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Soluble (pro)renin receptor via β-catenin enhances
urine concentration capability as a target of liver
X receptor
Xiaohan Lu
a,b,c,1
, Fei Wang
c,a,b,1
, Chuanming Xu
c
, Sunny Soodvilai
a,b
, Kexin Peng
a,b,c
, Jiahui Su
c
, Long Zhao
a,b
,
Kevin T. Yang
a,b
, Yumei Feng
d,e
, Shu-Feng Zhou
f
, Jan-Åke Gustafsson
g,2
, and Tianxin Yang
a,b,c,2
a
Department of Internal Medicine, University of Utah, Salt Lake City, UT 84112;
b
Veterans Affairs Medical Center, Salt Lake City, UT 84108;
c
Institute of
Hypertension, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China;
d
Department of Pharmacology,
University of Nevada School of Medicine, Reno, NV 89557;
e
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno,
NV 89557;
f
Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33620; and
g
Department of Biology
and Biochemistry, University of Houston, Houston, TX 77004
Contributed by Jan-Åke Gustafsson, February 12, 2016 (sent for review February 1, 2016; reviewed by Guanping Chen and Jia L. Zhuo)
The extracellular domain of the (pro)renin receptor (PRR) is cleaved
to produce a soluble (pro)renin receptor (sPRR) that is detected in
biological fluid and elevated under certain pathological conditions.
The present study was performed to define the antidiuretic action
of sPRR and its potential interaction with liver X receptors (LXRs),
which are known regulators of urine-concentrating capability. Wa-
ter deprivation consistently elevated urinary sPRR excretion in
mice and humans. A template-based algorithm for protein–protein
interaction predicted the interaction between sPRR and frizzled-8
(FZD8), which subsequently was confirmed by coimmunoprecipita-
tion. A recombinant histidine-tagged sPRR (sPRR-His) in the nano-
molar range induced a remarkable increase in the abundance of
renal aquaporin 2 (AQP2) protein in primary rat inner medullary
collecting duct cells. The AQP2 up-regulation relied on sequential
activation of FZD8-dependent β-catenin signaling and cAMP–PKA
pathways. Inhibition of FZD8 or tankyrase in rats induced polyuria,
polydipsia, and hyperosmotic urine. Administration of sPRR-His
alleviated the symptoms of diabetes insipidus induced in mice by
vasopressin 2 receptor antagonism. Administration of the LXR ag-
onist TO901317 to C57/BL6 mice induced polyuria and suppressed
renal AQP2 expression associated with reduced renal PRR express-
ion and urinary sPRR excretion. Administration of sPRR-His re-
versed most of the effects of TO901317. In cultured collecting duct
cells, TO901317 suppressed PRR protein expression, sPRR release, and
PRR transcriptional activity. Overall we demonstrate, for the first
time to our knowledge, that sPRR exerts antidiuretic action via
FZD8-dependent stimulation of AQP2 expression and that inhibi-
tion of this pathway contributes to the pathogenesis of diabetes
insipidus induced by LXR agonism.
soluble (pro)renin receptor
|
liver X receptor
|
aquaporin-2
|
frizzled-8
|
β-catenin
Full-length (Pro)renin receptor (PRR), a 350-amino acid
transmembrane receptor for prorenin and renin, is subjected
to protease-mediated cleavage to produce a 28-kDa protein of
the N-terminal extracellular domain, the soluble (pro)renin re-
ceptor (sPRR), and the 8.9-kDa C-terminal intracellular domain
called “M8.9”(1, 2). Before the cloning of full-length PRR in
mesangial cells as an integral 39-kDa membrane protein (3),
M8.9 was identified as a truncated protein associated with the
vacuolar H
+
-ATPase (V-ATPase) from bovine chromatin gran-
ules (4). The cleavage occurs in Golgi apparatus through furin
(5) or ADMA19 (6). An sPRR ELISA kit has been developed to
detect sPRR in plasma and urine samples (7)
.
With this assay,
increased serum sPRR levels have been demonstrated in patients
with heart failure (8), kidney disease (9, 10), hypertension (11),
and preeclampsia (2). Moreover, serum sPRR is positively as-
sociated with serum creatinine, blood urea nitrogen, and urine
protein and is inversely associated with the estimated glomerular
filtration rate in patients with chronic kidney disease caused by
hypertension and type 2 diabetes (9). However, serum sPRR was
not correlated with serum renin, prorenin, or aldosterone in
healthy subjects or in patients with diabetes and hypertension
(12). To our knowledge no prior studies have reported a bi-
ological function of sPRR in any condition.
Within the kidney, PRR is expressed abundantly in the distal
nephron, particularly in intercalated cells of the collecting duct
(CD) (13, 14). A functional role of PRR in regulating renal
aquaporin 2 (AQP2) expression and urine-concentrating capa-
bility has been revealed by analysis of mice with conditional
deletion of PRR in the nephron (15) and the CD (14). However,
whether the antidiuretic action of CD PRR is conferred by sPRR
remains elusive.
Liver X receptors (LXRs) are activated by oxidized choles-
terol derivatives and belong to a family of nuclear receptors that
form heterodimers with the retinoid X receptor to regulate tran-
scription of target genes governing cholesterol, fatty acid, and
glucose metabolism (16, 17) (18). LXRs consist of two isoforms,
LXRα, which is abundantly expressed in liver, small intestine,
kidney, macrophages, and adipose tissue, and LXRβ,whichis
expressed more ubiquitously (19). LXRs have an established role
in reverse cholesterol transport which leads to cholesterol efflux
from peripheral tissues to the liver (20). Interestingly, emerging
Significance
The soluble (pro)renin receptor (sPRR) is produced by protease-
mediated cleavage of PRR and is elevated under certain path-
ological conditions. To our knowledge, no prior studies have
reported the biological function of sPRR in general or the
antidiuretic function of the soluble protein in particular. Here
we describe a previously unreported role of sPRR in the en-
hancement of renal aquaporin 2 (AQP2) expression and urine-
concentrating capability. We further show that sPRR acts via
frizzled class receptor 8-depdendent β-catenin signaling to in-
crease AQP2 expression in the collecting duct cells. These find-
ings offer an unreported insight into the physiological role of
sPRR in regulating fluid homeostasis. In addition, we found that
liver X receptor activation by TO901317 resulted in diabetes
insipidus because of the inhibition of renal PRR expression.
Author contributions: S.-F.Z., J.-Å.G., and T.Y. designed research; X.L., F.W., C.X., S.S., K.P.,
J.S., L.Z., and K.T.Y. performed research; Y.F. contributed new reagents/analytic tools; and
J.-Å.G. and T.Y. wrote the paper.
Reviewers: G.C., Emory University School of Medicine; and J.L.Z., University of Mississippi.
The authors declare no conflict of interest.
1
X.L. and F.W. contributed equally to this work.
2
To whom correspondence may be addressed. Email: jgustafsson@uh.edu or Tianxin.
Yang@hsc.utah.edu.
www.pnas.org/cgi/doi/10.1073/pnas.1602397113 PNAS Early Edition
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MEDICAL SCIENCES PNAS PLUS
evidence suggests a potential role of LXRs in the regulation of
the renin-angiotensin system (RAS) and renal transporters such
as Na-Pi transporters (21), OAT1 (22), and epithelial Na
+
channel
(ENaC) (23). In the present study we attempted to define a bi-
ological function and signaling of sPRR in the regulation of fluid
homeostasis and to test PRR/sPRR further as a target of LXRs
in the kidney.
Results
Initial Evidence for a Biological Function of sPRR. A clue indicating a
potential biological function of sPRR came from immunostain-
ing of rat kidneys using antibodies against different domains of
PRR. We examined the cellular localization of renal PRR in rats
using two different antibodies: the antibody raised against the
C-terminal domain of PRR (termed “anti–PRR-C antibody”;
Abcam) and another antibody against the N-terminal domain in
the sPRR sequence (termed “anti–PRR-N antibody”) (24). After
the cleavage, sPRR is recognized by anti–PPR-N but not by anti–
PRR-C. Anti–PRR-C antibody labeled AQP2
−
CD cells, e.g., the
intercalated cells (Fig. 1C) (13, 25). Unexpectedly, the labeling
with anti–PRR-N was found predominantly on the apical mem-
brane of CD principal cells (Fig. 1 Aand B), overlapping with
AQP2 (Fig. 1 Dand E), confirming the localization in principal
cells but not in intercalated cells. The images from colabeling
with anti-PRR antibody and anti-AQP2 antibody were merged
(Fig. 1 G–I). We performed a competition assay to validate the
specificity of anti–PRR-N antibody. The signal from this anti-
body disappeared after preincubation with a recombinant sPRR
protein, sPRR-His (Fig. 1 Jand K) or with the immunizing peptide
(Fig. 1L) or with the omission of primary antibody (Fig. 1M).
These immunostaining results raised the intriguing possibility
that sPRR derived from intercalated cells or other tubules may
act on an as yet unknown membrane receptor in principal cells to
regulate tubular transport function. To test this possibility, a
recombinant rat sPRR was generated using a mammalian cell-
expressing system. This protein contained sPRR with a secretion
signal in the N terminus and an eight-histidine tag in the C terminus
(termed “sPRR-His”) and appeared as a single 29.6-kDa band on
12% SDS/PAGE gel (Fig. 2A). Primary cultures of rat inner
medullary collecting duct (IMCD) cells in Transwells exposed to
10 nM sPRR-His for 24 h exhibited a remarkable increase in
AQP2 expression at both mRNA and protein levels (Fig. 2 Band
C). In a separate experiment, murine immortalized cortical col-
lecting duct (mpkCCD) cells were transiently transfected by a
luciferase reporter construct, pGL3-AQP2. The transfected cells
were treated for 24 h with 10 nM sPRR-His or vehicle. The
sPRR-His treatment induced a 5.5-fold increase in luciferase
activity (Fig. 2D). The in vitro findings confirmed a direct stim-
ulatory effect of sPRR-His on AQP2 expression.
sPRR Activation of the Wnt–β-Catenin Pathway in the CD. Search tool
for the retrieval of interacting gene and proteins (STRING) is a
template-based algorithm for protein–protein structure pre-
diction. We used STRING to identify proteins that interact with
sPRR. Among 10 candidate proteins, three are related to Wnt–
β-catenin pathway: frizzled-8 (FZD8), low-density lipoprotein
receptor-related protein 6 (LRP6), and Wnt-3a (Fig. 2E). Be-
cause FZD8 is a receptor component in the Wnt–β-catenin
pathway, subsequent studies were focused on interaction be-
tween FZD8 and sPRR. Coimmunoprecipitation experiments
using membrane fraction proteins prepared from the rat inner
medulla demonstrated that sPRR binds to FZD8 (Fig. 3A).
Immunostaining showed that at low magnification (Fig. 3B),
FZD8 labeling was found predominantly in the outer and
inner medulla with sporadic labeling in the cortex. Colabeling
with AQP2 (the marker of CD principal cells) and labeling of
Fig. 1. Distinct labeling with antibodies against different domains of PRR. (A–C) Immunostaining on consecutive rat kidney sections using anti–PRR-N an-
tibody (magnification: 200×in A; 400×in B) recognizing the N-terminal domain of PRR (e.g., the sPRR sequence) and the anti–PRR-C antibody recognizing the
C-terminal domain of PRR (C; magnification: 400×). (D–F) The PRR antibodies were coincubated with anti-AQP2 antibody. (G–I) Merged images. Arrows in B
and Edenote principal cells; arrows in Cand Fdenote intercalated cells. The labeling with anti–PRR-N antibody was localized mostly to the apical membrane
of principal cells, contrasting with the labeling with anti–PRR-C antibody in the intercalated cells. (J–M) To validate the specificity of the labeling, immu-
nostaining was performed with anti–PRR-N antibody preincubated with an expressed sPRR (J), sPRR-His (K), or its immunizing peptides (L) or without the
primary antibody (M). The images shown are representative of three to six animals per group.
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consecutive sections for the electroneutral sodium, potassium and
chloride cotransporters (NKCC2), a marker of thick ascending limb
cells (Fig. 3C), confirmed FZD8 staining in the thick ascending
limb and the CD. In the CD, FZD8 staining was detected in both
principal and intercalated cells. The comparison between FZD8
and PRR labeling was made in the inner medulla where the two
signals were roughly colocalized to the CD. In principal cells,
both FZD8 and PRR were detected on the apical membrane
although FZD8 labeling was relatively diffuse (Fig. 3D).
The functional role of FZD8 in mediating sPRR signaling in
primary rat IMCD cells was assessed by using FZD8 siRNA and
an FZD8 inhibitor, OMP-54F03 (OMP). The efficacy of FZD8
siRNA was validated by immunoblotting analysis of FZD8 pro-
tein expression (Fig. 4A). Exposure of rat IMCD cells to 10 nM
sPRR-His for 24 h induced the activity of Wnt-responsive lu-
ciferase activity as assessed by using the Cignal TCF/LEF Re-
porter Assay kit (Qiagen), which was blunted by FZD siRNA
(Fig. 4B). Consistent with this result, the sPRR-His treatment
remarkably induced AQP2 protein expression, which was blun-
ted by both FZD8 siRNA (Fig. 4C) and OMP (Fig. 4D) as well as
by a tankyrase inhibitor XAV939 (XAV) (Fig. 4E) (26). Tankyrase
belongs to the poly (ADP-ribose) polymerase family responsi-
ble for the transfer of ADP ribose from NAD
+
to acceptor
proteins (27) and also for the activation of the Wnt–β-catenin
pathway through the stabilization of the axin–β-catenin com-
plex (26). Our results suggest that sPRR signals through FZD8
to activate the Wnt/β–catenin pathway, leading to increased
AQP2 expression.
Arginine vasopressin (AVP) is known to induce AQP2
trafficking to the apical membrane acutely (within minutes) by
increasing phosphorylation of AQP2 and chronically (within hours)
by stimulating AQP2 transcription, both through the cAMP–PKA
pathway (28, 29). We found that the rapid rise of cAMP and the
redistribution of AQP2 from the cytosol to the membrane in
response to a 30-min exposure to AVP was unaffected by XAV
treatment (Fig. 5 A–C); the trafficking event was evaluated by
examining the abundance of AQP2 protein in the fractionated
cell samples. Immunoblotting detected AQP2 protein as multi-
ple bands of 35–45 kDa and 29 kDa, reflecting the glycosylated
and nonglycosylated forms, respectively. In contrast, at 24 h the
increases in the cAMP level (Fig. 5D) and total protein abun-
dance (Fig. 5E) and mRNA expression (Fig. 5F) of AQP2 were
all effectively attenuated by XAV treatment. These results sug-
gest that the Wnt–β-catenin pathway may specifically target
AQP2 gene transcription but not AQP2 trafficking via selective
coupling with the late but not early cAMP production after
AVP treatment.
The in Vivo Role of β-Catenin Signaling in Rats During Antidiuresis. To
probe the in vivo role of β-catenin signaling in fluid homeostasis,
we administered OMP and XAV to Sprague–Dawley (SD) rats
under basal conditions and during 48-h water deprivation (WD)
and evaluated their impact on water balance. Under basal con-
ditions, the administration of OMP and XAV over 48 h similarly
induced polyuria, polydipsia, and hypoosmotic urine (Fig. 6 A–C).
During 48-h WD, these treatments consistently increased urine
volume and decreased urine osmolality, and these effects were
accompanied by exaggerated weight loss (Fig. 6 D–F) and greater
increases in plasma osmolality and hematocrit (Hct) (Fig. 6 Gand
H). Immunoblotting showed that the abundance of β-catenin
protein was increased in the nuclear fraction from both cortex and
inner medulla following WD, indicating the activation of β-catenin
signaling (Fig. 7 Aand B). AQP2 protein abundance in both renal
cortex and inner medulla was increased remarkably following WD;
this increase was attenuated significantly by both OMP and XAV
(Fig. 7 C–F).
Fig. 3. Renal FZD8 expression and its interaction with sPRR. (A) Coimmu-
noprecipitation analysis of the interaction between sPRR and FZD8. The
membrane fraction of rat renal medulla was immunoprecipitated with anti–
PRR-N antibody and probed with anti-FZD8 antibody or vice versa. (B)Immu-
nolabeling of FZD8 in rat kidney at low magnification. (C) Immunolabeling of
FDZ8, AQP2, and NKCC2 in the outer medulla at high magnification. Colab-
eling for AQP2 was performed on the same section, and labeling for NKCC2
was performed on consecutive sections. CD, collecting duct; PT, proximal
tubule; TL, thick ascending limb. (D) Immunolabeling of FDZ8 and PRR in the
rat inner medulla. The consecutive sections were stained with anti-FZD8
antibody and anti–PRR-N antibody. The same section was colabeled with
anti-AQP2 antibody. The images shown are representative of three to six
animals per group.
0
2
4
6
8
10
12
CTR sPRR-His
AQP2 mRNA/GAPDH
55
72
95
130
kDa
43
36
26
15
Marker E1 E2 E3
sPRR-His
29.6 kDa
E4
sPRR-His
AQP2
β-Actin
CTR
1.0f0.02 3.08f0.25 *
35~45 kDa
29 kDa
43 kDa
ABC
D
0
2
4
6
8
10
12
14
16
18
pGL3-blank pGL3-AQP2 pGL3-AQP2 + sPRR-His
Luminescence (RLU/µg protein)
E
p<0.01
Fig. 2. Characterization of sPRR function. A recombinant PRR, sPRR-His, was
expressed in a mammalian cell-expressing system as a fusion protein that con-
tained sPRR, a histidine tag in the C terminus, and a secretion signal peptide in
the N terminus. (A) sPRR-His was purified from the medium as a single
29.6-kDa band in 12% SDS/PAGE gel. E1–4 are sequential fractions from the
elution. (Band C) Primary rat IMCD cells grown in Transwells were exposed
to 10 nM sPRR-His for 24 h, and AQP2 expression was analyzed by quanti-
tative RT-PCR and immunoblotting (n=5 per group). (D) In a separate ex-
periment, the cultured CD cells were transfected with an AQP2-luciferase
construct, were allowed to grow to confluence, and then were treated with
vehicle or 10 nM sPRR-His for 24 h, and luciferase activity was assayed (n=5
per group). (E) STRING is a template-based algorithm for predicting protein–
protein structure. We used STRING to identify proteins that interact with
sPRR. This analysis revealed 10 hits: CTSG (cathepsin G), ACE2 (angiotensin
1-converting enzyme 2), CMA1 (chymase 1), MME (membrane metallo-endo-
peptidase), ACE (angiotensin 1 converting enzyme 1), CTSZ (cathepsin Z),
ENPEP (glutamyl aminopeptidase), FZD8 (frizzled family receptor 8), WNT3A
(wingless-type MMTV integration 3A), and LRP6 (low density lipoprotein
receptor-related protein 6). CTR, control.
Lu et al. PNAS Early Edition
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Therapeutic Potential of sPRR-His for Treatment of Nephrogenic
Diabetes Insipidus. Nephrogenic diabetes insipidus (NDI) is
commonly caused by mutations of the vasopressin 2 receptor
(V2R) gene; a specific therapy for this disease is lacking (30, 31).
We explored the therapeutic potential of sPRR-His in a mouse
model of NDI induced with a V2R antagonist, OPC. sPRR-His
was chronically infused via a catheter placed in the jugular vein
driven by an osmotic minipump. After 7 d of sPRR-His infusion,
OPC was given via gavage at 30 mg·kg
−1
·d
−1
for 3 d. Adminis-
tration of the V2R antagonist resulted in symptoms of NDI,
including polydipsia, polyuria, and hypoosmotic urine, all of
which were attenuated by sPRR-His treatment (Fig. 8 A–C).
This result supports the therapeutic potential of sPRR for
management of the symptoms of NDI. In the OPC-treated mice,
urinary sPRR excretion was suppressed significantly (Fig. 8D),
providing a rationale for the use of exogenous sPRR to treat
NDI. This result also suggests that the production of renal sPRR
may be under the direct control of the AVP–V2R pathway. In
support of this notion, we have shown that exposure of the CD
cells to AVP stimulates the release of sPRR (14). Fig. 8Epro-
vides a schematic illustration of the mechanism of action of
sPRR in the CD principal cells. Our data suggest that sPRR
binds FDZ8, leading to the activation of β-catenin that pro-
motes chronic cAMP accumulation, ultimately enhancing
AQP2 transcription.
Induction of Diabetes Insipidus and Suppression of Renal PRR by LXR
Agonism. Besides their well-recognized role in regulating lipid
and glucose metabolism, LXRs are potential regulators of the
RAS (32) and fluid balance (33). We tested the hypothesis that
LXRs may affect renal PRR expression and local RAS and
hence fluid homeostasis. In the initial experiment, we examined
the effects of the LXR agonist TO901317 on fluid homeostasis
and on renal PRR and urinary renin levels in mice. A 7-d treat-
ment with TO901317 in C57BL6 mice reduced body weight
(32.36 ±0.63 vs. 28.61 ±0.36 g, P<0.05) and food intake (4.08 ±
0.19 vs. 3.54 ±0.15 g, P<0.05), as previously reported (34),
although to a lesser extent. This treatment led to severe polyuria
(Fig. 9A) and hypoosmotic urine (Fig. 9B), indicating a urine-
concentrating defect. The expression of renal PRR protein and
the excretion of urinary sPRR were examined by immunoblotting
and ELISA, respectively. Both renal PRR expression (Fig. 9 C
and D) and urinary sPRR excretion (Fig. 9E) were suppressed
consistently by TO901317. This experiment suggested that the
suppressed renal PRR level may be responsible for TO901317-
induced diabetes insipidus (DI).
We performed cell-culture experiments to examine the direct
effect of TO901317 on PRR expression. mpkCCD cells were
exposed to TO901317 or vehicle for 24 h, and PRR protein ex-
pression was determined by immunoblotting. TO901317 treat-
ment reduced PRR protein expression by 85% (Fig. 9 Fand G)
and medium sPRR by 60% (Fig. 9H). In a separate experiment,
mpkCCD cells were transiently transfected by a pGL3-PRR
construct. The transfected cells were treated with TO901317 or ve-
hicle for 24 h. TO901317 treatment suppressed luciferase activity by
76% (Fig. 9I). The in vitro findings confirmed that TO901317
has a direct inhibitory effect on PRR expression.
sPRR-His Attenuates TO901317-Induced DI in Mice. In the subsequent
experiment, we performed more detailed analysis of TO901317-
induced DI and further examined the causal role of suppressed
renal PRR in this phenomenon. This experiment comprised
three groups: control, TO901317-treated, and TO901317 +sPRR-
His–treated mice. TO901317-treated mice displayed polyuria, poly-
dipsia, decreased water balance, hypoosmotic urine, and plasma
volume contraction (as reflected by the rise in Hct), confirming
the urine-concentrating defect (Fig. 10). All these parameters were
improved significantly in mice treated withTO901317 +sPRR-His
Fig. 5. The distinct role of the Wnt/ β–catenin pathway in the acute and
chronic responses to AVP in primary rat IMCD cells. The IMCD cells were
pretreated for 1 h with XAV and were treated for 30 min or 24 h with 10 nM
AVP. (A) At 30 min of AVP treatment, the medium was assayed for cAMP
using ELISA (n=6 per group). (Band C) Membrane fraction (n=6 per group)
(B) and cytosolic fraction (n=6 per group) (C) were subjected to immuno-
blotting analysis of AQP2. (D–F) At 24 h, medium cAMP was determined (n=
6 per group) (D), whole-cell lysates were subjected to immunoblotting
analysis of AQP2 (n=6 per group) (E), and total RNA was subjected to
quantitative RT-PCR analysis of AQP2 mRNA (n=4 per group) (F). Data are
shown as mean ±SE; *P<0.05 vs. control;
#
P<0.05 vs. AVP alone.
Fig. 4. Analysis of the in vitro role of the Wnt/β–catenin pathway in the
regulation of AQP2 expression in response to sPRR-His treatment. (A) Vali-
dation of FZD8 knockdown by siRNA (n=3 per group). Primary rat IMCD
cells were transfected with or without FZD8 siRNA. FZD8 protein expression
was evaluated by immunoblotting. (B) Effect of sPRR-His on Wnt-response
luciferase activity in the presence or absence of FZD8 siRNA (n=12 per
group). The IMCD cells were transfected with or without FZD8 siRNA, fol-
lowed by sPRR-His treatment at 10 nM for 24 h. The Cignal reporter system
was used to evaluate the activity of the Wnt/β–catenin pathway, and the
data are presented as relative response ratio. (C–E) Rat IMCD cells were
transfected with FZD8 siRNA, pretreated with OMP or XAV-939 for 1 h, and
treated with 10 nM sPRR for 24 h. AQP2 protein expression was determined
by immunoblotting analysis. Densitometric values are shown underneath
the blots. (C) Effect of FZD8 knockdown on sPRR-Induced AQP2 protein
expression (n=6 per group). (D) Effect of OMP on sPRR-His-induced AQP2
protein expression (n=6 per group). (E) Effect of XAV on sPRR-His-induced
AQP2 expression (n=6 per group). Data are shown as mean ±SE; *P<0.05
vs. control;
#
P<0.05 vs. sPRR-His.
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(Fig. 10). Of note, water balance was determined by subtracting
urine volume from water intake. AQP2, a major water channel
on the apical membrane of the CD, plays a key role in de-
termining urine-concentrating capability. Immunoblotting dem-
onstrated that TO901317 significantly reduced renal AQP2
expression, which was partially restored by sPRR-His (Fig. 11 A
and B). We suspected that sPRR-His may affect the intrarenal
RAS, and this effect can be reflected by urinary renin activity
(35). Urinary renin activity, as assessed by measuring the gen-
eration of angiotensin I (AngI), was suppressed by TO901317,
and the suppression was completely reversed by sPRR-His (Fig.
11C). We also performed ELISA to determine the prorenin/renin
concentration in the urine. Although urinary prorenin/renin ex-
cretion was suppressed by TO901317, it was unaffected by sPRR-
His (Fig. 11D). This result is compatible with the concept that
sPRR regulates renin primarily at its activity level (3). Fig. 11E
provides a schematic illustration of the mechanism by which
TO901317 induces DI. Upon binding to TO901317, LXRs func-
tion as a transcriptional repressor to inactivate PRR transcription.
The reduced PRR/sPRR levels down-regulate AQP2 expression,
leading to DI.
Discussion
sPRR is generated by protease-mediated cleavage in the Golgi
apparatus and is released to plasma or urine. Serum sPRR levels
are elevated in various pathological states. To our knowledge, no
prior studies have reported a biological function of sPRR. We
recently used pharmacological and conditional gene-knockout
approaches to demonstrate that CD PRR has an essential role in
determining renal AQP2 expression and urine-concentrating
capability (14). In the present study, we discovered that sPRR
acts via FZD8-dependent activation of β-catenin signaling that
leads to increased AQP2 expression and thus enhanced urine-
concentrating capability. In addition, we found that LXR ago-
nism with TO901317 induced DI by inhibiting the renal PRR and
the intrarenal RAS.
Probably the most striking finding of this study was the re-
markable stimulatory effect of sPRR-His on AQP2 expression in
cultured rat IMCD cells grown in Transwells. In this experiment,
sPRR-His was used at 10 nM, which is likely a physiological
concentration. Moreover, we vigorously examined the signaling
mechanism of sPRR-His up-regulation of AQP2, revealing the
involvement of FZD8-dependent β-catenin signaling. A clue
suggesting potential interaction between sPRR and FZD8 was
obtained by a template-based algorithm for predicting protein–
protein structure. The physical interaction of the two proteins in
the membrane fraction of the rat inner medulla was confirmed
by coimmunoprecipitation. The interaction also was demon-
strated at a functional level, because inhibition of FZD8 by siRNA
and pharmacological approaches effectively attenuated the stim-
ulatory effect of sPRR-His on AQP2 in cultured CD cells. The
functional role of FZD8 in regulating renal AQP2 expression and
urine-concentrating capability was confirmed in vivo by using a
FZD8 inhibitor, OMP. The antidiuretic function of FZD8 was
supported by the similar effect of a general Wnt/β-catenin in-
hibitor, XAV. Immunostaining demonstrated that FZD8 is lo-
calized to the CD and thick ascending limb, as is consistent with
the nephron-distribution pattern of PRR. Within the CD, FZD8
labeling was found in both principal and intercalated cells, a
pattern not exactly same as sPRR labeling. We suspect that
FZD8 may serve a function beyond its association with sPRR.
These results agree with a previous report that β-catenin sig-
naling mediated AVP-induced AQP2 expression in mpkCCDc14
cells (36). It is evident that β-catenin signaling is actively involved
in the physiological regulation of fluid homeostasis through cou-
pling with sPRR.
A large body of evidence has demonstrated that PRR serves as
a component of the Wnt receptor complex to regulate embryo-
genesis in low vertebrates in which the RAS does not exist (37,
38). It is evident that PRR acts in a renin-independent manner in
low vertebrates (1, 37, 39-41). PRR appears to be similarly
involved in the regulation of embryogenesis in mammals, as
evidenced by the lethal phenotype in mice with systemic or tis-
sue-specific deletion of PRR (1, 4). A large number of stud-
ies have challenged the physiological function of PRR and its
Fig. 7. The in vivo role of the Wnt/β–catenin pathway in the regulation of
renal AQP2 expression during antidiuresis in rats. (Aand B) To assess renal
β-catenin activation by WD, the nuclear fraction of renal cortex (n=4rats
per group) (A) and the inner medulla (n=4 rats per group) (B) from control
and dehydrated rats were subjected to immunoblotting analysis of β-cat-
enin. (C–F) Immunoblotting analysis of AQP2 expression was performed on
the renal cortex and the inner medulla of rats treated with vehicle or WD
with (C) or without (D) OMP or with (E) or without (F) XAV (n=5 rats per
group). Data are shown as mean ±SE; *P<0.05 vs. control;
#
P<0.05 vs. WD.
CO, cortex; IM, inner medulla.
Fig. 6. The role of the Wnt/β–catenin pathway in urine-concentrating ca-
pability in rats. SD Rats were administered vehicle, OMP, or XAV and were
placed in metabolic cages for assessment of the state of water metabolism at
basal condition (A–C) or after 24-h WD (D–H)(n=5 rats per group). At basal
the condition, water intake (A), urine volume (B), and urine osmolality
(C) were determined. (D–F) During 24-h WD, urine volume (D), urine osmo-
lality (E), and body-weight (BW) changes (F) were monitored. (Gand H)Atthe
end of the experiment plasma osmolality (G)andHct(H) were measured. *P<
0.05 vs. control;
#
P<0.05 vs. WD alone.
Lu et al. PNAS Early Edition
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MEDICAL SCIENCES PNAS PLUS
relationship with the RAS (1, 37, 39–41). Whether an intrinsic
linkage between the RAS and the developmental β-catenin path-
way occurs in mammals in settings of development or physiology is
unknown. For the first time, to our knowledge, our results link
prorenin/sPRR to the β-catenin pathway in the kidney during
physiological regulation of fluid homeostasis.
The cAMP–PKA pathway is the principle mediator of AVP-
induced AQP2 trafficking and transcription (28, 42, 43). An issue
arises as to whether the Wnt–β-catenin pathway interacts with
the cAMP–PKA pathway during AVP-induced signaling. We
found that inhibition of the Wnt–β-catenin pathway did not af-
fect AQP2 trafficking to the membrane fraction or cAMP pro-
duction induced by 30-min exposure to AVP. In contrast, this
maneuver did block AQP2 expression and cAMP production
induced by 24-h AVP treatment. These results suggest that ac-
tivation of the Wnt–β-catenin pathway may be involved primarily
in sustaining the cAMP production during prolonged AVP
treatment to increase AQP2 transcription. This mechanism does
not appear to be required for the regulation of AQP2 trafficking,
which is a rapid response to AVP. There is a wealth of in-
formation regarding the role of the cAMP–PKA pathway in this
acute response to AVP, but relatively little is known about this
pathway in a chronic setting of AVP treatment. To our knowl-
edge, the present study is the first to delineate a unique role for
β-catenin signaling in chronic but not acute regulation of the
cAMP–PKA–AQP2 axis.
Multiple previous studies, as well as this one, consistently
demonstrate that, within the CD, PRR is detected in intercalated
cells by using antibody against the C terminus of PRR (anti–
PRR-C antibody) (13, 25). A question arises as to how interca-
lated cell-derived PRR up-regulates AQP2 expression in the
principal cells. This regulation may be through a paracrine mech-
anism, and there is an intriguing possibility that sPRR may serve
as a mediator of the communication between the two cell types
in the CD. In support of this possibility, an antibody against
the epitope in the sPRR, anti–PRR-N antibody, stained only
principal cells but not intercalated cells or other non-CD tubules
that are known to express PRR. More direct evidence came from
cell-culture experiments showing that exposure of rat IMCD cells
to sPRR-His in the nanomolar range induced a remarkable in-
crease in AQP2 expression mimicking the effect of prorenin (44).
This result strongly suggests that sPRR has a physiological function.
The current therapy for DI is suboptimal. Although supple-
mentation of AVP is effective for central DI (45), no specific
therapy is currently available for nephrogenic DI. The present
study demonstrates, for the first time to our knowledge, the
therapeutic potential of sPRR-His in a mouse model of nephrogenic
DI induced by V2R antagonism. Because sPRR acts downstream of
V2R, it also should be effective for treatment of central DI.
A large body of evidence has established a link between energy
metabolism and fluid balance. A high-energy state, such as
obesity, is associated with disturbance of electrolytes and fluid
balance and hypertension (46, 47), whereas a low-energy state,
such as fasting, induces natriuresis and diuresis (48, 49). Along
this line, activation of PPARγ, a key regulator of glucose me-
tabolism and adipogenesis, causes body weight gain and plasma
volume expansion (50, 51). In the present study, we discovered
that LXR against TO901317 exerted a profound diuretic action
in mice as suggested by polyuria, polydipsia, hypoosmotic urine,
and contraction of plasma volume. AQP2, a major water channel
on the apical membrane of the CD, was remarkably suppressed,
likely conferring the diuretic action of TO901317.
In light of sPRR’s action as a key regulator of AQP2 expression
and urine-concentrating capability, we suspected that PRR/sPRR
Fig. 8. Antidiuretic action of sPRR-His in a mouse model of NDI. Male C57/
BL6 mice were infused for 7 d with sPRR-His via a catheter implanted in the
jugular vein and then received oral administration of either vehicle or the
V2R antagonist OPC31260 (OPC) for 3 d. Mice were placed in metabolic cages
for assessment of daily water intake and urine output. (A) Daily water intake
(n=4 mice per group). (B) Daily urine output (n=4 mice per group).
(C) Urine osmolality (n=4 mice per group). (D) Urinary sPRR excretion (n=4
mice per group). Data are shown as mean ±SE. In A–C,
#
P<0.05 vs. OPC
alone; in D*P<0.05 vs. control. (E) Schematic illustration of the mechanism
of action of sPRR. In the lumen of the distal nephron, sPRR binds FZD8 in a
receptor complex on the apical membrane of principal cells, resulting in the
activation of β-catenin, which promotes cAMP accumulation, ultimately
leading to increased AQP2 transcription and enhanced urine concentration.
43 kDa
TO901317CTR
PRR
β-Actin 43 kDa
Luminescence
(RLU/µg protein)
0
0.2
0.4
0.6
Cell medium sPRR content
(pg/μg protein)
CTR TO901317
p<0.05
400
800
1200
1600
Urinary sPRR excretion
(pg/24h)
0
CTR TO901317
p<0.05
Urinary osmolality
(mOsmo/kg.H2O)
Urine volume (ml/24h)
0
0.4
0.8
1.2
CTR TO901317
PRR densitometry
TO901317CTR
PRR
β-Actin
43 kDa
43 kDa
0
0.4
0.8
1.2
CTR TO901317
PRR densitometry
p<0.05
p<0.05
A
D
G
B
E
H
C
F
I
0
1000
2000
3000
CTR TO901317
p<0.05
4000
0
1.0
2.0
3.0
pGL3-blank pGL3-PRR pGL3-PRR +
TO901317
4.0
0
1.0
2.0
CTR TO901317
p<0.05
3.0
p<0.05
Fig. 9. Initial characterization of diuretic and PRR-inhibitory actions of
TO901317 in vivo and in vitro. Male C57/BL7 mice received oral administra-
tion of TO901317 with or without i.v. infusion of sPRR-His for 7 d. The mice
receiving vehicle treatment served as a control. At the end of experiment,
24-h urine collection was performed, followed by analysis of urinary sPRR
excretion by ELISA and renal PRR expression by immunoblotting. (A) Urine
output (n=30 mice per group). (B) Urinary osmolality (n=15 mice per
group). (C) Immunoblotting analysis of renal PRR expression (n=15 mice per
group). (D) Densitometric analysis of the immunoblot in C.(E) ELISA analysis
of urinary sPRR (n=8 mice per group). (F–I) The effect of TO901317 on PRR
expression in cultured CD cells. mpkCCD cells were exposed to vehicle or
10 μM TO901317 for 24 h. The cell lysates were subjected to immunoblotting
analysis of PRR protein expression, and the medium was assayed for sPRR
concentration by using ELISA and normalized by protein content. In a sep-
arate experiment, the cells were transfected with a PRR-luciferase construct,
allowed to grown to confluence, and then were treated with vehicle or
10 μM TO901317 for 24 h. (F) Immunoblotting analysis of PRR (n=15 per
group). (G) Densitometric analysis of the immunoblot in A.(H) ELISA analysis
of medium sPRR (n=10 per group). (I) The luciferase assay (n=5 per group).
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www.pnas.org/cgi/doi/10.1073/pnas.1602397113 Lu et al.
may be a molecular target of LXRs in the kidney. In vivo data
showed a significant reduction of renal PRR expression and
urinary sPRR excretion in TO901317-treated mice, and the
administration of sPRR-His completely rescued TO901317-induced
DI. Consistent with this observation, in vitro data demonstrated
that TO901317 had a direct inhibitory effect on PRR expression
and PPR-luciferase activity in cultured CD cells. These results
demonstrate, for the first time to our knowledge, that LXRs may
function as a transcriptional repressor of the PRR gene. It is
interesting that LXRβ
−/−
mice display central DI caused by the
impairment of AVP production (33). This phenotype suggests an
antidiuretic action of LXRβ, which is the opposite of the diuretic
action of TO901317. Therefore we speculate that diuretic action
of TO901317 may be conferred mainly by LXRα. This possibility
needs to be validated in future studies using LXRα-null mice.
It has been shown that acute LXR activation induces a tran-
sient increase in renin transcription in the juxtaglomerular cells
(32), but its chronic activation remarkably suppresses the ex-
pression of renin, AT1R, and angiotensin 1-converting enzyme 1
(ACE) in the heart and kidney following isoproterenol treatment
(52). In agreement with the inhibitory effect of LXRs on local
RAS, we found that TO901317 treatment remarkably suppressed
urinary renin, an index of the intrarenal RAS (35, 53). Although
LXRs may function as a negative regulator of renin expression at
the juxtaglomerular apparatus in the acute setting, they appear
primarily to suppress PRR and the local RAS to elicit a diuretic
response.
In summary, the present study reports, for the first time to our
knowledge, a biological function of sPRR in regulating fluid
homeostasis. sPRR is associated with FZD8 to activate β-catenin,
which interacts with the cAMP–PKA pathway to induce AQP2
expression and enhance the urine-concentrating capability. Be-
cause intercalated cells are the potential source of sPRR, and
principal cells are the site of its action, it seems reasonable to
speculate that sPRR mediates the communication between the
two cell types in the CD. Last, we discovered the diuretic action
of the LXR agonist TO901317, which is conferred by inhibition
of the renal PRR/sPRR system.
Methods
Animals. Male 10- to 12-wk-old SD rats and C57BL6 mice were purchased from
Charles River Laboratories and the Jackson Laboratory, respectively. All an-
imals were maintained in a temperature-controlled room with a 12:12-h light:
dark cycle, with free access to tap water and standar d rat chow. Animals were
randomized into different experimental groups. Animal protocols were
approved by the Animal Care and Use Committees at the University of Utah
and the Sun Yat-sen University.
Rat Experiments. Under isoflurane inhalation, rats were s.c. implanted with an
osmotic minipump delivering a tankyrase inhibitor, XAV939 (XAV; Med-
chemExpress) at 5 mg·kg
−1
·d
−1
or received a FZD8 inhibitor, OMP54F03
(OMP) (a gift from John Lewicki, OncoMed Pharmaceuticals, Redwood City,
CA), at 25 mg/kg via daily i.p. injection. All animals were acclimatized to
metabolic cages for 7 d. After collection of baseline data for 2 d, rats were
water deprived for 48 h but had free access to chow diets. At the end of the
experiment, under isoflurane anesthesia, blood was withdrawn from the
vena cava, one kidney was cut into cortex and inner medulla and snap-
frozen, and the other kidney was fixed and paraffin embedded.
Plasmid Construction and sPRR Protein Purification. The cDNA of PRR (Gen-
Bank accession no. NM_001007091.1; also known as “ATP6AP2”)was
subcloned into the pMD-18T vector (Takara). sPRR, a solubilized form of
sPRR (residues 17–274) lacking the transmembrane domain at the C termi-
nus, was combined with an eight-histidine tag in the C terminus (sPRR-His),
was generated by PCR from the PRR expression construct, and then was
cloned into pcDNA3.1. sPRR-His was generated by using a mammalian 293 cell
system and was purified by binding to IDA-Sephadex G-25 (GE Healthcare)
(XBIO, Shanghai, China).
Mouse Experiments. Male C57/BL6 mice received chronic i.v. infusion of vehicle
or sPRR-His at 30 μg·kg
−1
·d
−1
as described above. After the surgery, the mice
were acclimatized to metabolic cages for 7 d and then were randomly di-
vided to receive vehicle or a V2R antagonist, OPC-31260 (Sigma), at
30 mg·kg
−1
·d
−1
via gavage for 3 d. Water intake, urine volume, and urine
osmolality were recorded daily during the entire experimental period. In
another experiment to test the effects of sPRR-His on TO901317-induced DI,
male C57/BL6 mice were treated for 7 d with vehicle or with TO901317 alone
or in combination with sPRR-His. TO901317 was added to the chow diet at
Fig. 10. Effect of sPRR-His on TO901317-induced DI in mice. Male C57/BL6
mice were treated with vehicle or with TO901317 alone or in combination
with sPRR-His for 7 d. (A) Urine output (n=8 mice per group). (B) Water
intake (n=8 mice per group). (C) Water balance (n=8 mice per group).
(D) Urinary osmolality (n=8 mice per group). (E) Hct (n=8 mice per group).
AQP2
β-Actin
29 kDa
43 kDa
35~45 kDa
Urinary renin activity (ng/24h)
0
0.05
0.10
0.15
0.20
CTR TO901317 TO901317 + sPRR-His
p<0.05 p<0.05
0.25
AQP2 densitometry
0
0.2
0.4
0.6
0.8
1.0
1.2
CTR TO901317 TO901317 + sPRR-His
p<0.05 p<0.05
Urinary total prorenin/ renin
excretion (ng/24h)
p<0.05
0
0.3
0.6
0.9
CTR TO901317 TO901317 + sPRR-His
A
C
E
B
D
CTR TO901317 TO901317 + sPRR-His
Repressor
PRR
Inactive transcription
LXR
TO
PRR/sPRR
AQP2
DI
Fig. 11. Effect of sPRR-His on renal AQP2 expression and urinary renin in
TO901317-treated mice. Male C57/BL6 mice were treated with vehicle or
with TO901317 alone or in combination with sPRR-His for 7 d. AQP2 ex-
pression was analyzed by immunoblotting and immunostaining. Urinary
renin activity was determined by measuring AngI generation, and urinary
prorenin/renin concentration was determined by ELISA. (A) Immunoblotting
analysis of AQP2 expression (n=15 mice per group). (B) Densitometric
analysis of the immunoblots in A.(C) Urinary renin activity (n=8 mice per
group). (D) Urinary prorenin/renin excretion (n=8 mice per group).
(E) Schematic illustration of the mechanism by which the LXR agonist
TO091317 (TO) suppresses PRR transcription and induces DI. LXRs bound to
TO091317 function as a transitional repressor for the PRR gene, leading to a
reduced PRR/sPRR level that decreases AQP2 expression, ultimately causing DI.
Lu et al. PNAS Early Edition
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MEDICAL SCIENCES PNAS PLUS
an estimated level of 50 mg·kg body weight
−1
·d
−1
. All mice were placed in
metabolic cages, and 24-h water intake and urine output were recorded and
collected at the end of the experiments.
Cell-Culture Experiments. For sPRR signaling studies, primary IMCD cells were
prepared from 4-wk-old SD rats as previously described (35). The cells were
grown in Transwell plates (catalog no. 29442-074; VWR International) with
DMEM/F-12 medium containing 10% (vol/vol) FBS, 0.5 μM 8-Br-cAMP,
130 mM NaCl, and 80 mM urea. Upon confluence, the cells were serum deprived
for 12 h and pretreated with an inhibitor (10 μMXAVor10μMOMP)fol-
lowed by 24-h treatment with AVP (10 nM) or sPRR-His (10 nM). At the end
of the experiments, the medium was collected for biochemical assays.
The effect of TO901337 on PRR expression was tested in mpkCCD cells.
These cells were grown to confluence in six-well plates. After 12-h serum
deprivation, the cells were treated with vehicle or 10 μM TO901317 for 24 h
and then were harvested for analysis of PRR expression and sPRR release. In
a separate experiment, mpkCCD cells at ∼50% density were transiently
transfected with a construct containing the luciferase gene under the con-
trol of the 2,016-bp 5′flanking region of the PRR gene. Upon confluence,
the transfected cells were treated for 24 h with vehicle or 10 μM TO901317
and then were harvested for analysis of luciferase activity.
Enzyme Immunoassay. Urinary or medium sPRR was determined using a com-
mercially sPRR enzyme immunoassay (EIA) kit (catalog no. JP27782; Immuno-
Biological Laboratories) according to the manufacturer’s instructions.
Immunofluorescence Staining. The tissues were fixed in 10% neutral buffered
formalin for 24 h and then were embedded in paraffin. After deparaffini-
zation, thin sections (4 μm) were processed for double-labeling with im-
munofluorescence. For antigen recovery, the slides were immersed in
Tris·HCl EDTA buffer (pH 9.0) at a high temperature (98 °C) for 12 min. The
slides were blocked in 1% (wt/vol) BSA for 1 h and then were incubated
overnight with primary antibody at 4 °C. After the primary antibody was
washed off, sections were incubated for 1 h at room temperature with
donkey anti–goat-IgG-FITC (Santa Cruz) or donkey anti-rabbit IgG-TRITC
(Life Technologies). Rabbit anti-PRR antibody from Abcam was raised
against residues 335–350 in the C terminus (termed “anti–PRR-C antibody”).
A second anti-PRR antibody used in the present study raised against residues
218–235 in the N terminus of PRR (termed “anti–PRR-N antibody”) was
generated in Y.F.’s laboratory (24). Goat anti-AQP2 antibody was purchased
from Santa Cruz. Rabbit anti-NKCC2 antibody was purchased from Stress-
Marq Biosciences Inc.
Immunoblotting. Renal tissues were lysed and subsequently sonicated. Protein
concentrations were determined using Coomassie reagent. Forty micrograms
of protein from each sample were denatured in boiling water, separated by
SDS/PAGE, and transferred onto nitrocellulose membranes. Blots were blocked
1 h with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by overnight
incubation with primary antibody. After washing with TBS, blots were incubated
with goat anti-rabbit/mouse HRP-conjugated secondary antibody and visualized
using ECL. The blots were quantitated by using Image-Pro Plus (Media Cyber-
netics). The primary antibodies were goat anti-AQP2 antibody, rabbit anti–PRR-N
antibody, and rabbit anti-FZD8 antibody (all from Santa Cruz), rabbit anti-V2R
antibody (Abcam), and goat anti–β-catenin antibody (Novus).
Quantitative RT-PCR. Total RNA was isolated from renal tissues and reverse
transcribed to cDNA. Oligonucleotides were designed using Primer3 software
(bioinfo.ut.ee/primer3-0.4.0/). Primers of AQP2 were 5′-gctgtcaatgctctccacaa-3′
(sense) and 5′-ggagcaaccggtgaaataga-3′(antisense); primers for GAPDH were
5′-gtcttcactaccatggagaagg-3′(sense) and 5′-tcatggatgaccttggccag-3′(antisense).
Cell Membrane and Cytoplasmic Protein Fraction Isolation. The membrane and
cytosolic fractions of proteins were extracted using a kit according to the
manufacturer’s instructions (catalog no. BSP002; Bio Basic Inc.).
Coimmunoprecipitation. For coimmunoprecipitation with PRR and FZD8, the
membrane fraction of rat renal inner medullary was performed using a kit
(catalog no. BSP002; Bio Basic Inc.). Anti–PRR-N (from Y.F.) or anti-FZD8 (Santa
Cruz) antibody was cross-linked with the magnetic beads (catalog no. 88805;
Pierce) and then incubated for 30 min with the renal medullary membrane
proteins. The beads were collected, washed, and eluted. The immunoprecipi-
tated samples were analyzed for the binding partner by immunoblotting.
siRNA or Plasmid Transfection in Primary Cultured IMCD Cells. IMCD cells were
transfected with FZD8 siRNA oligonucleotides (Invitrogen) or the luciferase
reporter plasmid (catalog no. CCS-018L; Qiagen) at a final concentration of
5 nM using HiPerFect transfection reagent (catalog no. 301702; Qiagen) for
72 h. The efficiency of FZD8 knockdown was validated by immunoblotting of
FZD8. For the luciferase assay, each sample consisted of a positive control, a
negative control, and a target luciferase construct, and the reporter activity
was calculated according to the manufacturer’s instruction.
Preparation of Luciferase Constructs. Genomic DNA was extracted from rat tail
using a Tissue DNA kit (D3396-01; Promega). A 2,016-bp fragment of the 5′
flanking region of the PRR gene (GenBank accession no. NM_001007091;
1,941 ±75 bp) was amplified from the rat genomic DNA by PCR and
subcloned to the pGL3-Basic reporter vector (Promega) using NheI and BgIII
restriction sites; this construct was termed “pGL3-PRR.”A 2,084-bp frag-
ment of the 5′flanking region of the AQP2 gene (GenBank accession no.
NM_012909; 2,000 ±84 bp) was cloned to the pGL3-Basic reporter vector by
a similar strategy; this construct was termed “pGL3-AQP2.”The identity of
these constructs was validated by sequencing.
Luciferase Assay. The mpkCCD cells were transfected with pGL3-AQP2 plasmid
or empty vector by using HiPerFect Transfection Reagent (catalog no. 301702;
Qiagen). Upon confluence, all cells were starved for 12 h; then pGL3-PRR- and
pGL3AQP2-transfected cells were treated for 24 h with TO901317 (10 μM) or
sPRR-His (10 nM), respectively. The vehicle-treated group served as a control.
The luciferase activities were measured using a luciferase assay system
(Promega), and luminescence was detected by using an illuminometer (BMG
FLUOstar OPTIMA).
Statistical Analysis. Data are summarized as means ±SE. Statistical analysis
was performed using ANOVA with the Bonferroni test for multiple com-
parisons or paired or unpaired Student’sttest for two comparisons. P<0.05
was considered statistically significant.
ACKNOWLEDGMENTS. We thank Dr. John Lewicki (OncoMed Pharmaceuti-
cals) for providing the FZD8 inhibitor OMP-54F03. This work was supported
by National Natural Science Foundation of China Grants 91439205 and
31330037; NIH Grants DK094956 and DK104072; National Basic Research
Program of China 973 Program 2012CB517600 Grant 2012CB517602; and
by the Veterans Administration Merit Review. T.Y. is a Research Career Sci-
entist in the Department of Veterans Affairs. J.-Å.G. was supported by
the Swedish Science Council and by Grant E-0004 from the Robert A.
Welch Foundation. Y.F. was supported by Grant NHLBI/R01HL122770
from the NIH.
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