Am J Physiol Cell Physiol 307: C597–C605, 2014.
First published June 18, 2014; doi:10.1152/ajpcell.00154.2014.
Methods In Cell Physiology
High-throughput chemical screening identifies AG-490 as a stimulator of
aquaporin 2 membrane expression and urine concentration
Naohiro Nomura,1,2 Paula Nunes,1,2 Richard Bouley,1,2 Anil V. Nair,1,2 Stanley Shaw,1 Erica Ueda,1
Nutthapoom Pathomthongtaweechai,1,2 Hua A. Jenny Lu,1,2 and Dennis Brown1,2
1
Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts;
and 2Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts
Submitted 22 May 2014; accepted in final form 13 June 2014
cell signaling; diabetes insipidus; drug screening; vasopressin; water
channels; EGFR and JAK-2 inhibitor
(DI) is characterized by a reduction in urine
concentrating ability; the main symptoms of DI are polyuria
with associated polydipsia, dehydration, and hypernatremia.
Both central DI [due to loss of vasopressin (VP)] or nephrogenic DI (NDI) can be hereditary or acquired. In adults,
acquired NDI is more common and it is most often due to
chronic lithium use or hypercalcemia. In patients with hereditary NDI, symptoms usually appear soon after birth; the most
severe complications are renal insufficiency and mental retar-
DIABETES INSIPIDUS
Address for reprint requests and other correspondence: D. Brown, Massachusetts General Hospital, Program in Membrane Biology and Division of
Nephrology, Simches Research Center, 185 Cambridge St, Suite 8.202, Boston, MA 02114 (email: [email protected]).
http://www.ajpcell.org
dation. Hereditary NDI is caused by mutations in two genes,
the V2 vasopressin receptor (V2R) and the aquaporin 2
(AQP2) water channel (11, 39). Ninety percent of hereditary
NDI is caused by inactivating mutations in V2R, and the other
10% results from AQP2 mutations (13, 34). While central DI
is usually manageable by administration of desmopressin
(modified vasopressin) by nasal spray, for example, treating
NDI patients is more problematic because of their resistance to
vasopressin. They are frequently submitted to salt restriction,
thiazide diuretics, and prostaglandin inhibitors (12, 22), but
these therapies have only limited benefit.
VP-induced trafficking and membrane accumulation of
the water channel AQP2 plays a critical role in maintaining
water balance. Our understanding of this process at the
cellular level has increased considerably over the past few
years and has informed directed attempts to bypass defective VP signaling to increase urinary concentrating ability in
DI. We now know, for example, that AQP2 recycles constitutively between cytosolic vesicles and the apical membrane, and the amount of AQP2 on the apical membrane at
any given time is determined by a balance of exocytosis and
endocytosis (7, 18, 24, 25, 27). We also know that phosphorylation of AQP2 is required to maintain this channel at the cell
surface following its exocytotic insertion. AQP2 membrane accumulation then allows water to permeate through the plasma
membrane, resulting in urine concentration.
Recent studies from our group and others have suggested
that bypassing the defective V2R (which accounts for 90%
of hereditary NDI cases) to activate AQP2 membrane accumulation in kidney principal cells is a feasible and promising therapeutic strategy. Some compounds, for example a
phosphodiesterase type 5 (PDE5) inhibitor (sildenafil) (5), a
phosphodiesterase type 4 (PDE4) inhibitor (rolipram) (35),
statins (simvastatin, fluvastatin, and lovastatin) (21, 32),
calcitonin (4), and selective agonists for E-prostanoid receptors (20, 30), successfully increased AQP2 plasma membrane accumulation. However, it remains uncertain whether
these compounds can be translated into humans as a therapeutic strategy. For example, use of rolipram did not help
patients with hereditary X-linked NDI (1). Therefore, additional approaches and new drugs are still needed to bypass
the defective V2R/VP signaling pathway in both acquired
and hereditary DI. The compounds discussed above were
identified rationally on the basis of our understanding of
known AQP2 signaling/trafficking pathways. However, this approach is limited and has tended to focus on mechanisms related
to the cAMP signaling pathway. In contrast, nonbiased screening
0363-6143/14 Copyright © 2014 the American Physiological Society
C597
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Nomura N, Nunes P, Bouley R, Nair AV, Shaw S, Ueda E,
Pathomthongtaweechai N, Lu HA, Brown D. High-throughput
chemical screening identifies AG-490 as a stimulator of aquaporin 2
membrane expression and urine concentration. Am J Physiol Cell
Physiol 307: C597–C605, 2014. First published June 18, 2014;
doi:10.1152/ajpcell.00154.2014.—A reduction or loss of plasma
membrane aquaporin 2 (AQP2) in kidney principal cells due to
defective vasopressin (VP) signaling through the VP receptor
causes excessive urine production, i.e., diabetes insipidus. The
amount of AQP2 on the plasma membrane is regulated by a
balance of exocytosis and endocytosis and is the rate limiting step
for water reabsorption in the collecting duct. We describe here a
systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds
that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a
high-throughput exocytosis fluorescence assay using LLC-PK1
cells expressing soluble secreted yellow fluorescent protein and
AQP2. Thirty-six candidate exocytosis enhancers were identified.
These compounds were then rescreened in AQP2-expressing cells
to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro.
Three compounds, AG-490, ␤-lapachone, and HA14-1 increased
AQP2 membrane accumulation in LLC-PK1 cells, and both AG490 and ␤-lapachone were also effective in MDCK cells and
principal cells in rat kidney slices. Finally, one compound, AG-490
(an EGF receptor and JAK-2 kinase inhibitor), decreased urine
volume and increased urine osmolality significantly in the first 2– 4
h after a single injection into VP-deficient Brattleboro rats. In
conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial
HTS followed by in vitro assays in cells and kidney slices, and
concluding with in vivo testing in an animal model.
Methods In Cell Physiology
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SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
lial cells, then in kidney slices in vitro, and finally using in
vivo whole animal studies.1
MATERIALS AND METHODS
High-throughput screening assay. This screening assay was developed from the exocytosis assay that was described in detail in a
previous study (29). LLC-PK1 cells expressing both AQP2 and
soluble secreted yellow fluorescent protein (ssYFP) were seeded at a
density of 2 ⫻ 103 cells/well on 384-well microplates using a
Multidrop Combi reagent dispenser (Thermo Scientific, Waltham,
MA) and then cultured until confluence (3 days). Cells were washed
1
This article is the topic of an Editorial Focus by Jeff M. Sands and Mitsi
A. Blount (33a).
Table 1. Candidate AQP2 exocytosis enhancer compounds
Compound Name
Neurotransmitter modulators
1) Salsoline
2) S(-)-lisuride
3) Ergonovine
4) Benserazide hydrochloride
5) Agroclavine
6) Fluphenazine
7) Trifluoperazine
8) Menadione
9) Dobutamine hydrochloride
Lipid receptor ligands
10) U-46619
11) Prostaglandin E2
Kinase inhibitors
12) Emodin
13) AG-490
14) Y-27632
15) Tyrphostin AG-825
16) RO31-8220
17) AG-213
18) GF-109203X
Other inhibitors
19) ␤-Lapachone
20) NSC-95397
21) Dipyridamole
Cytotoxic/apoptosis-inducing compounds
22) 1,4-Naphthoquinone
23) Dalbergione
24) Quinalizarin
25) Shikonin
26) Pyrromycin
27) Aklavine hydrochloride
28) Antimycin a
29) HA14-1
30) Doxorubicin hydrochloride
Other natural products
31) Brazilin
32) Brazilein
33) Epigallocatechin 3,5-digallate
34) Purpurin
35) 7-Hydroxy-2-methoxyisoflavone
36) 10Dimenthyl bicycle [7:2:0] undecane
Final Concentration, ␮M
NEx ⫾ SD
Description
IF Result
50
10
10
7.77
10
10
10
50
50
1.37 ⫾ 0.09
1.45 ⫾ 0.03
1.45 ⫾ 0.03
1.54 ⫾ 0.01
1.59 ⫾ 0.02
1.64 ⫾ 0.03
1.65 ⫾ 0.81
1.78 ⫾ 0.08
1.85 ⫾ 0.04
Dopamine-related compound
MAO-A inhibitor, serotonin
Dopamine/serotonin agonist
AAAD decarboxylase inhibitor, antiparkinsonian
D2 agonist, D1 antagonist, serotonin agonist
Adrenergic or serotonergic receptor
Calmodulin inhibitor, antiadrenergic/dopaminergic
DOPA decarboxylase inhibitor
A-␤-2 agonist
NA
NA
NA
NA
NA
CT
AK
AF
NC
5
5
1.41 ⫾ 0.18
1.65 ⫾ 1.22
Thromboxane TP receptor agonist
Prostaglandin EP receptor agonist
NC
AK
50
85
100
63
54.5
115
60.5
1.46 ⫾ 0.12
1.46 ⫾ 0.06
1.46 ⫾ 0.20
1.55 ⫾ 0.26
1.66 ⫾ 0.11
1.97 ⫾ 0.01
2.96 ⫾ 0.12
Tyrosine kinase inhibitor, CK2 inhibitor, natural
JAK-2 kinase and EGFR inhibitor
ROCK-1 kinase inhibitor
HER-1,2 tyrosine kinase inhibitor
PKC inhibitor
EGFR tyrosine kinase inhibitor
PKC inhibitor
AF
PH
AK
NC
AF
NC
AF
105
80.5
3.96
1.46 ⫾ 0.03
1.75 ⫾ 0.28
2.09 ⫾ 0.09
Topoisomerase I inhibitor, apoptosis inducer
CDC25 phosphatase inhibitor
Phosphodiesterase inhibitor
PH
CT
AK
50
50
50
86.5
50
50
50
61
3.68
1.36 ⫾ 0.06
1.45 ⫾ 0.09
1.49 ⫾ 0.04
1.55 ⫾ 0.07
1.58 ⫾ 0.03
1.60 ⫾ 0.07
1.73 ⫾ 0.05
1.76 ⫾ 0.06
1.97 ⫾ 0.06
Antibacterial, anticancer
Severe skin irritant, anti-cancer
Cytotoxic dye, inhibitor of CK2, induces apoptosis
Apoptosis inducer, p53-dependent
Anthracycline antibiotic, inhibits RNA synthesis
Cytotoxic activityagainst multidrugresistant cells
Induces apoptosis, inhibits mitochondria function
Bcl-2 ligand induces apoptosis
DNA-intercalating agent
NA
NA
NC
AF
NA
NA
NC
PH
AF
50
50
50
50
50
50
1.48 ⫾ 0.05
1.50 ⫾ 0.01
1.54 ⫾ 0.01
1.67 ⫾ 0.06
1.88 ⫾ 0.06
2.68 ⫾ 0.14
Haematoxylin-like may protect from oxidative stress
Anti-inflammatory, lowers cytokines TNF-␣, IL-6
Tea extract polyphenol, inhibits insulin secretion
Red/yellow dye, unknown
Isoflavonoid, unknown
Unknown
NA
NA
PH
AF
NA
NA
Thirty-six candidate enhancers of aquaporin 2 (AQP2) exocytosis were identified from primary screening of 3,464 compounds with a soluble secreted yellow
fluorescent protein (ssYFP) exocytosis assay. These candidates were evaluated by secondary screening with immunofluorescence (IF). NEx, normalized
exocytosis score; NA, not available commercially; CT, cytotoxic; AK, already known as enhancer of AQP2 membrane accumulation (compounds 7, 11, 14, 21);
AF, autofluorescence; NC, no change of localization of AQP2; PH, positive hit with immunofluorescence (compound 13, 19, 32). AK and PH compounds are
highlighted in bold. AAAD, aromatic L-amino acid decarboxylase; EGFR, epidermal growth factor receptor; ROCK-1, Rho-associated protein kinase 1.
AJP-Cell Physiol • doi:10.1152/ajpcell.00154.2014 • www.ajpcell.org
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of chemical libraries using appropriate assays may provide unexpected novel therapeutic strategies.
In the present report we have, therefore, established and
validated a nonbiased high-throughput chemical screening
method which, when coupled to additional downstream
assays, was designed to identify compounds that might
modulate principal cell water permeability by affecting
AQP2 membrane accumulation. Initial chemical screening
was performed by adapting our fluorescence-based exocytosis assay (29) to a high-throughput format. The purpose of
this screening was to quickly identify compounds that modulate exocytosis in renal epithelial cells. From the initial
candidates we next identified those compounds that regulate
specifically AQP2 trafficking first in cultured renal epithe-
Methods In Cell Physiology
SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
C599
Fig. 1. Flow chart showing the screening and testing procedures used in this
study. The boxes show the number of compounds that remained after each
screening step. From an initial pool of 3,464, only one compound remained
that increased urine concentration in rats while activating membrane accumulation of aquaporin 2 (AQP2) in vivo. ssYFP, soluble secreted yellow fluorescent protein.
with Hank’s balanced salt solution HBSS (containing 20 mM HEPES
and 2 g/l glucose) using a BioTek ELx 405 microplate washer
(BioTek US, Winooski, VT) and then incubated in HBSS for 1 h
before chemical delivery. Compounds were from the Prestwick
Chemical Library of agency-approved (including the FDA), off-patent
drugs (Prestwick Chemical, Illkirch, France), Spectrum Collection
(Microsource Discovery Systems, Gaylordsville, CT), and the supplemented ICCB (Harvard Institute of Chemistry and Cell Biology)
known bioactives collection libraries. Compounds were delivered to
each well using the 100 nl 384-pin head attachment of a CyBio-Well
Vario robotic pipettor (CyBio US, Woburn, MA). After pinning, the
cells were incubated for 30 min at 37°C, then the extracellular
medium was removed and delivered to 384 black low-volume nonbinding surface microplates (Corning, Tewksbury, MA). Plates were
spun at 100 g for 1 min to collect medium at the bottom of the plates,
and fluorescence was read on a SafireII multiwavelength fluorimeter
(Tecan Systems, San Jose, CA). Concomitantly, the remaining cells
were lysed by lysis buffer (20 mM Tris·HCl, 2 mM EGTA, 2 mM
EDTA, 30 mM NaF, 30 mM Na4O7P2, 2 mM Na2VO4, 1% Triton
Fig. 2. AQP2 accumulates on the plasma membrane after treatment of LLCPK1 cells with candidate compounds. A: representative AQP2 staining in
LLC-PK1 cells stably expressing AQP2 after treatment with AG-490 (40 ␮M),
␤-lapachone (␤-lap, 50 ␮M), epigallocatechin gallate (EGCG, 50 ␮M),
HA14-1 (60 ␮M), and lysine-vasopressin/forskolin (VP/FK, 10 nM/10 ␮M)
for 30 min. B: quantification of membrane AQP2 accumulation in LLC-PK1
cells. Mean fluorescence intensity in the plasma membrane was divided by
mean fluorescence intensity in the cytosol. Data are shown as means ⫾ SE;
n ⫽ 4; NS, not significant. *P ⬍ 0.05 vs. control. The images presented here
were all subjected to enhancement and sharpening of the entire field using the
levels command and/or the high-pass filter in Adobe Photoshop. Images for
quantification were all used without any enhancement.
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X-100, 0.03% SDS, pH 7.4, Roche complete-mini cocktail) and the
well fluorescence was measured. The exocytosis (Ex) score was
defined as the secreted ssYFP fluorescence divided by the sum of the
secreted ssYFP fluorescence and the ssYFP fluorescence remaining in
the lysates. Ex scores were then normalized by the DMSO (or HBSS for
those compounds dissolved in aqueous solutions) to give the normalized
exocytosis score (NEx) score. The NEx scores of the triplicate wells were
averaged. Z-scores are defined as follows: Z ⫽ [(NEx ⫺ ␮o)/␴o], where
␮o and ␴o are the DMSO NEx population mean and standard deviation,
respectively. Positive hits listed in Table 1 were chemicals for which the
absolute value of the Z-score was greater than 2.5.
Immunofluorescence staining. Immunofluorescence with LLC-PK1
cells and Madin Darby canine kidney (MDCK) cells stably expressing
AQP2 (17, 42) was performed as previously described (40, 41).
LLC-PK1 cells and MDCK cells were cultured on glass coverslips
and polyester filters (Corning), respectively. The MDCK cells were
treated with 50 ␮M indomethacin overnight as previously described
(10, 41). After 2 h of serum starvation, the cells were incubated with
compounds for 30 min and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100-PBS, and blocked in 1% BSA-PBS.
Goat anti-AQP2 C-17 antibody (0.5 ␮g/ml, Santa Cruz Biotechnology, Dallas, TX) and donkey anti-goat IgG conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA) were used as a primary
antibody and a secondary antibody, respectively. Images were acquired with a Zeiss Radiance 2000 confocal laser-scanning microscope or a Nikon 80i microscope. AG-490 was obtained from Selleck
Chemicals (Houston, TX). ␤-Lapachone and epigallocatechin gallate
Methods In Cell Physiology
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SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
(EGCG) were obtained from Enzo Life Sciences (Farmingdale, NY)
and Sigma-Aldrich (St. Louis, MO), respectively.
Animal experiments. Animal experiments were approved by the
Massachusetts General Hospital Institutional Committee on Research
Animal Care in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. For in vitro
kidney slice experiments, kidney slices were prepared as described
previously (4, 5). Briefly, adult Sprague-Dawley rats were anesthe-
tized using isoflurane. Kidneys were harvested, and slices of ⬃0.5 mm
were cut using a Stadie-Riggs microtome. All of the sliced kidneys
were incubated at 37°C for 15 min in equilibrated HBSS (pH 7.4, with
5% CO2). After equilibration, the slices were incubated in HBSS
containing chemicals (100 ␮M arginine vasopressin and 10 ␮M
forskolin, 40 ␮M AG-490, 50 ␮M ␤-lapachone, or 0.1% DMSO as
control) for 30 min. After incubation, all of the samples were immersed in 4% paraformaldehyde-lysine-periodate (PLP) fixative. For
Fig. 4. AQP2 accumulates on the apical
plasma membrane in rat kidney slices after
treatment with candidate compounds in
vitro. Kidney slices from a Sprague-Dawley
rat were incubated for 30 min with VP/FK
(10 nM/10 ␮M), AG-490 (40 ␮M), or ␤-lapachone (50 ␮M). A: in inner medulla,
AQP2 accumulated on apical membranes
after VP/FK or AG-490 treatment. ␤-Lapachone had no detectable effect. B: in cortical
collecting duct, AQP2 accumulated on apical membranes of principal cells (arrows)
after VP/FK, AG-490, or ␤-lapachone treatment. Scale bars, 20 ␮m. C and D: quantitative analysis of AQP2 in inner medulla (C)
and cortex (D). Fluorescence intensity of the
apical area was divided by fluorescence of
the whole cell area. Data are shown as
means ⫾ SE; n ⫽ 3. *P ⬍ 0.05 vs. control.
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Fig. 3. Confocal immunofluorescence localization of AQP2 in Madin-Darby canine kidney (MDCK) cells stably expressing AQP2.
The cells were cultured on filters. Top: apical
slice of cells (i.e., the apical plasma membrane domain). Middle: middle portion of
the cells. Bottom: xz-stack slice. AQP2 was
located in the cytosol under basal conditions.
After 30 min of incubation with VP/FK,
␤-lapachone, and AG-490, AQP2 accumulated at the apical pole (compare top panels
with control). The concentrations of compounds were as same as in Fig. 2A with
LLC-PK1 cells. The images presented here
were all subjected to enhancement and
sharpening of the entire field using the levels
command and/or the high-pass filter in
Adobe Photoshop.
Methods In Cell Physiology
SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
throughput screening of chemical libraries. Such studies are
reported below, and a flow chart of the entire screening and
testing procedure is illustrated in Fig. 1. The chart shows the
number of compounds surviving each step of the procedure.
Testing candidate compounds on cultured cells and kidney
tissue slices in vitro. The ssYFP exocytosis assay indicated
whether a given compound could stimulate or inhibit the
accumulation of ssYFP in the culture medium, but it did not
prove directly its effect on membrane accumulation of AQP2.
“False positives” can occur if a compound is autofluorescent or
causes cell death or lysis. Therefore, we performed immunofluorescence studies to determine AQP2 localization in cultured cells exposed to candidate enhancer compounds. We
excluded 4 drugs (prostaglandin E2, dipyridamole, trifluoperazine, and Y-27632) that were already known to affect AQP2
trafficking, and 13 drugs were not commercially available.
Finally, we tested 19 enhancer candidates by immunofluorescence with AQP2 expressing LLC-PK1 cells. Treatment with
50 ␮M ␤-lapachone, 40 ␮M AG-490, and 60 ␮M HA14-1
caused significant plasma membrane accumulation of AQP2
(Fig. 2A). Epigallocatechin gallate (EGCG, 50 ␮M) caused
some apparent membrane accumulation by visual inspection,
but the effect did not reach statistical significance upon quantification (Fig. 2B), even after increasing the concentration of
EGCG to 200 ␮M (data not shown).
Seven compounds initially identified (menadion, emodin,
RO31-8220, GF-109203X, shikonin, doxorubicin hydrochloride, and purpurin) were autofluorescent and had no effect on
AQP2 trafficking (Table 1). Two compounds (fluphenazine
and NSC-95397) were cytotoxic (Table 1). Six others (dobutamine hydrochloride, U-46619, tyrphostin AG-825, AG-213,
quinalizarin, and antimycin A) had no significant effect on the
localization of AQP2 in LLC-PK1 cells (Table 1).
We further examined the effect of candidate compounds
using MDCK cells stably expressing AQP2 to determine the
generality of the effect of enhancer compounds on AQP2
trafficking. Both ␤-lapachone and AG-490 caused a marked
RESULTS
High-throughput screening assay. The total amount of
ssYFP secreted into the extracellular medium was measured in
LLC-PK1 cells expressing both ssYFP and AQP2 (LLCAQP2-ssYFP cells) (29) in the presence of 3,464 bioactive
molecules. We identified 36 candidate exocytosis enhancers
that increased ssYFP medium fluorescence (Table 1). In our
unbiased study, we also detected some compounds already
known to regulate AQP2 trafficking: prostaglandin E2 (20, 30),
dipyridamole (5, 35, 36), and Y-27632 (37). These results,
which serve as positive controls, indicate that our screening
system worked effectively. However, one of the compounds
picked up as an exocytosis enhancer was trifluoperazine, which
has been reported to inhibit VP-dependent water permeability
and AQP2 trafficking (8). This indicates the need to perform
careful confirmatory follow-up experiments after initial high-
Fig. 5. Effect of acute in vivo treatment with AG-490 and ␤-lapachone. A and
B: cumulative urinary volume (A) and time course of change in urinary
osmolality (B) in rats housed in metabolic cages. Brattleboro rats were injected
with AG-490 (35 mg/kg), ␤-lapachone (17 mg/kg), or vehicle intraperitoneally. Urine was collected every 2 h. Data are shown as means ⫾ SE; n ⫽ 3.
*P ⬍ 0.05 vs. vehicle. The experiment was performed twice, and the average
of the two experiments was used for each rat. AG-490 treatment caused a
highly significant reduction in urine volume and an increase in urine osmolality
2 and 4 h after injection.
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metabolic cage studies, each adult Brattleboro rat (from the Rat
Resource and Research Center, Columbia, MO) was placed individually in a metabolic cage. Before the experiment, rats were allowed to
acclimate to metabolic cages for 5 days. AG-490, ␤-lapachone, or the
same amount of control solution (50% DMSO and 50% ethanol) was
injected into rats intraperitoneally. The initial dose of each drug was
determined by reference to previous studies (9, 19, 26, 38). We used
different doses of chemicals and finally we chose a dose that had no
significant side effects on animal behaviors such as food intake,
grooming behavior, and general appearance of well-being. The metabolic cage study was repeated two times with a 10 days break period
between treatments. Data from each rat were, therefore, determined by
the average of two independent experiments. For blood sampling and
kidney fixation, rats were anesthetized with isoflurane 2 h after drug
injection. Blood was collected from the abdominal vena cava, and
kidneys were immediately perfused through the left ventricle with
PBS then with the PLP fixative. Cryosections were prepared as
described previously (4). These specimens were stained with antiAQP2 antibody (see above). Serum and urine were analyzed at the
Massachusetts General Hospital Clinical Pathology Laboratory facility. Urine osmolality was analyzed with a Vapor Pressure Osmometer
5520 (Wescor, Logan, UT).
Quantification of AQP2 plasma membrane accumulation. The
fluorescence of AQP2 labeling at the plasma membrane was quantified using Volocity software (PerkinElmer, Santa Clara, CA). For the
quantification, LLC-PK1 cells were costained with wheat germ agglutinin (WGA 2 ␮g/l; Lectin Kit, Vector Labs, Burlingame, CA) to
clearly delineate the plasma and nuclear membranes. The mean
fluorescence intensity of at least 30 regions of plasma membrane and
cytosol from each of 5 different images from each sample taken at
⫻40 magnification were analyzed. The plasma membrane fluorescence was divided by the mean cytosolic value of each sample to
normalize the values among different experiments. For quantification
in rat kidney tissue, the fluorescence level of apical AQP2 and total
cellular AQP2 was determined by measuring the intensity levels of a
region of interest (ROI) encompassing only the apical membrane, and the
whole cell area including the apical membrane domain, respectively. At
least four tubules and three cells in each tubule were measured in each of
three different areas of kidney (cortex, outer medulla, and inner medulla).
Each experiment was repeated at least three times.
cAMP and cGMP assays. Total cellular cAMP and cGMP levels
were measured with the BioTrak EIA system (GE Healthcare Life
Sciences, Piscataway, NJ) as described in detail previously (4, 5).
Cells treated with VP (10 nM) or sodium nitroprusside (NTP; 1 mM)
for 10 min were used as positive controls. Each assay was performed
in triplicate and repeated three times independently.
Statistical analysis. Statistical analyses were performed using the
unpaired t-test. P values ⬍0.05 were considered statistically significant.
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accumulation of AQP2 on the apical membrane that mimicked
the effect of VP (Fig. 3). However, the effect of EGCG and
HA14-1 on AQP2 membrane accumulation was not significant
in MDCK cells (data not shown).
We then tested these compounds on rat kidney slices in
vitro. After AG-490 treatment, AQP2 accumulated on the
apical membrane of principal cells in both inner medulla
(Fig. 4, A and C) and cortex (Fig. 4, B and D). ␤-Lapachone
treatment induced AQP2 accumulation on the apical membrane
in the cortex only, and not in the medulla. We also applied
EGCG and HA14-1 to kidney tissue slices, but as in MDCK
cells, these compounds had no significant effect on AQP2
localization (data not shown).
Effect of compounds on urinary concentration in VP-deficient Brattleboro rats. To evaluate the effect of compounds on
urine volume and osmolality in vivo, VP-deficient Brattleboro
rats were housed individually in metabolic cages. These rats,
which model central DI, have been used in the past to identify
drugs that can increase urine concentration in the complete
absence of the antidiuretic hormone VP. Rats were injected
intraperitoneally with AG-490 (35 mg/kg), ␤-lapachone (17
mg/kg), or control solution. After AG-490 injection, urine
volume was significantly decreased and urine osmolality
was significantly increased within 2 h after injection (Fig. 5,
A and B). However, this effect of AG-490 did not continue
after 6 h, and urine volume and osmolality after 8 h was
almost the same as in control rats (Fig. 5). Despite a potent
effect on AQP2 trafficking in all of the in vitro systems we
Table 2. Twenty-four hour metabolic cage study data
Time 0
Control
AG-490
␤-Lap
Control
AG-490
␤-Lap
Control
AG-490
␤-Lap
24 h
Food intake, g
17.4 ⫾ 0.4
16.7 ⫾ 0.7
14.5 ⫾ 2.1
14.7 ⫾ 1.8
14.3 ⫾ 3.5
10.9 ⫾ 2.6
Feces, g
8.6 ⫾ 1.2
10.0 ⫾ 2.5
7.8 ⫾ 2.2
7.4 ⫾ 1.9
6.4 ⫾ 0.4
6.4 ⫾ 2.3
Body wt, g
468 ⫾ 20
463 ⫾ 19
475 ⫾ 24
472 ⫾ 25
494 ⫾ 29
485 ⫾ 29
Ratio
0.96 ⫾ 0.04
1.02 ⫾ 0.12
0.75 ⫾ 0.18
1.17 ⫾ 0.29
0.94 ⫾ 0.24
1.00 ⫾ 0.36
0.99 ⫾ 0.04
0.99 ⫾ 0.05
0.98 ⫾ 0.06
Values are means ⫾ SE; n ⫽3. Experiments were repeated twice, and an
average for each rat was obtained. Ratio was defined as after-treatment (24 h)
values divided by before-treatment (time 0) values. There were no significant
differences between ratio values of treated animals compared with control
untreated animals. ␤-Lap, ␤-lapachone.
AJP-Cell Physiol • doi:10.1152/ajpcell.00154.2014 • www.ajpcell.org
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Fig. 6. A: immunofluorescence staining of
AQP2 in Brattleboro rat kidney. At 2 h after
injection, rats were perfused via the left
ventricle and kidneys were fixed. In cortical
collecting ducts (top), AQP2 accumulated on
apical membranes of principal cells after
AG-490 treatment. Arrows indicate AQP2
apical membrane accumulation. However,
there was no significant difference in medullary collecting ducts (bottom). AQP2 localization after ␤-lapachone treatment was
not different from the controls. Scale bars,
40 ␮m. B: quantification of AQP2 fluorescence intensity. Fluorescence intensity of the
apical area was divided by fluorescence of
the whole cell area. Data are shown as
means ⫾ SE; n ⫽ 3. *P ⬍ 0.05 vs. control.
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SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
Table 3. Serum biochemistries show no significant differences after treatment
Control
AG-490
␤-Lapachone
BUN
Creatinine
TP
Glucose
ALT
ALP
CCr
17.5 ⫾ 2.7
15.4 ⫾ 5.3
15.9 ⫾ 0.8
0.3 ⫾ 0.1
0.3 ⫾ 0.1
0.3 ⫾ 0.0
5.3 ⫾ 0.1
5.0 ⫾ 0.1
5.2 ⫾ 0.3
217 ⫾ 57
187 ⫾ 7
187 ⫾ 45
39 ⫾ 7
32 ⫾ 7
42 ⫾ 8
277 ⫾ 19
239 ⫾ 51
293 ⫾ 105
3.8 ⫾ 0.7
3.5 ⫾ 1.8
4.3 ⫾ 0.3
Values are means ⫾ SD; n ⫽ 3. BUN, blood urea nitrogen (mg/dl); creatinine (mg/dl); TP, total protein (g/dl); glucose (mg/dl); ALT, alanine aminotransferase
(U/l); ALP, alkaline phosphatase (U/l); CCr, creatine clearance (mg/min). CCr was obtained from 4 h urine collection after injection of compounds.
assays (Fig. 7). AG-490 increased exocytosis in LLC-AQP2ssYFP cells, but it did not increase exocytosis in LLC-PK1
cells that do not express AQP2. This indicates that the increase
in exocytosis induced by AG-490 requires expression of
AQP2. These data are similar to those seen after VP treatment
(Fig. 7) which, as we previously showed, increases exocytosis
only in cells that express AQP2 (29, 41). Interestingly, ␤lapachone, a compound that increased AQP2 membrane accumulation in cell cultures and in tissue slices (see above), also
required AQP2 expression to stimulate ssYFP exocytosis. This
again indicates that its apparent lack of effect in vivo could
result from a bioavailability problem.
AG-490 does not increase cAMP or cGMP levels. The
significant effect of AG-490 on AQP2 trafficking prompted us
to look at the effect of AG-490 on cAMP and cGMP levels to
determine whether its effect on AQP2 trafficking might occur
via the canonical cAMP/PKA pathway or the alternative cGMP
Fig. 7. AG-490 significantly increases exocytosis only in AQP2-expressing
cells. Exocytosis assay with LLC-PK1 cells stably expressing ssYFP is shown.
In AQP2-expressing cells (AQP2 ⫹ ssYFP), exocytosis of ssYFP was increased after treatment with VP/FK, AG-490, and ␤-lapachone. In cells
without AQP2 (ssYFP only), exocytosis was not significantly increased after
treatment with any of the compounds. Data are shown as means ⫾ SE; n ⫽ 4.
*P ⬍ 0.05 vs. control.
Fig. 8. AG-490 does not increase cAMP or cGMP levels in LLC-PK1 cells.
There was no significant elevation of cAMP in response to 10 min of treatment
with 40 ␮M AG-490, in contrast to the large increase with 10 nM vasopressin
(top). Similarly, AG-490 did not increase cGMP levels, whereas 1 mM sodium
nitroprusside (NTP) had a highly significant effect (n ⫽ 3). Data are expressed
in femtomoles of cAMP or cGMP generated per 106 cells and are shown as
means ⫾ SE. P ⬍ 0.05 vs. control for VP and NTP effects.
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examined, ␤-lapachone failed to show any significant effect
on urine volume and osmolality in the time period tested
(Fig. 5).
We next determined the subcellular distribution of AQP2
after in vivo drug treatment. Kidneys from each group of
treated Brattleboro rats were harvested 2 h after injection and
were immunostained for AQP2 (Fig. 6A). There was a significant accumulation of AQP2 at the apical membrane in the
cortex of AG-490-treated rats, in contrast to the more diffuse
distribution in vehicle-treated rats (Fig. 6, A and B). However,
there was no significant enhancement of AQP2 apical localization in principal cells from the medulla in vivo, in contrast
to the effect on kidney tissue slices in vitro. ␤-Lapachone had
no detectable effect on AQP2 localization in any area of the
kidney in vivo. These observations suggest that there are
potential differences in bioavailability and drug delivery to
target cells in vivo and in vitro.
Potential side effects of these chemicals were also examined.
After the treatment, the rats looked normal and showed normal
behaviors. There were no significant differences in food intake
and body weight change after normalization compared with the
control group (Table 2). There was no significant difference in
blood chemistries, including kidney and liver function tests
(Table 3). Finally, there was no significant difference in creatinine clearance (Table 3), suggesting adequate renal function
in these animals. Therefore, the reduction in urine volume seen
in AG-490-treated animals was unlikely to have resulted from
a reduction of the glomerular filtration rate.
AG-490 and ␤-lapachone increase exocytosis only when
AQP2 is expressed. To determine the specificity of AG-490 on
AQP2 exocytosis, we performed additional ssYFP exocytosis
Methods In Cell Physiology
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SCREENING FOR AQUAPORIN 2 EXOCYTOSIS ENHANCERS
pathway that we have previously described (3, 5). As shown in
Fig. 8, AG-490 treatment with LLC-PK1 cells did not induce
a measurable elevation of cAMP or cGMP levels, in contrast to
the expected and strong effect of VP and NTP, respectively.
DISCUSSION
ACKNOWLEDGMENTS
We thank Maggie Ma for technical assistance, and Dr. S. Sasaki and Dr. S.
Uchida (Tokyo Medical and Dental University) for providing MDCK clones.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK) Grant DK-096586 (to D. Brown). H. A. Lu was
supported by NIDDK Grants DK-075940 and DK-092619 and an MGH/ECOR
interim support fund. P. Nunes was supported by a doctoral-level scholarship
from the Natural Sciences and Engineering Research Council of Canada. N.
Nomura was supported by a grant for “Strategic Young Researcher Overseas
Visits Program for Accelerating Brain Circulation program” from Japan
Society for the Promotion of Science. Additional support for the Program in
Membrane Biology Microscopy Core comes from the Boston Area Diabetes
and Endocrinology Research Center (DK-57521) and the MGH Center for the
Study of Inflammatory Bowel Disease (NIDDK Grant DK-43351). N. Pathomthongtaweechai was supported during his stay in D. Brown’s laboratory by the
Medical Scholar’s Program of the Faculty of Science, Mahidol University,
Bangkok, Thailand.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
N.N., P.N., R.B., A.V.N., S.S., N.P., H.A.J.L., and D.B. conception and
design of research; N.N., P.N., R.B., A.V.N., E.U., and N.P. performed
experiments; N.N., P.N., R.B., A.V.N., S.S., E.U., N.P., H.A.J.L., and D.B.
analyzed data; N.N., P.N., R.B., A.V.N., S.S., N.P., H.A.J.L., and D.B.
interpreted results of experiments; N.N., A.V.N., N.P., and D.B. prepared figures;
N.N. and D.B. drafted manuscript; N.N., P.N., R.B., A.V.N., S.S., E.U., H.A.J.L.,
and D.B. edited and revised manuscript; N.N., P.N., R.B., A.V.N., S.S., E.U., N.P.,
H.A.J.L., and D.B. approved final version of manuscript.
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We describe here an unbiased screening procedure to identify novel chemicals that stimulate VP-independent AQP2
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