZ motif
6 1 P
2 G S K A Ubiquitylation
Q S
L PR Phosphorylation
269
270
264

Fig. 2 | AQp2 topology and structure. a | Aquaporin 2 (AQP2) exists as a tetramer in the plasma membrane and each
monomer contains a single pore through which water molecules pass in a single file. b | Simple topography of an
AQP2 monomer. A single monomer of an AQP2 channel consists of six membrane-spanning α-helices and short intracellular
amino and carboxy termini. Almost all AQPs have two highly conserved Asn-Pro-Ala (NPA) motifs in each monomer.
c | The NPA sequences of each monomer have been proposed to interact in the plasma membrane to form a pathway for
translocation of water across the plasma membrane. d | Carboxy-terminal tail of AQP2, highlighting the phosphorylation
sites and ubiquitylation sites that are most relevant to AQP2 trafficking. Adapted with permission from ref.152, American
Physiological Society.

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Urine

H2O H2O H2O H2O Apical plasma H2O

AQP2 membrane

Ub P P P P P H2O P P

CHIP–NEDD4
Ubiquitylation Stabilization SIPA1-like
protein 1
P P
Endocytosis Actin
depolymerization
Ub Recycling

Ub Ub USP4 P

Ub P P
Degradation Proteasome
P
Actin
filaments
Golgi
P
cAMP–PKA
independent P
Degradation
Lysosome
Native
Misfolded AQP2 P P
AQP2 RhoA
PKA and/or
CHIP

Ub ER other kinases

AQP2
cAMP

Basolateral Ca 2+

plasma H2O H2O

AQP3 Cytosol
membrane

AQP4 V2R
Blood H2O H2O Vasopressin

Stimulation P Ser256 phosphorylation Ub Ubiquitin

Inhibition P Ser269 phosphorylation

Fig. 3 | Main mechanisms underlying the regulation of AQp2 trafficking. The figure illustrates some of the major
pathways of aquaporin 2 (AQP2) regulation, including simplified depictions of the roles of phosphorylation and
ubiquitylation. Natively folded AQP2 traffics to the Golgi apparatus, is organized into tetramers and stored in transport
vesicles. In the classical pathway, vasopressin stimulation of the vasopressin V2 receptor (V2R) causes an increase in the
second messenger cAMP, which activates protein kinase A (PKA), and leads to phosphorylation of AQP2 at Ser256 and
redistribution of AQP2-containing vesicles to the apical plasma membrane. However, activation of this pathway is not an
absolute requirement for AQP2 trafficking and cAMP–PKA-independent mechanisms exist, including the phosphorylation
of AQP2 by other protein kinases, such as AMP-activated protein kinase (AMPK), and Rho-dependent remodelling of the
actin cytoskeleton. In the apical plasma membrane, phosphorylation at Ser269 stabilizes AQP2 and limits its interaction
with the signal-induced proliferation-associated 1-like (SIPA1-like) protein 1, which reduces SIPA1-like protein 1-mediated
AQP2 endocytosis. In the plasma membrane, AQP2 can be ubiquitylated by the E3 ubiquitin ligases CHIP and NEDD4,
which promotes its endocytosis. Following AQP2 internalization, activity of the deubiquitylase ubiquitin carboxyl-terminal
hydrolase 4 (USP4) determines whether AQP2 is deubiquitylated and recycled to the plasma membrane, or targeted for
lysosomal degradation. ER, endoplasmic reticulum. Adapted with permission from ref.152, American Physiological Society.

K63-linked polyubiquitin
chain
A chain of ubiquitin molecules
that are linked to each other on
Lys63 (one of seven lysine
residues within ubiquitin) to
form a long polyubiquitin
chain; these chains can mark
the ubiquitylated protein for
endocytosis.
however, the role of Ser269 in this interaction is
unclear49. Signal-induced proliferation-associated 1-like
protein 1 (SIPA1-like protein 1) also includes a PDZ
domain45 and its interaction with AQP2 is inhibited by
Ser269 phosphorylation, which reduces SIPA1-like pro-
tein 1-mediated AQP2 endocytosis and might explain
why Ser269 phosphorylation prolongs AQP2 retention
in the plasma membrane (Fig. 3).
Ubiquitylation of AQP2 at Lys270 (Fig. 2d) with one
K63-linked polyubiquitin chain enhances AQP2 endocy-
tosis to MVBs, after which it is either targeted to lyso-
somes for degradation or excreted in exosomes50. Several
ubiquitin-ligating enzymes (E3 ligases), including
NEDD4 and NEDD4-like (also known as NEDD4-1 and
NEDD4-2, respectively)51–53, and the carboxyl terminus
of heat shock protein 70 (HSP70)-interacting protein
CHIP54–56 (encoded by Stub1), plus the deubiquitylase
ubiquitin carboxyl-terminal hydrolase 4 (USP4)57, have
been implicated in ubiquitin-dependent AQP2 degrada-
tion (Fig. 3). In cultured mpkCCD14 cells treated with for-
skolin (an adenylate cyclase activator) to enhance AQP2
membrane accumulation, removal of forskolin increased
AQP2 ubiquitylation before AQP2 endocytosis50. A sim-
ilar increase in AQP2 ubiquitylation was observed in

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F-actin
A filamentous polymer
composed of soluble G-actin
monomers; it is the most
abundant component of the
cytoskeleton of eukaryotes.
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mpkCCD14 cells stimulated with d-amino d-arginine
vasopressin (dDAVP; a synthetic vasopressin ana-
logue that acts mainly on the V2R) followed by dDAVP
removal58, but not in another study, where AQP2 ubiq-
uitylation levels (and AQP2 membrane accumulation)
were highest in the presence of dDAVP59. Taken together,
although these studies suggest that deubiquitylation of
AQP2 can reduce its endocytosis and degradation dur-
ing dDAVP stimulation, whether ubiquitylation has a role
in AQP2 internalization processes after dDAVP removal
is unclear. Furthermore, whether increased AQP2 ubiq-
uitylation is a process to remove AQP2 from the plasma
membrane in vivo when vasopressin levels are no longer
raised (plasma osmolality is normal) is unclear.
Of note, AQP2 phosphorylation and ubiquityla-
tion seem to be interconnected. Phosphorylation at
Ser256 and/or Ser269 seemingly counterbalances the
endocytosis-promoting effects of polyubiquitylation,
whereas low Ser261 phosphorylation is associated with
low polyubiquitylation and high cellular and membrane
abundance of AQP2 (refs58,60). Our understanding of the
physiological roles of V2R-mediated post-translational
modifications of AQP2 would benefit from in vivo val-
idation experiments using CRISPR–Cas9 approaches,
to eliminate one or more of the phosphorylation or
ubiquitylation sites.
The cytoskeleton and plasma membrane targeting
Principal cells have a dense cortical actin network that
limits docking of AQP2-containing vesicles; however,
vasopressin stimulation causes actin depolymeriza-
tion in both the apical cell cortex and the basal stress
fibres61. Under baseline conditions, actin polymeriza-
tion is maintained by the small GTPase RhoA, but this
effect is inhibited by V2R stimulation62–64 (Fig. 3). AVPR2
mutations that render V2R constitutively active (R137L
and R137C) and increase the osmotic water permea-
bility of AQP2-expressing cells65 have been identified
in humans66. Their effects on water permeability are
PKA-independent but Rho-associated protein kinase
(ROCK)-dependent, and are associated with increased
phosphorylation of AQP2 at Ser269 compared with
non-mutated (wild-type) V2R. Although this obser-
vation suggests that Ser269 phosphorylation might be
required for Rho-mediated and ROCK-mediated AQP2
plasma membrane insertion, increased ROCK activity
was not important for V2R-mediated Ser269 phosphory­
lation or vasopressin-stimulated water permeability in
wild-type cells. These observations suggest that altered
ROCK activity might only be important for AQP2
plasma membrane targeting in pathological conditions.
The trace metal manganese (Mn) can enhance the
polymerization of G-actin to form F-actin by binding
directly to a metal-binding site on G-actin. In vitro,
exposure of LLC-PK1 cells that stably expressed AQP2
or rat IMCD cells that expressed AQP2 to MnCl2 inhib-
ited AQP2 targeting to the plasma membrane and
promoted its internalization, which resulted in intra-
cellular accumulation67. Accordingly, MnCl2 admin-
istration to mice reduced the accumulation of AQP2
in the plasma membrane of principal cells relative to
vehicle-treated controls and led to a vasopressin-resistant
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urine-concentrating defect. MnCl 2-induced intra-
cellular accumulation of AQP2 was associated with
decreased phosphorylation (and thus increased acti-
vation) of RhoA and increased F-actin polymerization.
Vasopressin-induced phosphorylation of AQP2 in vitro
at Ser256, Ser264 and Ser269 was also significantly
decreased by MnCl2, but whether this effect was due to
effects of MnCl2 on V2R-mediated signalling pathways,
or the altered cellular distribution of AQP2 (for example,
reduced Ser269 phosphorylation owing to reduced levels
of AQP2 in the plasma membrane) remains unclear42.
The internalization of AQP2 from the plasma mem-
brane involves ezrin68, which is a member of the ezrin,
radixin and moesin (ERM) family of proteins that
interact with both the plasma membrane and F-actin69.
Ezrin is also a member of the A-kinase anchoring pro-
tein (AKAP) family of proteins70. AKAPs are structurally
diverse scaffolding proteins that tether the regulatory
subunit of PKA (and other components such as phos-
phodiesterases) to specific cellular compartments, and
thereby spatially direct cAMP signalling71 (discussed
below). In vitro and in vivo, ezrin colocalized with AQP2
during vasopressin stimulation, and ezrin knockdown in
cells reduced endocytosis and increased apical plasma
membrane accumulation of AQP2 in the absence of
AVP stimulation, whereas no changes were observed in
cAMP or Ser256 phosphorylation levels, or in the degree
of AQP2 exocytosis68. The physiological role of ezrin is
unclear, as increased ezrin at the plasma membrane
during vasopressin stimulation would be expected to
enhance AQP2 endocytosis. One possible explanation
for these seemingly paradoxical observations is that dur-
ing vasopressin stimulation, despite the close association
between ezrin and AQP2 at the apical plasma mem-
brane, their interaction is limited by their phosphoryla-
tion status. However, upon vasopressin withdrawal, their
concomitant phosphorylation status might promote
their interaction and subsequent endocytosis.
Collectively, these findings suggest that cytoskeletal
remodelling is a key component of AQP2 targeting to the
plasma membrane, but several important unanswered
questions remain. For example, whether actin depolym-
erization is a rate-limiting step in V2R-mediated AQP2
regulation, and whether cytoskeleton-mediated modu-
lation of AQP2 trafficking requires Ser269 phosphoryl-
ation are unclear. Notably, Ser269 phosphorylation does
not seem to be required for V2R-mediated AQP2 tar-
geting to the plasma membrane. If neither cytoskeletal
remodelling nor AQP2 phosphorylation is rate-limiting
for AQP2 plasma membrane targeting, then alternative
rate-limiting components must exist.
Role of cAMP and PKA in plasma membrane
targeting
The canonical signalling pathway downstream of GSPCR
stimulation involves the activation of adenylyl cyclases,
which leads to a rise in cAMP levels and enhanced
activity of basophilic protein kinases, including PKA.
For many years, this response was considered the only
mechanism underlying the effects of V2R stimulation on
AQP2 plasma membrane targeting72–74 and is often the
easiest and simplest mechanism for explaining general
Reviews
AQP2 trafficking. However, new findings have high-
lighted that this model cannot explain all the molecular
effects of vasopressin and several pathways independent
of cAMP and/or PKA must exist, as summarized below.
PKA. The Ser256 site of AQP2 is located within
a PKA consensus sequence. Early studies show-
ing that N-[2-p-bromocinnamylamino-ethyl]-
5-isoquinolinesulphonamide (H89), which was initially
thought to be a PKA-specific inhibitor, limited AQP2
membrane accumulation, suggested that PKA-mediated
phosphorylation of AQP2 at Ser256 was a key event in
AQP2 trafficking75. However, subsequent studies showed
that H89 can affect the activity of protein kinases other
than PKA, including AMP-activated protein kinase
(AMPK) and ROCK, and therefore responses to H89
should not be regarded as sufficient evidence of PKA
involvement in AQP2 trafficking76. In mice expressing
dominant-negative PKA77, AQP2 abundance, Ser256
phosphorylation and apical plasma membrane target-
ing were reduced and renal water handling was defec-
tive compared with wild-type controls. However, these
effects could be partially rescued by dDAVP, suggesting
either a residual effect of the endogenous PKA, or the
existence of a V2R-mediated but PKA-independent
mechanism for modulation of AQP2 and urine concen-
tration. By contrast, experiments that used mpkCCD14
cells with a complete CRISPR–Cas9-mediated double
knockout (dKO) of the genes encoding the PKA catalytic
subunits PKA-Cα and PKA-Cβ (PKA-dKO)78 suggested
that the dDAVP-mediated increase in AQP2 protein
expression requires PKA activity. In these cells, AQP2
mRNA and protein were not detectable in whole-cell
lysates following dDAVP stimulation, whereas their
levels increased in control cells. Subsequent transfec-
tion of these knockout cells with AQP2 showed that the
PKA-dKO did not significantly affect baseline Ser256
and Ser269 phosphorylation, although dDAVP-induced
AQP2 plasma membrane targeting and phosphorylation
at Ser269 seemed to be reduced compared with control
cells using immunocytochemistry. Importantly, the sim-
ilar dDAVP-induced increase in AQP2 phosphorylation
at Ser256 in PKA-dKO and wild-type cells transfected
with AQP2 (ref.79) indicates that other protein kinases
can be active at this site. Of note, findings on the role
of PKA in cells with PKA defects should be interpreted
with caution because they might reflect the potential
role of PKA in the maintenance of a wide range of cel-
lular biological processes such as vesicular trafficking,
microtubule organization and actin cytoskeleton (de)
polymerization, rather than a direct involvement of PKA
in dDAVP-induced AQP2 phosphorylation and plasma
membrane targeting.
Stable-isotope labelling with amino acids in cell
culture followed by quantitative phosphoproteom-
ics demonstrated an approximately sixfold reduction
in the number of proteins with significant alterations in
phosphorylation status following dDAVP stimulation
in PKa-dKO cells compared with PKA-intact cells79.
However, dDAVP-dependent but PKA-independent
changes in protein phosphorylation were still observed
in PKA-dKO cells at sites predicted to be targeted by
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basophilic protein kinases. A similar increase in putat­
ive basophilic protein kinase sites in PKA-intact cells sug-
gests that the V2R signals through a range of pathways,
several of which might be involved in AQP2 plasma
membrane targeting. Of particular interest, three of the
sites in which phosphorylation increases after exposure
to dDAVP in PKA-dKO cells are known AMPK targets;
other members of the AMPK-related subfamily, such as
serine/threonine-protein kinases Mark2 and Mark3, and
NUAK family SNF1-like kinase 2, have been proposed
to phosphorylate AQP2 at Ser256 (ref.80).
AMPK is an energy-sensitive kinase that is stimulated
by high AMP and/or low ATP levels and is important
for hypertonicity-induced increases in IMCD water
permeability81. The AMPK s­ti­mu­la­tor 5­-a­mi­no­im­id­az­ol­e-­
4-­c a­r b­o xamide-1-β-d-ribofuranoside (AICAR)
increased the osmotic water permeability of rat IMCDs
within 30 min compared with vehicle controls, which
corresponded with increased phosphorylation of AQP2
at Ser256 (ref.81). However, another study found that
AICAR inhibited dDAVP-mediated Ser256 phospho-
rylation but not plasma membrane targeting of AQP2
in mpkCCD14 cells82. The difference in these findings
might reflect different responses to AMPK activation in
different cell types (that is, in cortical versus IMCD cells)
or it might be explained by time-dependent effects of
AMPK activation (short-term activation increases AQP2
membrane abundance and water permeability81, whereas
prolonged activation has the opposite effect82). The anti-
diabetic drug metformin, which activates AMPK by
increasing the cellular AMP to ATP ratio, also increased
AQP2 phosphorylation and plasma membrane targeting
in isolated rat IMCDs (Fig. 4) and acutely increased urine
osmolality in AVP2R-knockout mice compared with
wild-type mice, and in rats treated with the V2R antag-
onist tolvaptan compared with untreated controls83,84.
However, metformin did not increase urine concentra-
tion in water-loaded healthy human volunteers85 and
hyponatraemia and oedema are not listed as common
side effects of metformin treatment according to the
FDA. Overall, these observations suggest that metformin
modulates water reabsorption predominantly in a setting
of inappropriate water diuresis (NDI models), perhaps
reflecting altered intracellular signalling mechanisms
under such conditions.
cAMP. levels and this process is generally believed to
be the signalling pathway that leads to AQP2 phos-
phorylation and/or plasma membrane targeting. This
hypothesis emerged mainly from observations that
cAMP or forskolin stimulation leads to AQP2 plasma
membrane accumulation and increases water per-
meability in the collecting duct (reviewed in ref.86).
Importantly, studies have established that although
cAMP is a diffusible molecule, it does not diffuse
freely within the cell but is instead buffered within
the cytosol owing to fluid-phase droplets formed by the
cAMP-dependent protein kinase type Iα regulatory
subunit; the formation of these droplets is enhanced
by cAMP87. However, whereas enhanced droplet for-
mation is sustained during >20 min of forskolin stim-
ulation, it is transient (<10 min) after stimulation of
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 Reviews

Urine

Apical plasma H2O H2O H2O H2O H2O H2O

AQP2
membrane

P P P P P P P P P P P P
Cytosol

Translocation Translocation

Actin
depolymerization
P P

P P
FMP-API-1 P P
P
A KA
Src Bosutinib Actin
filaments
A
PK
Actin P
polymerization
P P
Calcineurin
RhoA
CAM

Ca2+ AMPK
Basolateral
plasma EGFR
membrane

Blood AG-490
V2R FZD Fluconazole
Wnt5a
Metformin Statin
Erlotinib

Stimulation P Ser256 phosphorylation Drug

Inhibition P Ser269 phosphorylation

Fig. 4 | Alternative regulators of AQp2 trafficking. The figure illustrates regulators of aquaporin 2 (AQP2) plasma
membrane targeting that are independent of the vasopressin V2 receptor (V2R; encoded by Avpr2) and/or cAMP–protein
kinase A (PKA)-independent. The A-kinase anchoring protein (AKAP)–PKA inhibitor FMP-API-1 increases PKA activity and
plasma membrane targeting of AQP2, and increases urine osmolality in mice with tolvaptan-induced nephrogenic diabetes
insipidus (NDI)98. The tyrosine-protein kinase Src inhibitor bosutinib increases Ser269 phosphorylation and plasma
membrane targeting of AQP2 independently of Ser256 phosphorylation103. The epidermal growth factor receptor (EGFR)
inhibitor erlotinib increases AQP2 plasma membrane targeting and urine concentration in mice with lithium-induced
NDI110. The EGFR–protein kinase JAK2 inhibitor A­G-­490 increases V2R-dependent and V­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­2­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­R­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­-­­­­­­­­­­­­­­­­­­­i­­­­­­­­­­­­­­­­­­­­­n­­­­­­­d­­­­­ep­­endent t­­­­­­­­­­­­­­­­­­­­­­­­­­a­­­­­­­­­­­­­r­­­­­­­­­­­­­­­­­g­­­e­­­­ting o­­­­f
A­­­­Q­­P­2 t­­­­o t­­h­e p­­l­a­­sma membrane in rats108. Wnt5a acts via Frizzled receptors (FZDs) to increase intracellular Ca2+ and induces
calmodulin (CAM) and calcineurin-dependent AQP2 plasma membrane targeting, as well as urine concentration in mice
with tolvaptan-induced NDI105. Metformin increases Ser256 phosphorylation via AMP-activated protein kinase (AMPK) and
plasma membrane targeting of AQP2 and increases urine concentration in rats with tolvaptan-induced NDI and in
Avpr2-knockout mice83,84. Statins107 and fluconazole60 inhibit RhoA activity and actin polymerization, which enables AQP2
plasma membrane targeting. The drugs depicted in the figure have shown promise in the treatment of autosomal dominant
polycystic kidney disease in animal studies or in clinical trials.

the β2 adrenergic receptor, a GSPCR coupled to ade- be important for V2R-mediated AQP2 plasma mem-
nylyl cyclase87. These observations highlight that the brane targeting because in resting conditions (AQP2
effects of administering either cell-permeable cAMP present in intracellular vesicles), localized effects of
or the general adenylyl cyclase activator forskolin do the cAMP-specific 3′,5′-cyclic phosphodiesterase 4D
not completely mimic the effect of GSPCR signalling, might prevent inappropriate translocation of AQP2 to
as there are differences in constraining the diffusion the plasma membrane as a result of small changes in
of cAMP throughout the cell when using the vari- intracellular cAMP levels89. The cellular localization
ous approaches. The low cytosolic concentration of and specificity of cAMP–PKA signalling are further
cAMP achieved by this ‘buffering’ of cAMP within ensured by the actions of various scaffolding proteins,
droplets allows for phosphodiesterase-mediated including AKAPs90,91, which are also associated with
compartmentalization 87,88. This mechanism might AQP2-bearing vesicles (see below).

Nature Reviews | Nephrology

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Reviews
Conceptual advances in GPCR signalling, such
as the discovery of β-arrestin and various alternative
pathways that can relay information to specific targets
within the cell (reviewed in ref.92) have brought the
widely accepted view of the role of cAMP in AQP2 phos-
phorylation and trafficking into question (reviewed in
ref.86). One example of how AQP2 plasma membrane
targeting can be achieved in the absence of cAMP
was demonstrated using mice with global AC6 defi-
ciency in which cAMP does not increase in response
to dDAVP stimulation18. Compared with control mice,
AC6-deficient mice have impaired AQP2 phosphoryla-
tion at Ser256 and Ser269 and reduced apical membrane
AQP2 accumulation in IMCD cells, which results in a
urine-concentrating defect. However, although urine
osmolality remained lower than that observed in con-
trol animals after water restriction, the fold change in
urine osmolality in AC6-deficient mice in response to
water restriction was greater (~2.5-fold) than in con-
trols (~1.5-fold). Urine osmolality increased ~10-fold in
response to dDAVP in knockout mice, almost match-
ing the ~12-fold increase observed in wild-type con-
trols. Furthermore, both dDAVP and water restriction
induced Ser256 phosphorylation in the inner medulla.
AC6-deficient mice were able to concentrate their urine
to >2,000 mOsm/kg, which cannot be explained by
residual AC6 activity in the cortex and must therefore
occur through cAMP-independent AQP2 regulation in
the IMCD18. Overall, this study highlights that AC6 and
cAMP are important for AQP2 phosphory­lation
and membrane abundance at baseline but are not
required for acute regulation of AQP2, including apical
plasma membrane targeting and Ser256 phosphoryla-
tion. This conclusion is supported by the observation
that stimulation of the V2R, or the prostaglandin recep-
tors EP2 or EP4, during pharmacological inhibition
of cAMP generation, enhances AQP2 targeting to the
plasma membrane93.
In summary, cAMP and PKA are not an absolute
requirement for Ser256 phosphorylation or AQP2
plasma membrane targeting. Although several alternative
kinases have been identified, their roles and the pathways
that lead to their activation are unclear. Furthermore,
whether cAMP or PKA activity is necessary for actin
depolymerization is unknown.
Novel regulators of AQP2 membrane targeting
New insights into the complex signalling networks and
regulatory steps that govern AQP2 trafficking have
identified promising pharmacological targets for the
treatment of water balance disorders (reviewed in ref.5).
Several of these novel compounds increase AQP2 plasma
membrane targeting independently of known V2R sig-
nalling pathways such as cAMP–PKA and extracellular
signal-regulated kinase (ERK; also known as MAPK), and
not only represent potential therapeutics but might also
help to determine the role of AQP2 in ADPKD. Some of
the newest approaches are detailed below (Fig. 4).
Inhibitors of AKAP-mediated events. Although the
intracellular levels of cAMP are increased by stim-
ulation of a variety of different hormone-coupled
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receptors, activation of these receptors can have dif-
ferent and sometimes opposing roles in physiology
and, occasionally, within the same cell71,94,95. Several
investigations have been aimed at uncovering how
compartmentalization of cAMP is achieved, includ-
ing the role of more than 30 canonical AKAPs87,88,91.
The modulation of specific AKAPs to target selected
PKA-regulated domains is an area of interest in the field
of precision pharmacology91. In the kidney, AKAP18δ
is mainly expressed in IMCDs and traffics to the plasma
membrane along with AQP2 (ref.96), suggesting that it
anchors PKA to AQP2-containing vesicles and controls
cAMP-mediated AQP2 phosphorylation at Ser256 and
plasma membrane targeting. In line with this hypoth-
esis, the general AKAP inhibitor Ht31 prevented
forskolin-mediated AQP2 plasma membrane target-
ing in primary cultured rat IMCD cells97, but it did not
inhibit PKA activity in an in vitro assay, suggesting that
the catalytic activity of PKA is not sufficient for AQP2
translocation. By contrast, another study showed that
Ht31 led to AQP2 plasma membrane accumulation in
mpkCCD14 cells independently of V2R activation98. The
discrepancies between the two studies might be due to
cell type-specific effects and further highlight that the
role of PKA in AQP2 plasma membrane targeting is not
fully understood. Exposure to another AKAP inhibitor,
FMP-API-1 (ref.99) (a low-molecular-weight compound
with enhanced plasma membrane permeability), also
increased apical AQP2 expression in mpkCCD14 cells
to similar levels to vasopressin alone98. Furthermore,
FMP-API-1 or its derivative FMP-API-1/27 potently
increased urine osmolality and reduced urine output in
tolvaptan-treated mice, highlighting them as a potential
novel treatment approach for NDI.
The effects of the inhibitors of the AKAP–PKA
interaction, Ht31 or FMP-API-1, on the biological
activity of PKA are complex. On the one hand, Ht31
decreases certain cAMP–PKA-dependent events, such as
forskolin-induced phosphorylation of cAMP-responsive
element-binding proteins (CREBs)100 and PKA-induced
inhibition of Rho activity101. Therefore, in some cases,
Ht31 inhibits local PKA activity, presumably by dis-
placing PKA from its anchored sites and substrates,
regardless of the intracellular concentration of active
PKA. Similarly, although FMP-API-1 increased PKA
activity in cardiac myocytes, as assessed by a PKA assay
and increased phosphorylation of selected targets, it did
not induce phosphorylation of PKA substrates globally99.
By contrast, Ht31 and FMP-API-1 increased the gen-
eral phosphorylation of PKA substrates in mouse corti-
cal collecting duct cells98, but despite this global effect,
Ht31 prevented AQP2 plasma membrane targeting in
response to forskolin stimulation97. This observation
suggests that PKA targets of relevance to cAMP-induced
AQP2 plasma membrane targeting are inhibited by
Ht31. Collectively, although these studies provide evi-
dence for V2R-independent AQP2 plasma membrane
targeting and a tool for studying PKA involvement, they
do not directly support a role for PKA in AQP2 plasma
membrane targeting in general.
Another AKAP shown to have an indirect role in
AQP2 plasma membrane targeting through cytoskeletal
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remodelling is AKAP13 (also known as lymphoid blast
crisis oncogene)102. This AKAP has guanine nucleo­
tide exchange factor (GEF) activity and activates RhoA.
Thus, AKAP13 inhibitors might increase AQP2 plasma
membrane targeting through inhibition of RhoA and
consequent actin depolymerization. A small-molecule
screening approach identified Scaff10-8 as a compound
that, by binding to RhoA and inhibiting AKAP13-
mediated RhoA activation, promotes actin depolym-
erization102. However, although treatment of primary
IMCD cultures with Scaff10-8 resulted in redistribution
of AQP2 towards the plasma membrane, AQP2 was
not inserted into the plasma membrane. These studies
suggest that, although actin depolymerization facilitates
AQP2-containing vesicular transport to the plasma
membrane, this process alone is not sufficient to increase
collecting duct water transport, and that other regulatory
processes, such as AQP2 phosphorylation, are required.
Src inhibition. Phosphorylation of the non-receptor
tyrosine kinase Src was enhanced in IMCD cells follow-
ing dDAVP stimulation of the V2R40. Src inhibition using
dasatinib or bosutinib increased AQP2 plasma mem-
brane targeting in AQP2–LLC-PK1 cells and in fresh
rat kidney tissue independently of changes in cAMP
levels or PKA activity103. Dasatinib increased AQP2
Ser269 phosphorylation in AQP2–LLC-PK1 cells inde-
pendently of phosphorylation at Ser256, and resulted in
increased AQP2 exocytosis and decreased endocytosis
compared with vehicle-treated AQP2–LLC-PK1 cells.
This increased AQP2 trafficking with dasatinib was not
apparent in cells expressing an AQP2-S269A mutant,
which indicates that Ser269 phosphorylation has an
important role in this effect. Future work should inves-
tigate the potential role of RhoA and/or ROCK in this
process. These results might also explain why hypon-
atraemia has been reported in patients treated with Src
inhibitors104.
Wnt5a. In mpkCCD14 cells, the endogenous frizzled
receptor ligand Wnt5a increased calcium signalling and
AQP2 protein expression, plasma membrane target-
ing and Ser269 phosphorylation compared with vehi-
cle controls105. Unlike dDAVP, Wnt5a did not increase
cAMP or PKA activity, and the increase in AQP2 phos-
phorylation induced by Wnt5a (but not dDAVP) could
be prevented by the calcineurin inhibitor cyclosporine A.
Furthermore, Wnt5a-induced AQP2 protein expression
was not inhibited by H89, indicating that the underlying
mechanism is not dependent on H89-sensitive kinases.
In mice with tolvaptan-induced polyuria, Wnt5a treat-
ment for 2 h also caused a minor increase in urine osmo-
lality compared with vehicle-treated mice. Collectively,
these results indicate that the signalling pathway acti-
vated by Wnt5a differs from that downstream of V2R
stimulation.
Fluconazole. Triazolpropenon, which was identified as
a modulator of AQP2 plasma membrane targeting in
screening assays that used kidney collecting duct cells
transfected with AQP2 (MCD4)106 (Table 1), is struc-
turally similar to the antimycotic agent fluconazole.
Nature Reviews | Nephrology
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Treatment of rat primary IMCD cultures with 10 μM
fluconazole prevented forskolin-mediated AQP2 plasma
membrane targeting106. However, in another study,
50 μM of fluconazole increased AQP2 plasma mem-
brane accumulation in IMCD cultures to levels similar
to those observed using forskolin or vasopressin, and
increased collecting duct water permeability60. The
reason for the contrasting effects of fluconazole at the
two concentrations is unknown. The effects of 50 μM
fluconazole were not accompanied by increases in PKA
activity or AQP2 phosphorylation at Ser256 or Ser269,
which suggests that they are independent of canonical
signalling pathways (cAMP–PKA)60. Compared with
vehicle-treated controls, treatment of mice with flucona-
zole for 96 h (at a dose that resulted in an extracellular
fluconazole concentration similar to that observed in
patients treated for fungal infections) decreased urine
output and increased urine osmolality60. Fluconazole
treatment of mice for 96 h also ameliorated the polyuric
effect of tolvaptan60. These effects of fluconazole were
associated with decreased RhoA activity and depoly­
merization of F-actin, but this can only partially explain
the observed functional effects because RhoA inhibition
alone does not have similar effects102.
Fluconazole functions as an antimycotic agent by
blocking the fungal lanosterol 14a-demethylase (LDM;
encoded by CYP51A1) and inhibiting ergosterol produc-
tion, which causes membrane defects. Native mamma-
lian LDM, which has an important role in the last steps of
cholesterol synthesis, is also inhibited by fluconazole.
Of note, statins, which are cholesterol-lowering drugs
that act at an early step of cholesterol synthesis (conver-
sion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic
acid), also increase AQP2 plasma membrane targeting
and can ameliorate NDI107. Similar to statins, fluconazole
might interfere with mevalonate conversion and there-
fore interrupt the formation of isoprenoids, which are
important for the membrane tethering and activation of
small GTPases such as Rho. Hence, fluconazole might
alter AQP2 function by altering both RhoA activity and
its subcellular distribution.
EGF receptor antagonists. AG-490 is an epidermal
growth factor receptor (EGFR) antagonist and a tyros-
ine protein kinase JAK2 inhibitor that was identified in
an LLC-PK1 cell-based screening assay as an enhancer
of AQP2 plasma membrane targeting108. These results
were verified in AQP2-transfected Madin–Darby canine
kidney cells (MDCK–AQP2; Table 1) and in kidney tis-
sue slices from Sprague–Dawley rats cultured in vitro.
In vasopressin-deficient Brattleboro rats (Table  1) ,
AG-490 increased AQP2 plasma membrane targeting
in cortical but not medullary collecting ducts108. This
observation suggests that in the IMCD, EGFR might
inhibit V2R-induced AQP2 plasma membrane tar-
geting, which is in accordance with the observation
that EGF enhances diuresis109. However, in the corti-
cal collecting duct, AG-490 might have an additional
vasopressin-independent effect on AQP2 plasma mem-
brane targeting. The involvement of EGFR in AQP2
plasma membrane targeting was further supported by
experiments showing that the FDA-approved EGFR
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CFTR corrector
A family of compounds that
facilitate the transport of
misfolded CFTR proteins
(produced owing to mutations
in CFTR) to the cell membrane
without being degraded.
antagonist erlotinib increased plasma membrane target-
ing of AQP2 in LLC-PK1 cells110 and in mice with lithium-
induced NDI; these mice also had increased urine
osmolality and reduced urine volume after erlotinib
treatment110.
AQP2 regulation and ADPKD therapy
The prevalence of ADPKD is 1 in 500–1,000 live births
and at least 45% of patients progress to kidney failure111,112.
The competitive V2R antagonist tolvaptan delayed the
onset of kidney failure in clinical trials7–9,113 and is now
approved by, among others, the FDA and the EMA for
this purpose. However, polyuria is a major side effect of
tolvaptan and leads to treatment discontinuation in ~8%
of patients7; moreover, treatment with tolvaptan requires
an intake of >2–3 l of water per day113. Importantly,
in the tolvaptan initiation phase, the drug should be
titrated to sustain a urine osmolality of <300 mOsm/kg
(normal range 400–900 mOsm/kg) and ensure suffi-
cient inhibition of V2R signalling113. Consequently, tol-
vaptan use might become one of the leading causes of
NDI in the future and novel discoveries in the field
of AQP2 research might have direct clinical benefits in
patients with ADPKD receiving tolvaptan treatment.
In this section, we provide a brief and focused overview of
ADPKD pathophysiology and discuss treatment options
for tolvaptan-induced polyuria based on novel insights
into AQP2 regulation. A focus is placed on those that
could be tested in clinical trials of patients treated with
tolvaptan, and we propose the novel hypothesis that
AQP2 dysregulation is an overlooked aspect of ADPKD
pathophysiology and requires investigation.
The pathophysiology of ADPKD
ADPKD is an adult-onset ciliopathy114 that is character-
ized by diffuse cyst development, and ~95% of ADPKD
cases are associated with mutations in PKD1 (encoding
polycystin 1 (PC1)) or PKD2 (encoding PC2). PC1 and
PC2 can form a complex that traffics to the primary
cilia of kidney collecting duct cells where it functions
as a cation channel115. Normally, bending the primary
cilium alters flow-induced calcium signalling116, which
modulates various signalling pathways, including induc-
ing alterations in cAMP. In the collecting duct, these sig-
nalling responses are amplified by ATP release through
connexin 30 channels situated on the apical plasma mem-
brane of β-intercalated cells, and P2Y2 receptors on the
apical plasma membrane of principal cells117. However, in
ADPKD, flow-induced calcium signalling is impaired118,
which leads to disproportionate GPCR signalling and a
myriad of cellular effects, including increased intracel-
lular cAMP and increased activity of the cystic fibrosis
transmembrane regulator (CFTR). These alterations
result in progressive fluid secretion into the cyst lumen,
which is widely thought to be a major pathophysiological
mechanism for cyst growth. Supporting this mechanism,
conditional inactivation of PC1 (ref.119) and PC2 (ref.120)
in mice leads to the development of kidney cysts and
small-molecule CFTR inhibitors slow cyst growth121 (dis-
cussed below). By contrast, mice deficient for connexin
30 or P2Y2 do not develop kidney cysts117,122. Thus, the
pathophysiology of ADPKD is not readily explained by
0123456789();:
this simplified mechanism and is likely to involve a range
of mechanisms, including changes in cellular prolifera-
tion and transformation of the epithelia from a reabsorp-
tive to a secretory phenotype123. A detailed description
of the intricate pathophysiology and cellular mechanism
underlying ADPKD is beyond the scope of this article
(but is available elsewhere124,125).
Cell proliferation and epithelial transport
Targeting a broad range of proteins, including the YAP
transcriptional coactivators, MYC, RhoA–ROCK and
the mechanistic target of rapamycin (mTOR), to con-
trol cellular proliferation in ADPKD has shown promise
in animal studies126–128. Of note, PC1 has been shown
to modulate RhoA activation and ROCK signalling129.
However, randomized controlled clinical trials using
mTOR inhibitors in patients with ADPKD have been
generally disappointing130,131. The discrepancies between
the effects observed in mouse models and in humans
might be related to drug dosage, because the doses tested
in animals were 35–70-fold higher than those that were
administered clinically (owing to potential side effects).
Moreover, mTOR inhibitors were primarily tested in
rodent models of early-onset or rapidly progressive dis-
ease at early stages of cyst development, which might not
reflect the complex and heterogeneous pathogenesis of
ADPKD in humans.
Another strategy for treating ADPKD involves tar-
geting the secretory phenotype of the epithelia because
formation of fluid-filled cysts from epithelial cells is the
hallmark of ADPKD, a process that is fuelled at least in
part by cAMP-mediated Cl− secretion through CFTR123.
Accordingly, treatment of neonatal, kidney-specific
Pkd1-knockout mice with small-molecule CFTR inhibi-
tors for 7 days slowed kidney enlargement and cyst expan-
sion, and partially preserved kidney function compared
with untreated knockout mice121. In Pkd1-knockout
mice (a model of ADPKD), CFTR was predominantly
detected in the apical plasma membrane (consistent with
its role in cAMP-dependent fluid secretion), which con-
trasts with the intracellular, and apical and basolateral
membrane localization observed in wild-type mice132.
However, the CFTR corrector VX-809 redirected CFTR
towards the basolateral plasma membrane, which pro-
moted a fluid absorption cyst phenotype, rather than
the fluid secretion phenotype observed in untreated
Pkd1-knockout mice. VX-809 also reduced the cyst
index and serum creatinine in Pkd1-knockout mice,
although a prominent cystic phenotype remained133.
Compared with control mice, Pkd1-knockout mice also
have reduced levels of the α-subunit of the epithelial Na+
channel (αENaC), which is highly associated with the
endoplasmic reticulum, rather than being expressed at
the apical plasma membrane as normal. Surprisingly,
VX-809 rescued the subcellular location defect of αENaC
but not its expression levels132. In line with these studies,
patients with ADPKD have higher fractional excretion
of Na+ than healthy individuals134, which is consistent
with lower apical expression of ENaC. These observa-
tions suggest that ADPKD is characterized by increased
cellular proliferation, increased epithelial Cl− secretion
and deficient epithelial Na+ reabsorption in the kidney.
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 Reviews

Urine and microcysts as early as 1 week postpartum135; induc-

ible Pkd1-knockout mice have a similar phenotype
Cl–
H2O H2O Cl– Cl– 4 weeks after induction of knockout when the mice are
Apical plasma membrane ‘pre-cystic’ (ref.136). However, whether Cl− secretion into
the lumen of cysts severely affects the osmotic gradient
and favours water transport into cysts, or whether water
Cytosol AQP2 CFTR is retained in the lumen owing to AQP2 dysregulation
remains unclear. The role of AQP2 in the pathophysiol-
Translocation ogy of ADPKD therefore requires thorough investiga-
tion. Below, we discuss the potential of novel regulators
of AQP2 that have slowed the progression of ADPKD in
animal models or clinical trials.

Multipronged approach to prevent kidney failure in

PKA
Src Pravastatin
Bosutinib
Metformin
Cell proliferation
RhoA cAMP
ERK
MYC
mTOR
Nucleus
Basolateral
plasma membrane
V2R
Blood Tolvaptan
Vasopressin
Stimulation Inhibition
Fig. 5 | Drug targets and novel treatment options for ADpKD. Cell proliferation and
cystic fibrosis transmembrane regulator (CFTR)-mediated Cl− secretion are established
contributors to cyst progression in autosomal dominant polycystic kidney disease
(ADPKD). Several novel drugs that target these processes have been tested in patients
with ADPKD. Metformin inhibits mechanistic target of rapamycin (mTOR), which is a
major regulator of MYC transcription and reduces cyst progression, as well as glomerular
filtration rate decline in ADPKD mouse models. A retrospective study of metformin in
patients with ADPKD supported these findings147; randomized controlled clinical trials
are ongoing. The tyrosine-protein kinase Src inhibitor bosutinib prevented cyst
progression in a randomized controlled clinical trial in patients with ADPKD142. Statins
can inhibit RhoA and pravastatin inhibited cyst progression in young adults with
ADPKD143. Statins, bosutinib and metformin also regulate aquaporin 2 (AQP2) plasma
membrane targeting (Fig. 3). Thus, in contrast to ADPKD drugs that target only cell
proliferation (such as everolimus), these newer and more promising drug options also
enhance the absorptive phenotype of the collecting duct epithelium. Tolvaptan, which is
a vasopressin V2 receptor (V2R) antagonist, inhibits extracellular signal-regulated kinase
(ERK)-mediated cell proliferation and cAMP-mediated CFTR activity, and delays kidney
failure in ADPKD7–9,113. PKA, protein kinase A.
Potential role for AQP2 in ADPKD
Compared with healthy individuals, urine osmolal-
ity is lower and urine volume higher in patients with
ADPKD, despite increased vasopressin levels. This
observation is consistent with the hypothesis that the
pathophysiology of ADPKD is partially due to reduced
absorptive capacity in cystic kidneys that do not respond
sufficiently to vasopressin123. In line with this hypothesis,
Aqp2-knockout mice display collecting duct expansion
patients with ADPKD. Patients with ADPKD might
benefit from a multipronged treatment approach that
targets cellular proliferation and transformation of the
epithelia from a reabsorptive to a secretory phenotype123
simultaneously (Fig. 5). Currently, tolvaptan is the only
drug approved by the FDA for ADPKD treatment and
is predicted to delay the progression to kidney failure by
up to 7.3 years7–9,113. The rationale for the use of tolvaptan
is that its inhibition of V2R signalling should reduce
cAMP-mediated CFTR-induced Cl − secretion and
ERK-mediated epithelial cell proliferation137. Animal
models suggest that cAMP formation in ADPKD occurs
through the calcium-sensitive adenylyl cyclases AC5
(ref.138) and AC6 (ref.139), which enhance cyst expansion,
whereas in vitro studies have also suggested a role for
AC3 (refs140,141). In general, V2R has a preference for AC6
(refs18,141) and we would predict that tolvaptan inhibits
cAMP formation mediated by AC6 but not AC5, and
only has an effect in portions of the nephron and the
Drug collecting duct where V2R is highly expressed. Given
its aforementioned side effects, the use of tolvaptan in
ADPKD might benefit from treatment in parallel with
agents that increase AQP2 plasma membrane targeting
and urine concentration, independently of the detri-
mental signalling pathways of V2R. Intriguingly, several
of these potential pathways can be targeted by drugs
shown to delay cyst progression in preliminary clinical or
preclinical trials.
Bosutinib was administered to patients with ADPKD
during a 24-month phase II randomized controlled clini-
cal trial142. Increases in total kidney volume were reduced
to 0.84% in patients treated with bosutinib (200–400 mg
daily) compared with 4.74% in patients who received
a placebo. Although the rationale for using Src inhib-
itors to treat ADPKD was to target their effect on cell
proliferation, Src inhibition might have also caused
vasopressin-independent targeting of AQP2 towards
the apical plasma membrane (discussed earlier), thereby
correcting the concentrating defect and restoring
epithelial fluid reabsorption.
Patients with ADPKD aged 8–22 years that were
being treated with the angiotensin-converting enzyme
inhibitor lisinopril were randomly assigned to receive
placebo or the statin pravastatin in a small randomized
controlled trial143. At the 3-year follow-up, the percen­
tage change in total kidney volume (adjusted for height,
age, sex and hypertension status) was significantly
decreased in the pravastatin group versus placebo

Nature Reviews | Nephrology

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Reviews

(23% ± 3% versus 31% ± 3%; P = 0.02). The patients in
these trials were young and, on average, normotensive
and normocholesterolaemic; therefore, the effect of
pravastatin is unlikely to be secondary to vascular ben-
efits. As discussed earlier, statins have a mechanism of
action similar to fluconazole; thus, they might not only
inhibit RhoA–ROCK-mediated MYC transcription and
cellular proliferation during ADPKD but they might also
enhance AQP2 plasma membrane targeting.
In addition to its aforementioned effects on AQP2,
which are probably due to its effects on AMPK, met-
formin inhibits the activity of CFTR and mTOR, and
has shown promise in preventing and/or reducing cyst
growth in miniature pig models of ADPKD and in some,
but not all, mouse models of ADPKD144–146. Randomized
controlled clinical trials are currently underway to inves-
tigate the effects of metformin in patients with ADPKD,
but preliminary retrospective studies have indicated
a slower decline in glomerular filtration rate over a
3-year period in patients with diabetes and ADPKD
who received metformin, compared with patients with
ADPKD without diabetes147. Whether this reduction in
glomerular filtration rate decline is due to the ability of
metformin to reduce blood sugar levels in patients with
diabetes or any direct beneficial effect on ADPKD is
unknown.
Erlotinib has been approved by the FDA for the
treatment of non-small-cell lung cancer. This drug
reduces mTOR and ERK signalling and reduced cyst
growth in Pkd1-knockout mice through an unidenti-
fied mechanism148. As discussed previously, erlotinib
also increases AQP2 plasma membrane targeting and,
although clinical trials in ADPKD are not available, it is
another candidate tolvaptan companion drug.
New treatment options for ADPKD
The aforementioned effects of fluconazole60 — enhancing
AQP2 plasma membrane targeting without increasing
cAMP or PKA levels, and decreasing activity of RhoA —
might be beneficial in ADPKD. Furthermore, this anti-
mycotic is generally well tolerated during long-term
administration, for example, in patients with recurrent
candidiasis149, and its urine-concentrating effects seem
to be sustained over several days60. Understanding the
effects of other AQP2 regulators such as Wnt5a, or other
frizzled receptors in ADPKD would be informative and
might uncover further alternative treatment options for
ADPKD. Finally, AKAP–PKA disrupters might also be
beneficial in ADPKD. Although the general AKAP–
PKA interaction inhibitors Ht31 and FMP-API-1
increased PKA activity in collecting duct cells98, which
is a known detrimental factor in ADPKD150, certain
physiological effects of PKA are attenuated in the con-
text of AKAP–PKA disruption; the general AKAP–PKA
interaction inhibitor Ht31 decreases forskolin-induced
phosphorylation of CREB (a sensor for PKA activity)100,
PKA-mediated Rho inhibition 101 and, importantly,
cAMP-induced CFTR activation151. We predict that
identifying specific AKAPs involved in PKA-mediated
cyst progression could lead to the development of
AKAP–PKA disrupters that target such selected AKAPs.
In contrast to tolvaptan, these would function inde-
pendently of specific receptors and tubular structures.
As discussed above, specific AKAP–PKA interaction
inhibitors might also rescue the concentrating defect
caused by tolvaptan treatment in ADPKD, as observed
with FMP-API-1 (ref.98).
Conclusions
Much is known about the molecular regulation of
AQP2, but several of the signalling pathways that link
receptor stimulation to AQP2 plasma membrane tar-
geting remain elusive. Furthermore, how these signal-
ling pathways interact or their potential crosstalk, and
whether one or more of these pathways is dominant
over another, are unknown. Nonetheless, the canonical
pathway of AQP2 targeting to the plasma membrane
— cAMP–PKA-mediated Ser256 phosphorylation — is
insufficient to explain multiple research findings. For
example, Ser256 phosphorylation is not a rate-limiting
step for plasma membrane targeting and can occur with-
out cAMP-stimulated PKA activity; cAMP and PKA
activity are important for AQP2 protein abundance, but
AQP2 plasma membrane targeting and urine osmolality
can increase independently from their activity; AMPK
might be a novel signalling node of PKA-independent
V2R signalling and AQP2 plasma membrane target-
ing, but the mechanism underlying AMPK activation
downstream of V2R activation is unknown.
In ADPKD, despite the known urine-concentrating
defect and the generation of large fluid-filled cysts that
derive from the tubular epithelium, the pathophysiolog-
ical role of AQP2 has never been investigated in detail.
This gap in knowledge is probably due to the complexity
of separating known factors that occur downstream of
V2R activation and that are known to be detrimental
in ADPKD, such as increased cAMP and ERK activa-
tion, from a direct role of AQP2. However, the emer-
gence of novel signalling pathways and compounds that
cause AQP2 plasma membrane targeting independently
from cAMP–PKA provides an opportunity to investi-
gate in detail the pathophysiological role of AQP2 in
ADPKD, potentially expanding the breadth of current
treatment options. Furthermore, novel compounds
that increase AQP2 plasma membrane targeting inde-
pendently of V2R, cAMP or PKA might be ideal com-
panion drugs to tolvaptan in the treatment of ADPKD
because they might ameliorate both cyst progression and
tolvaptan-induced polyuria. Clinical trials are warranted
to develop such a multipronged approach.
Published online xx xx xxxx

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Acknowledgements
The authors’ research is supported by the Danish Medical
Research Council, The Novo Nordisk Foundation, The Leducq
Foundation and the Carlsberg Foundation.
Author contributions
All authors researched data for the article, made substantial
contributions to discussions of the content, wrote the
manuscript, and reviewed or edited the manuscript before
submission.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Nephrology thanks T.-H. Kwon, V. Torres and
the other, anonymous, reviewer(s) for their contribution to the
peer review of this work.
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