BASIC RESEARCH www.jasn.

org

Epithelial Vasopressin Type-2 Receptors Regulate

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Myofibroblasts by a YAP-CCN2–Dependent

BASIC RESEARCH
Mechanism in Polycystic Kidney Disease
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Nidhi Dwivedi,1,2 Shixin Tao ,1,2 Abeda Jamadar,1,2 Sonali Sinha,1,2 Christianna Howard,1,2
Darren P. Wallace ,1,2 Timothy A. Fields,1,3 Andrew Leask,4 James P. Calvet,1,5 and
Reena Rao1,2
1
The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas
2
Department of Medicine, University of Kansas Medical Center, Kansas City, Kansas
3
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas
4
School of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada
5
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas

ABSTRACT
Background Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney
disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic
epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys.
Methods We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and
evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the
effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibro-
blast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased
significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in
ADPKD as well as that of yes-associated protein (YAP).
Results V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM
deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibro-
blast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct–specific gene deletion of
CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP
regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys.
Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys.
Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human
ADPKD cystic epithelial cells.
Conclusions Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofi-
broblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell sig-
naling pathway may present a potential therapeutic target for fibrosis in ADPKD.

JASN 31: 1697–1710, 2020. doi: https://doi.org/10.1681/ASN.2020020190

Autosomal dominant polycystic kidney disease
(ADPKD) is characterized by the growth of fluid-
filled cysts and is the most common inherited kid-
ney disorder, affecting over 12.5 million people
worldwide.1 Cysts in ADPKD commonly originate
from renal collecting ducts and distal tubular seg-
ments.2,3 Progressive tubulointerstitial fibrosis ac-
Received February 18, 2020. Accepted April 13, 2020.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Reena Rao, 5040 WHE, The Jared Gran-
tham Kidney Institute, University of Kansas Medical Center, 3901
Rainbow Boulevard, Kansas City, KS 66160-3018. Email: rrao@
kumc.edu

companies cyst expansion in ADPKD and is Copyright © 2020 by the American Society of Nephrology

JASN 31: 1697–1710, 2020 ISSN : 1046-6673/3108-1697 1697

 BASIC RESEARCH www.jasn.org
thought to be a major contributor to the decline in renal function
and the final path to ESKD.4,5 The minimal interstitial space
around renal tubules is normally occupied by blood capillaries,
small vessels, and interstitial cells and is important for proper
kidney function. Interstitial expansion due to excessive extracel-
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lular matrix (ECM) production, reduced degradation, and
changes in the ECM composition, together with increased in-
flammatory infiltrates, disrupts this delicate architecture, leading
to loss of renal function in ADPKD.4–6 Myofibroblasts are highly
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contractile cells that express a-smooth muscle actin (a-SMA),
migrate, proliferate, and persistently secrete large amounts of
ECM.7 Renal myofibroblasts are known to originate by activation
and differentiation of resident fibroblasts and pericytes.8 As in
other CKDs, increased myofibroblast numbers, indicated by high
a-SMA expression, are known to occur in human and mouse
polycystic kidney disease (PKD) kidneys.4 However, it is currently
unknown whether the cystic epithelium has a role in myofibro-
blast activation and in modifying the pericystic microenviron-
ment to promote fibrosis.
In this study, we tested whether an epithelial-specific stimulus
from cystic epithelial cells can regulate interstitial myofibroblast
activation in ADPKD kidney. This hypothesis was on the basis of
our serendipitous finding that short-term stimulation of the va-
sopressin type-2 receptor (V2R) significantly increased renal fi-
brosis in ADPKD kidneys. V2R is normally expressed on renal
tubular epithelial cells of the collecting ducts, connecting tubules
and thick ascending limbs,2 and V2R-mediated cell signaling
promotes cystic epithelial cell proliferation in PKD.9,10 It is
unknown if V2R plays any role in renal fibrosis in PKD. Yes-
associated protein (YAP) of the Hippo signaling pathway regu-
lates the proliferation of cystic epithelial cells in ADPKD mouse
kidneys11 and autosomal recessive PKD cystic liver cells in vitro.12
YAP can be activated by ECM stiffness, which can stimulate pro-
duction of profibrotic factors and ECM proteins by fibroblasts
and increase proliferation of epithelial cells.13 However, the role
of the Hippo pathway in either V2R-mediated cell signaling or
fibrosis in PKD has not been reported.
METHODS
ADPKD Mouse Studies
Mouse Models
(1) One model was Pkd1f/fPkhd1cre (Pkd1 gene knockout
[Pkd1KO]) orthologous ADPKD mouse model with collecting
duct–specific - Pkd1 gene deletion.14 Wild-type (WT) mice were
Pkd1f/f mice without Cre. (2) Another model is collecting duct–
specific YAP knockout Pkd1f/fYapf/fPkhd1cre mice (Yapf/f mouse is
Yap1tm1.1Dupa/J; stock no. 027929; The Jackson Laboratory, Bar
Harbor, ME). (3) The third model is collecting duct–specific
CCN2 knockout Pkd1f/fCCN2f/fPkhd1cre (Pkd1-CCN2KO).
CCN2 exon 2 floxed mice15 were received from A.L.
Desmopressin Study
Pkd1KO mice were administered desmopressin (dDAVP;
Sigma-Aldrich, St. Louis, MO; 1 mg/kg body wt daily
1698 JASN
Significance Statement
In autosomal dominant polycystic kidney disease (ADPKD), pro-
gressive fibrosis contributes to renal failure, leading to ESKD. The
vasopressin type-2 receptor (V2R) helps to regulate renal water
homeostasis and stimulates cyst expansion in ADPKD. We discov-
ered a novel pathogenic pathway behind V2R regulation of fibrosis
in ADPKD kidneys. Epithelial V2R stimulation activates interstitial
myofibroblasts, in a paracrine manner, in Pkd1 gene knockout (KO)
mice. Pharmacologic inhibition and gene knockout studies in-
dicated that V2R regulates myofibroblast activation by a yes-
associated protein (YAP)– and connective tissue growth factor
(CCN2)–dependent mechanism. The V2R-YAP-CCN2 molecular
axis may present novel pharmacologic targets for control of fibrosis
in ADPKD.
intraperitoneally) on postnatal days P18–P20. The treatment
time was selected on the basis of the findings that by P18,
Pkd1KO mouse kidneys are highly cystic and that cAMP levels
are already high.16 In a separate study, Pkd1KO mice and YAP
gene knockout Pkd1f/fYapf/fPkhd1cre (Pkd1-YapKO) mice were
administered dDAVP (1 mg/kg body wt intraperitoneally daily
from P18 to P21).
Verteporfin Study
Pkd1KO mice were administered vehicle or verteporfin
(75 mg/kg body wt intraperitoneally) on P10, P12, P14, and
P16 and euthanized on P18. Verteporfin was dissolved in ve-
hicle (50% corn oil 125% methanol 125% DMSO), vortexed
vigorously, and sonicated before injection.
Short-Term dDAVP Treatment
On P18, WT and Pkd1KO mice were given two dDAVP injec-
tions (1 mg/kg body wt intraperitoneally) at 8 AM and 12 PM
and euthanized at 4 PM of the same day.
All animal studies were carried out according to the pro-
tocols approved by the University of Kansas Medical Center
Institutional Animal Care and Use Committee.
Human Tissues and Cells
Primary culture ADPKD and normal human kidney (NHK)
cells and kidney tissue from deidentified patients were from
the PKD Biomaterials Core at the University of Kansas. NRK-
49F (ATCC CRL-1570) cell lines were also used.
Quantification of Cysts and Tissue Fibrosis
Kidney tissue sections (5 mm) were stained with hematoxylin
and eosin, and images were captured using a microscope con-
nected to a digital camera (Leica Microsystems, Buffalo Grove,
IL). An observer blinded to the sample identity recorded and
quantified the number of cysts, cystic area, and total kidney
area using ImageJ (Fiji, Madison, WI).
BUN Levels
BUN levels were measured in serum as described previously14
using the QuantiChrom Urea Assay Kit from BioAssay Sys-
tems (Hayward, CA).
JASN 31: 1697–1710, 2020

Urinary Osmolality
Spot urine samples were collected before euthanizing the
mouse, and urinary osmolality was measured using the VAPRO
vapor pressure osmometer (Model 5600; ELITech Benelux).
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Western Blot
Mouse kidneys were homogenized in SDS Laemmli buffer
and loaded onto 10% or 4%–20% gradient SDS-PAGE gels
essentially as described before.14,17 Primary antibodies for YAP
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(sc-101199; Santa Cruz Biotechnology, Inc.), CCN2 (sc-101586;
Santa Cruz Biotechnology, Inc.), Lamin B (sc-365214; Santa
Cruz Biotechnology, Inc.), glyceraldehyde-3-phosphate dehy-
drogenase (sc-32233; Santa Cruz Biotechnology, Inc.), pERK
(sc-7383; Santa Cruz Biotechnology, Inc.), extracellular signal–
regulated kinase (ERK; sc-514302; Santa Cruz Biotechnology,
Inc.), SMAD (9513S; Cell Signaling Technology, Inc.), pSMAD
(9520S; Cell Signaling Technology, Inc.), a-SMA (ab5694; Ab-
cam, Cambridge, MA), V2R (V5514; Millipore Sigma, St. Louis,
MO), and type 1 collagen (203002; MD Bioproduct, Oakdale,
MN) were used. Secondary antibodies, both anti-mouse
(P0447) and anti-rabbit (P0448), were purchased from Dako
and ECL reagent (PerkinElmer).
Immunohistochemistry/Immunofluorescence
Fixed and paraffin tissue sections were processed as described
before.14,17 The following primary antibodies were used:
a-SMA, Type 1 collagen, CCN2, YAP, V2R, E-Cadherin (sc-
7870; Santa Cruz Biotechnology, Inc.), and DBA (RL-1032;
Vector Laboratories). For immunohistochemistry, secondary
antibodies were applied followed by incubation with Strepta-
vidin HRP conjugate (Invitrogen), and slides were developed
with DAB (Vector Laboratories), counterstained with Harris
Haematoxylin, dehydrated, and mounted with Permount
(Fisher Scientific, Fail Lawn, NJ). For immunofluorescence,
goat anti-rabbit IgG fluor and goat anti-mouse IgG Texas
red (Invitrogen) secondary antibodies were applied, incu-
bated, washed with PBST, and stained with 49,6-diamidino-
2-phenylindole. Slides were mounted with Flour-G (Invitrogen)
and sealed with nail polish. All images were captured using a
Nikon 80i upright microscope (Tokyo, Japan) in the KUMC
imaging center.
Quantitative Real-Time PCR
RT-PCR using cDNA prepared from RNA isolated from
whole-kidney and cultured cells was carried out as described
previously.14 Primer sequences are provided in Supplemental
Table 1.
Conditioned Media Collection
Primary culture human ADPKD cystic epithelial cells were
grown in 100-mm plates in DME/F-12 (13) media (catalog
no. SH30023.01; HyClone). When confluent, the media were
replaced with serum-free media. Conditioned media (CM)
were collected after 48 hours, centrifuged to remove debris,
and used directly for studies.
JASN 31: 1697–1710, 2020
www.jasn.org BASIC RESEARCH
Fibroblast to Myofibroblast Differentiation
NRK-49F cells were grown in 100-mm plates in DMEM media
(ATCC 30–2002). When 50% confluent, NRK-49F cells were
exposed to serum-free CM from ADPKD cells for 48 hours.
CM were changed every 24 hours. Cells were lysed, and
a-SMA levels were measured by immunoblotting.
Wound Closure Assay
NRK-49F or human ADPKD renal myofibroblasts were
seeded in six-well plates and grown until confluent. A sterile
pipette tip was used to place a scratch (wound) in the cell
monolayer followed by washing with PBS to remove dislodged
cells. The wounds were then photographed at the same posi-
tion at 0 hours and at different time intervals to measure
wound closure. A separate set of plates with similar treatment
was used to assess cell viability to adjust percentage wound
closure to cell viability. Cells were grown in 10% FBS contain-
ing medium.
To determine the effect of CCN2 on NRK-49F cell differ-
entiation and wound closure, recombinant CCN2 (connective
tissue growth factor, full-length peptide, catalog no.
SRP4702–20UG; Sigma Aldrich) was used.
Cell Viability Analyses
Briefly, exponentially growing cells were seeded in 24-well
plates. When cells were 40%–50% confluent, they were
washed with PBS, and media were replaced with serum-free
CM for 48 hours. Following this, cells were incubated in
5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium solution for 2 hours, and intracellular purple formazan
was solubilized in DMSO and quantified by spectrophotom-
etry at 540 nM.
Nuclear-Cytoplasmic Extraction
The cytoplasmic and nuclear fractions were prepared using
the NE-PER Nuclear Cytoplasmic Extraction Reagent kit
(Thermo Fischer Scientific) following the manufacturer’s
protocol.
Statistical Analyses
Values were expressed as mean 6 SEM and 6 SD for in vivo
and in vitro studies, respectively. Data were analyzed by two-
tailed unpaired t test with Welch correction or one-way
ANOVA followed by the Dunnett multiple comparison test
using GraphPad Prism software (Version 5.0d). Probability
value of P#0.05 was considered significant.
RESULTS
Epithelial-Specific V2R Stimulation Increases the
Interstitial Myofibroblast Population, ECM Deposition,
and Renal Fibrosis in ADPKD Kidneys
In human ADPKD kidneys, immunostaining for a-SMA re-
vealed a dense myofibroblast population in the pericystic area,
Epithelial V2R Regulates Fibrosis 1699
 BASIC RESEARCH www.jasn.org
in close proximity to cystic epithelial cells (Figure 1A). V2R
expression was localized to tubular epithelial cells, including
cyst-lining cells, but not in the a-SMA–expressing cells
(Figure 1A). Human ADPKD kidneys also showed signifi-
cantly higher a-SMA mRNA levels compared with normal
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control kidneys (Figure 1B). To determine the effect of a tu-
bular epithelial–specific stimulus on interstitial myofibroblast
activation and fibrosis, Pkd1f/fPkhd1cre (Pkd1KO) mice14,16
were treated with dDAVP, a V2R-selective agonist,18 from
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postnatal day P18 to P20 and euthanized on P21
(Figure 1C). In Pkd1KO mice, dDAVP treatment significantly
upregulated the myofibroblast population and expression of
ECM proteins, compared with vehicle treatment, as indicated
by higher a-SMA, collagen-1a, collagen-IIIa, and fibronectin
mRNA levels (Figure 1D). Collagen-1a and a-SMA protein
levels were also higher in dDAVP-treated Pkd1KO mice
(Figure 1E, Supplemental Figure 1). Immunostaining for
collagen-1a and a-SMA was also increased in dDAVP-treated
Pkd1KO kidneys (Figure 1F). Compared with vehicle treat-
ment, a 17% increase in kidney-body weight ratio and 40%
increase in BUN levels were observed in dDAVP-treated
Pkd1KO mice (Figure 1, G and H). As expected, dDAVP treat-
ment significantly reduced urine osmolality in both WT and
Pkd1KO mice, supporting its V2R agonistic activity
(Supplemental Figure 2). Notably, in WT mice, dDAVP treat-
ment did not affect a-SMA and collagen-1a expression,
kidney-body weight ratio, or BUN (Figure 1, D, E, G, and
H). The above results demonstrate that epithelial V2R activa-
tion can stimulate interstitial myofibroblast activation in PKD
kidneys. The results also suggest that renal cystic epithelial
cells could regulate a profibrotic response.
Secreted Factors from ADPKD Renal Tubular Epithelial
Cells Can Activate Myofibroblasts
To determine whether ADPKD cystic epithelial cells regulate
myofibroblasts, we tested the effect of their CM on myofibro-
blast activation (fibroblast to myofibroblast differentiation),
viability, and migration. CM were collected from primary cul-
ture human ADPKD and NHK epithelial cells and tested on
primary culture myofibroblasts from human ADPKD kidneys
and undifferentiated NRK-49F rat renal fibroblasts. Exposure
to ADPKD-CM significantly increased cell viability
(Figure 2A) and faster wound closure (Figure 2B) of both
human ADPKD myofibroblasts and NRK-49F cells compared
with NHK-CM. The human myofibroblasts expressed high
levels of a-SMA at baseline, which did not further change
upon exposure to ADPKD-CM (Supplemental Figure 3A).
However, when NRK-49F fibroblasts were exposed to
ADPKD-CM, a-SMA expression increased significantly com-
pared with NHK-CM (Figure 2, C and D, Supplemental
Figure 3B). These results suggest that secreted factors from
ADPKD epithelial cells can stimulate myofibroblast activa-
tion, migration, and viability.
To determine how V2R stimulation activates myofibro-
blasts, we examined the renal mRNA expression of known
1700 JASN
secreted and myofibroblast-activating factors in Pkd1KO
mice treated with dDAVP for 3 days as in Figure 1. Of the 25
secreted profibrotic factors examined (Supplemental Table 1),
renal mRNA levels of CCN2, TGFb, amphiregulin (AREG),
PAI-1, TNFa, IL1b, IL6, IL10, CCL2, and CCL3 were signif-
icantly higher in the dDAVP treatment group when compared
with the vehicle-treated group (Figure 2E). To further test if
V2R signaling can directly stimulate expression of these se-
creted factors, we performed a short-term study in which
dDAVP was administered to WT and Pkd1KO mice, and they
were euthanized after only 8 hours. Of the above-mentioned
ten secreted factors, only CCN2 mRNA levels increased sig-
nificantly by twofold in Pkd1KO mice (Figure 2F,
Supplemental Figure 3, C and D). The CCN2 mRNA levels
remained unaffected by dDAVP treatment in WT mice
(Figure 2F). Furthermore, recombinant CCN2 protein in-
duced a-SMA expression and cell migration in a dose-
dependent fashion in NRK-49F cells (Supplemental
Figure 4, A and B), suggesting that CCN2 could be an impor-
tant V2R-stimulated secreted profibrotic factor in ADPKD
kidneys.
Renal Tubular CCN2 Gene Deletion Reduced
Interstitial Myofibroblast Activation, ECM, and Cyst
Growth in ADPKD Mouse Kidneys
CCN2 is a matricellular protein that is known to be profibrotic
in the kidney,19,20 secreted by myofibroblasts in mouse CKD
models,21–26 and immunolocalize to human cystic tubular
epithelial cells in ADPKD.27 It is unknown if CCN2 plays a
pathogenic role in cyst growth or fibrosis in PKD. We found
eight- and threefold higher CCN2 mRNA levels, respectively,
in human ADPKD kidneys and primary culture ADPKD ep-
ithelial cells compared with NHK controls (Figure 2G).
Importantly, CCN2 was localized to the cyst-lining epithe-
lial cells in human ADPKD kidneys (Figure 2H), especially in
cysts of collecting duct origin, a tubule segment that expresses
V2R (Figure 2H). CCN2 was also detected in cyst-lining epi-
thelium in Pkd1KO mice (Supplemental Figure 4C).
To determine the role of cystic epithelial CCN2 in interstitial
myofibroblast activation and fibrosis, we generated a collecting
duct–specific CCN2 gene knockout Pkd1f/fCCN2f/fPkhd1cre
mouse (Pkd1-CCN2KO). CCN2 expression in collecting duct
cystic epithelial cells was reduced in Pkd1-CCN2KO kidneys,
whereas other pericystic cells still expressed CCN2
(Supplemental Figure 5A). The Pkd1-CCN2KO kidneys were
smaller than Pkd1KO kidneys (Figure 3A) and showed reduced
kidney-body weight ratio (32% reduction), BUN (22% reduc-
tion), and cystic index (23% reduction) (Figure 3, B–D). Cyst
numbers were not significantly different between Pkd1-
CCN2KO and Pkd1KO kidneys (Supplemental Figure 5B).
Compared with Pkd1KO kidneys, the mRNA levels of
a-SMA, collagen-1a, and collagen-IIIa were significantly re-
duced in the Pkd1-CCN2KO kidneys (Figure 3E). The Pkd1-
CCN2KO kidneys also showed significant reduction in a-SMA
protein levels (Figure 3, F and G) and reduced immunostaining
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A
* *

* *
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* *
*

α-SMA,DAPI V2R,E-cadherin,DAPI V2R, α-SMA, DAPI

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B 40 **
C
relative to GAPDH

30
α-SMA mRNA

WT/
Pkd1KO dDAVP
20
P0 P18,P19,P20,P21
10

(Sacrifice)
0
Control ADPKD

Human kidney tissue

D
14
** 14 * 8 * 14 *
Collagen-IIIa mRNA
Collagen-1a mRNA

12

relative to GAPDH
12
relative to GAPDH

relative to GAPDH
relative to GAPDH

12
α-SMA-mRNA

10 6 10

FN1 mRNA
10
8 8 8
4
6 6 6
4 4 2 4
2 2 2
0 0 0 0
Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP

WT Pkd1KO WT Pkd1KO WT Pkd1KO WT Pkd1KO

E Pkd1KO
WT Pkd1KO F WT Vehicle dDAVP
Vehicle dDAVP Vehicle dDAVP

α-SMA
Collagen-1a, DAPI

Collagen-1a

GAPDH

G H 140 **
**
Kidney/ BWt. ratio (%)

14
120
12
BUN (mg/dl)

100
10
80
α-SMA, DAPI

8
6 60
4 40
2 20
0 0
Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP

WT Pkd1KO WT Pkd1KO

Figure 1. Activation of V2R increases fibrosis in ADPKD kidneys. (A) In human ADPKD kidney tissue sections, immunostaining for
a-SMA (green) and 49,6-diamidino-2-phenylindole (DAPI; blue), V2R (red) and E-cadherin (green), or V2R (red), and a-SMA (green).
Scale bar 5 50 mm. *Cysts. (B) a-SMA mRNA in human normal control (n58) and human ADPKD kidneys (n59) relative to glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. (C) Scheme of vehicle or dDAVP (1 mg/kg body wt per day intraperitoneally
on P18–P20) treatment in WT and Pkd1KO mice. (D) a-SMA, collagen-1a, collagen-IIIa, and fibronectin (FN1) mRNA relative to
GAPDH for WT (n56) and Pkd1KO (n58) mice. (E) Immunoblot for a-SMA and collagen-1a. (F) Immunostaining. Scale bar 5 50 mm.
(G) Two kidney-body weight ratio (percentage; n58). (H) Plasma BUN levels (n58). *P,0.05 versus control or vehicle by t test;
**P,0.01 versus control or vehicle by t test.

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 BASIC RESEARCH www.jasn.org

A ***
B 120 NHK-CM NHK-CM
***
*** 100

Migration (% Closure)
160 ADPKD-CM ADPKD-CM

Migration (% Closure)
100 ***
80 ***
Cell Viability (%)
140
80
120 ** 60 **
60
100 40
40
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80
20 20
60
0 0
40 0h 12h 24h 36h
0h 16h 20h
NHK-CM ADPKD-CM NHK-CM ADPKD-CM
Human myofibroblasts NRK-49F cells
Human myofibroblasts NRK-49F cells (Time exposed to CM) (Time exposed to CM)
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D ** E **
C 3 ** PKd1KO-Vehicle
*
Relative band density

mRNA relative to GAPDH

PKd1KO-dDAVP
M

*
-C

αSMA/GAPDH
M
D
l

*
tro

PK

2
K-

*
on

3
H
AD

**
C

** *
1 2 *
α-SMA

0 1
β-actin
l

M
tro

-C

0
on

K-
D
C

H
PK

CCN2 TGFβ AREG PAI-1 TNFα IL-1β IL-6 IL-10 CCL2 CCL3
AD

F G
15
**
18 ** 6 **
Relative to GAPDH

16
Relative to GAPDH

Relative to GAPDH
14
CCN2 mRNA

CCN2 mRNA

10

CCN2 mRNA
12 4
10
8
5 6 2
4
2
0 0
0 NHK ADPKD NHK ADPKD
Vehicle dDAVP Vehicle dDAVP
Human Kidney Tissue Human Cells
WT Pkd1KO

H
Human ADPKD Kidney

DBA CCN2

Figure 2. Secreted factors from ADPKD cystic epithelial cells can induce myofibroblast activation. (A) The 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium assay in human primary culture ADPKD fibroblasts and NRK-49F rat fibroblasts incubated in NHK-CM or
ADPKD-CM (72 hours exposure to CM): n53 biologic replicates and n53–4 technical replicates. (B) Wound healing (percentage wound
closure in a scratch assay): n52 biologic replicates and n54 technical replicates each. (C) Immunoblot for a-SMA in NRK-49F rat renal
fibroblasts incubated for 48 hours with CM from primary culture NHK or ADPKD cells. (D) Densitometry of a-SMA relative to glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH): n53 biologic replicates and n51–3 technical replicates. (E) mRNA levels of secreted
factors relative to GAPDH mRNA in vehicle or dDAVP-treated Pkd1KO mouse kidneys: n56. (F) CCN2 mRNA in WT and Pkd1KO mice
after 8 hours of dDAVP treatment (1 mg/kg body wt intraperitoneally): n56. (G) CCN2 mRNA in NHK (n57) and ADPKD (n510) kidney
tissue and in primary culture human NHK (n58) and ADPKD (n57) renal epithelial cells. (H) Immunostaining in human ADPKD kidneys
for CCN2 (green) and DBA (red; collecting duct). Each data point represents one individual human sample. *P,0.05 versus NHK by
one-way ANOVA followed by the Dunnett multiple comparison test for (B) and by t test for all others; **P,0.01 versus NHK by one-way
ANOVA followed by the Dunnett multiple comparison test for (B) and by t test for all others; ***P,0.001 versus NHK by one-way
ANOVA followed by the Dunnett multiple comparison test for (B) and by t test for all others. Scale bar 5 50 mm

for a-SMA and collagen-1a (Figure 3H, Supplemental Renal Tubular Epithelial YAP Regulates CCN2 and
Figure 5C) compared with Pkd1KO kidneys. These results sug- Fibrosis in ADPKD Kidneys
gest that renal tubular epithelial–specific CCN2 is important To determine how CCN2 is regulated in the cystic epithelium,
for the development of interstitial fibrosis in ADPKD. we examined the role of YAP, a transcriptional regulator of

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 www.jasn.org BASIC RESEARCH

A WT Pkd1 KO Pkd1-CCN2KO
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B C D
*** ** 80
**
Kidney/ BWt ratio (%)

15 80

Cystic index (%)

70

BUN (mg/dl)
60
10
60
40
5 50
20

0 0 40
WT CCN2 Pkd1 Pkd1- WT CCN Pkd1 Pkd1- Pkd1 Pkd1-
KO KO CCN2KO 2KO KO CCN2KO KO CCN2KO
E
10 ** *
10 6 *
relative to GAPDH

Collagen-1a mRNA

Collagen-IIIa mRNA
8
relative to GAPDH

relative to GAPDH
8
α-SMA mRNA

6 4
6
4 4
2
2 2
0 0 0
WT CCN2 Pkd1 Pkd1- WT CCN2 Pkd1 Pkd1- WT CCN2 Pkd1 Pkd1-
KO KO CCN2KO KO KO CCN2KO KO KO CCN2KO

F H Pkd1 KO Pkd1-CCN2KO
CCN2 Pkd1 Pkd1-
WT KO KO CCN2KO

α-SMA
α-SMA

GAPDH

G 5
**
relative band density

4
α-SMA/GAPDH

3
Collagen-1a

0
WT CCN2 Pkd1 Pkd1-
KO KO CCN2KO

Figure 3. Renal tubular epithelial–specific gene deletion of CCN2 reduced cyst growth and myofibroblast population in ADPKD
kidneys. (A) In WT, CCN2f/fPkhd1cre (CCN2KO), Pkd1KO, and CCN2f/fPkd1f/fPkhd1cre (Pkd1-CCN2KO) mice, hematoxylin and eosin
staining is shown. (B) Kidney-body weight (BWt) ratio. (C) BUN levels. (D) Cystic index. (E) mRNA levels of a-SMA and collagen-1a and

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A NHK-Vehicle NHK-Verteporfin ADPKD-Vehicle ADPKD-Verteporfin

@@@

@@@
3
@@

C D

@@@
mRNA relative to GAPDH

40
**

@@
***

relative to GAPDH
30

Collagen-1a mRNA
relative to GAPDH
2

a-SMA mRNA
30
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20
20

***

***
1 10
***

***

10

***

***
***

***
***
0
***

0
Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert
0
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CCN2 PAI-1 AREG CYR61 CCL2 IL8 IL-6 IL1β TSP1 TNFα TGFβ ICAM1 WT Pkd1KO WT Pkd1KO

B Pkd1KO Pkd1KO E F
Vehicle Vert Vehicle Vert 20 *** 25 ***

Collagen-IIIa mRNA

relative to GAPDH
relative to GAPDH
20

FN1 mRNA
15
15
10
Collagen-1a

10
α-SMA

5 5
0 0
Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert

WT Pkd1KO WT Pkd1KO

WT Pkd1KO
H
G Vehicle Vert Vehicle Vert

20 *** 10 *** * α-SMA

relative to GAPDH

20
relative to GAPDH

relative to GAPDH
CCN2 mRNA

8
AREG mRNA

15
PAI-1 mRNA

15
6 Collagen-1a
10 10
4
5 5 CCN2
2
0 0 0
Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert pSMAD-3
WT Pkd1KO WT Pkd1KO WT Pkd1KO SMAD-3

GAPDH

Figure 4. YAP regulates CCN2 in PKD cystic epithelium. (A) mRNA levels in human NHK and ADPKD cells treated with vehicle or
verteporfin (Vert; 2.5 mM for 16 hours): n53 biologic replicates and n52 technical replicates each. WT or Pkd1KO mice were treated
with vehicle or Vert (75 mg/kg body wt intraperitoneally on P10, P12, P14, and P16) and euthanized on P18. (B) Immunostaining for
a-SMA and collagen-1a. mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for (C) a-SMA, (D) collagen-1a,
(E) collagen-IIIa, and (F) fibronectin (FN1). (G) Quantitative PCR showing mRNA levels of CCN2, AREG, and PAI-1. (H) Immunoblot of
kidney tissue lysates. *P,0.05 versus NHK-vehicle by t test for (A) and versus vehicle by t test in all others; **P,0.01 versus NHK-
vehicle by t test for (A) and versus vehicle by t test in all others; ***P,0.001 versus NHK-vehicle by t test for (A) and versus vehicle by
t test in all others; @@P,0.01 versus ADPKD-vehicle; @@@P,0.001 versus ADPKD-vehicle. Scale bar 5 50 mm

CCN2.28 Although YAP is known to regulate cystic epithelial transcriptional targets of YAP as well as other profibrotic fac-
cell proliferation in ADPKD mouse models,11,12 its role in tors in ADPKD cells (Figure 4A). Although some of these
fibrosis in PKD is unknown. Nuclear YAP expression was de- factors were also reduced in verteporfin-treated NHK cells,
tected in the cyst-lining epithelium of human ADPKD and CCN2 mRNA levels were unaffected (Figure 4A).
Pkd1KO mouse kidneys (Supplemental Figure 6A). We first To examine the role of YAP in myofibroblast activation and
determined the role of YAP in CCN2 expression by treating renal fibrosis in ADPKD mouse kidneys, we tested the effect of
human ADPKD and NHK epithelial cells with verteporfin, an pharmacologic YAP inhibition or renal collecting duct–
inhibitor of YAP transcriptional enhanced associate domain specific YAP gene deletion. WTand Pkd1KO mice were treated
interaction that is shown to reduce cancer cell proliferation.29 with vehicle or verteporfin from P10 to P18 and euthanized.
Verteporfin treatment for 16 hours significantly reduced Verteporfin-treated Pkd1KO mouse kidneys showed reduced
mRNA levels of CCN2, PAI-1, AREG, and CCL2, known immunolabeling of a-SMA and collagen-1a (Figure 4B),

collagen-IIIa levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. (F) Immunoblot of kidney tissue lysate and
(G) quantitation of band density. (H) Immunostaining of kidney for a-SMA (brown) and collagen-1a (green). *P,0.05 versus Pkd1KO by
t test; **P,0.01 versus Pkd1KO by t test; ***P,0.001 versus Pkd1KO by t test. Scale bar 5 50 mm

1704 JASN JASN 31: 1697–1710, 2020

 www.jasn.org BASIC RESEARCH

B ***
15 * 12 8 **
A

Collagen-1a mRNA
relative to GAPDH

relative to GAPDH

relative to GAPDH
10

α-SMA mRNA
Pkd1 KO Pkd1-YapKO

CCN2 mRNA
10 6
8
6 4
5 4
Collagen-1a

2
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0 0 0
WT Yap Pkd1 Pkd1- WT Yap Pkd1 Pkd1- WT Yap Pkd1 Pkd1-
KO KO YapKO KO KO YapKO KO KO YapKO

C D E
***
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WT 20 120 **

Kidney/ BWt ratio (%)

Pkd1 KO Pkd1-YapKO
100
15

BUN (mg/dl)
PD
80
α-SMA

10 60

YAPKO 40
5
20
0 0
WT Yap Pkd1 Pkd1- WT Yap Pkd1 Pkd1-
KO KO YapKO KO KO YapKO

F WT Pkd1KO G 15 ***
H 80 **
Kidney/BWt ratio (%)

Vehicle Verteporfin Vehicle Verteporfin 70

60

BUN (mg/dl)
10
50
40
5 30
20
10
0 0
Vehicle Vert Vehicle Vert Vehicle Vert Vehicle Vert

WT Pkd1KO WT Pkd1KO

Figure 5. Effect of tubular epithelium–specific YAP gene knockout on renal fibrosis in PKD mice. WT, Yapf/fPkhd1cre (Yap KO),
Pkd1KO, and Yapf/fPkd1f/fPkhd1cre (Pkd1-YapKO) mice. (A) Immunostaining for collagen-1a and a-SMA. (B) mRNA levels of collagen-
1a, a-SMA, and CCN2 relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; n56). (C) Hematoxylin and eosin staining of
WT, Yapf/fPkhd1cre (Yap KO), Pkd1KO, and Yapf/fPkd1f/fPkhd1cre (Pkd1-YapKO) mice kidney sections at P18. (D) Kidney-body weight
(BWt) ratio (percentage). (E) BUN levels. (F) H&E staining of WT or Pkd1KO mice treated with vehicle or verteporfin (Vert) kidney
sections at P18. (G) Kidney-BWt ratio and (H) BUN levels. *P,0.05 by t test for WT or Pkd1KO mice; **P,0.01 by t test for WT or
Pkd1KO mice; ***P,0.001 by t test for WT or Pkd1KO mice. Scale bar 5 50 mm

whereas no notable changes were observed in verteporfin- was observed in both verteporfin-treated Pkd1KO and in
treated WT mice (Supplemental Figure 6B). Verteporfin treat- Pkd1-YapKO mice (Figure 5, C and F). Renal cyst number
ment significantly reduced mRNA levels of a-SMA and ECM and cyst index were also significantly reduced in the Pkd1-
proteins (Figure 4, C–F), YAP-regulated genes (Figure 4G), YapKO kidneys and verteporfin-treated Pkd1KO kidneys
and proinflammatory and profibrotic factors (Supplemental compared with the Pkd1KO kidneys (Supplemental
Figure 6C) compared with vehicle treatment in Pkd1KO mice. Figure 8, B–E). In both of the above studies, neither verte-
Verteporfin treatment also significantly reduced protein levels porfin treatment nor renal collecting duct–specific YAP gene
of a-SMA, collagen-1a, CCN2, and pSMAD-SMAD ratio in knockout in WT mice caused alterations in renal structure,
Pkd1KO mice (Figure 4H, Supplemental Figure 7). function, or fibrosis.
Similarly, in collecting duct–specific Pkd1-YapKO mouse
kidneys, collagen-1a and a-SMA immunostaining V2R Signaling via ERK1/2 Regulates YAP Expression
(Figure 5A, Supplemental Figure 8A) and mRNA levels of and Activity in the Cystic Epithelium of ADPKD Kidneys
collagen-1a, a-SMA, and CCN2 were significantly reduced To determine the mechanism by which V2R signaling regu-
when compared with Pkd1KO mouse kidneys (Figure 5B). lates YAP, we examined YAP expression in the kidneys of
At P18, the Pkd1-YapKO mice had significantly smaller kid- Pkd1KO mice treated with dDAVP as in Figure 1C. Pkd1KO
neys (Figure 5C) and showed a 60% reduction in kidney-body kidneys showed high YAP protein levels compared with WT
weight ratio and a 55% reduction in BUN levels compared kidneys, which was further significantly increased by dDAVP
with Pkd1KO mice (Figure 5, D and E). Similarly, treatment (Figure 6, A and B). The dDAVP-treated Pkd1KO
verteporfin-treated Pkd1KO mice also showed smaller kid- mice also showed increased activated ERK1/2 (pERK) levels
neys, reduced kidney-body weight ratio, and BUN (Figure 5, (Figure 6, A and B). V2R-cAMP-protein kinase A (PKA)–
F–H). Compared with Pkd1-CCN2KO mouse kidneys mediated ERK1/2 activation is known to promote cystic epi-
(Figure 3A), more sparing of the renal cortical parenchyma thelial cell proliferation and cyst-filling fluid secretion in

JASN 31: 1697–1710, 2020 Epithelial V2R Regulates Fibrosis 1705

 BASIC RESEARCH www.jasn.org

A B

pERK/GAPDH:ERK/GAPDH
WT Pkd1KO
6 * 2.5
*

YAP/GAPDH relative

relative band density

Vehicle dDAVP Vehicle dDAVP
2.0

band density
4
YAP 1.5
1.0
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0.5
pERK 0.0
0
Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP Vehicle dDAVP
ERK
WT Pkd1KO WT Pkd1KO
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C NHK ADPKD
Vehicle Forskolin Vehicle Forskolin

Forskolin
D Vehicle UO126 H89

YAP

GAPDH

E 1.5 ns F dDAVP treated

Relative band density

*** Pkd1KO Pkd1-YapKO Pkd1KO + Vert

YAP/GAPDH

1.0
α-SMA

0.5
Collagen-1a

0.0
Vehicle UO126 H89 GAPDH
Forskolin

G 2.0 *** *** H

*** * *** ***
Band density relative

2500 *** ***

1.5
to GAPDH

ns
2000
Urine Osmolality
(mOsm/KgH2O)

1.0
1500
0.5 ns
1000
0.0
500
t

t
KO

KO

O
er

er
pK

pK
+V

+V
d1

d1
Ya

Ya
KO

KO
Pk

Pk

0
d1

d1
d1

d1
Pk

Pk

WT Yap Pkd1 Pkd1- Veh Vert Veh Vert

Pk

Pk

KO KO Yap WT Pkd1KO
dDAVP
KO
α-SMA Collagen-1a

Figure 6. V2R-mediated myofibroblast activation in ADPKD kidneys is dependent on ERK1/2 and YAP. (A) Immunoblot of kidney tissue
of mice described in Figure 1C: n53 WT and n54 Pkd1KO. (B) Quantitation of band density of YAP and pERK-ERK ratio relative to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C) YAP expression (green) in NHK or ADPKD cells serum deprived (16 hours)
and treated with vehicle or forskolin (5 mM for 2 hours). (D) Immunoblot and (E) quantitation of band density for YAP in ADPKD cells

1706 JASN JASN 31: 1697–1710, 2020


PKD,9,10,30 and ERK1/2 regulates YAP expression in tumor
cells.31,32 Hence, we tested if V2R activation stimulates YAP
expression and YAP transcriptional activity in PKD kidneys in
an ERK1/2-dependent fashion. In vitro, YAP was detected even
in confluent monolayers of primary culture human ADPKD
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cells, and forskolin, an adenylate cyclase agonist, increased
YAP nuclear accumulation (Figure 6C). In ADPKD cells,
forskolin-induced YAP protein levels were significantly re-
duced by UO126, an inhibitor of ERK1/2 (Figure 6, D and
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8K2+Ya6H515kE= on 04/27/2023
E, Supplemental Figure 9). Although YAP levels showed a re-
ducing trend in cells treated with H-89, a PKA inhibitor, it was
not significant (Figure 6, D and E, Supplemental Figure 9).
These results suggest that the V2R-cAMP-ERK–dependent
mechanism could regulate YAP expression in ADPKD cystic
epithelial cells.
To further confirm that YAP plays a critical role in V2R-
mediated myofibroblast activation, the effect of dDAVP was
tested on vehicle or verteporfin-treated Pkd1KO mice and on
Yap-Pkd1KO mice. Although dDAVP treatment increased
a-SMA and collagen-1a expression in Pkd1KO mice, neither
verteporfin-treated Pkd1KO mice nor Pkd1-YapKO mice
showed an increase in a-SMA or collagen-1a (Figure 6, F
and G). Urine osmolality in Pkd1KO mice was 80% lower
than WT mice (Figure 6H). However, neither collecting ducts
YAP gene deletion nor YAP systemic inhibition reduced urine
osmolality further in Pkd1KO mice (Figure 6H). Overall, these
results suggest that YAP inactivation can reduce myofibroblast
activation, fibrosis, and cyst expansion in ADPKD kidneys
without the complication of further affecting urine concen-
trating ability.
DISCUSSION
This study demonstrates a novel mechanism by which V2R-
YAP-CCN2–mediated cell signaling in tubular epithelium ac-
tivates interstitial myofibroblasts and fibrosis in ADPKD
kidneys. Our novel observations include the finding that (1)
V2R activation increases myofibroblast population; (2) CCN2
is an important V2R-stimulated secreted factor that activates
myofibroblasts; (3) YAP activity is key to V2R-mediated CCN2
production and interstitial fibrosis in ADPKD; and (4) unlike
V2R inhibition, YAP inhibition does not reduce urine concen-
trating ability in mice.
This study provides the first example of a new pathologic
role for V2R in which stimulation of V2R in ADPKD cystic
epithelial cells increases the myofibroblast population in the
pericystic microenvironment. Normal V2R activity is essential
treated with forskolin (5 mM) and vehicle, H-89 (5 mM), or UO126 (20 mM) for 16 hours. Experiment was repeated three times. (F)
Immunoblot of kidney tissue lysate of dDAVP-treated Pkd1KO mice, dDAVP 1 verteporfin (Vert)–treated Pkd1KO mice, and dDAVP-
treated Pkd1-YapKO mice. (G) Quantitation of band density for a-SMA and collagen-1a relative to GAPDH and (H) urine osmolality
measurements in spot urine samples: n55 for WT and n56–8 for Pkd1KO mice as shown. *P,0.05 versus vehicle by t test; ***P,0.001
versus vehicle by t test.
JASN 31: 1697–1710, 2020
www.jasn.org BASIC RESEARCH
for the urine concentrating function of the kidney collecting
ducts. However, V2R plays a key pathogenic role in PKD by
upregulating the cAMP-PKA-ERK1/2 MAPK pathway to drive
cell proliferation and cyst expansion.9,14 Arginine vasopressin
deficiency or V2R antagonists are known to reduce cyst
growth, whereas dDAVP treatment induces or accelerates
cyst growth in rodent models of PKD.33–37 Importantly, tol-
vaptan, a V2R antagonist, is the only Food and Drug Admin-
istration (FDA)–approved drug for the treatment of
ADPKD.38
Although V2R activation has a clear mitogenic effect on
ADPKD epithelial cells, this study demonstrates an additional
novel role in myofibroblast activation and fibrosis. We found
dense populations of a-SMA–expressing myofibroblasts
around V2R-expressing cystic epithelium in human ADPKD
kidneys. V2R agonist significantly increased profibrotic fac-
tors, myofibroblast population, and ECM proteins in cystic
mouse kidneys. Importantly, conditioned culture media
from human ADPKD cystic epithelial cells stimulated activa-
tion, migration, and viability of fibroblasts in vitro. Renal fi-
brosis is recognized as a major pathology linked to loss of renal
function in ADPKD, and TGFb, inflammatory cells, and ma-
tricellular proteins are thought to enhance this fibrosis.4,5,39–43
Activated stromal myofibroblasts, also called cancer-
associated fibroblasts in the tumor microenvironment, are
known to enhance tumor cell proliferation and metastasis,
whereas tumor cells in turn can stimulate myofibroblast acti-
vation and fibrosis.44 Our study now demonstrates that cystic
epithelial cells can regulate myofibroblasts by a V2R-
dependent mechanism.
This study also provides evidence that V2R-stimulated
myofibroblast activation is mediated by YAP. YAP and its ho-
molog—transcriptional coactivator with PDZ-binding motif—
are transcriptional coactivators of transcriptional enhanced
associate domain that regulate cell proliferation, differentia-
tion, and apoptosis.45 Activation of the Hippo signaling cas-
cade leads to phosphorylation, nuclear exclusion, cytoplasmic
sequestration, and proteolytic degradation of YAP and tran-
scriptional coactivator with PDZ-binding motif.46–48 YAP
protein is elevated and promotes fibrosis in mouse CKD mod-
els.48 YAP was detected in the cystic epithelium of human and
mouse PKD kidneys,27 and YAP gene deletion reduced cyst
growth in ADPKD mouse models.11 However, the role of YAP
in fibrosis in PKD had not been previously examined. We
demonstrated that in response to dDAVP treatment, renal
YAP levels and YAP target gene expression were increased in
PKD mouse models. Moreover, pharmacologic YAP inhibi-
tion and renal tubule–specific YAP gene deletion significantly
Epithelial V2R Regulates Fibrosis 1707
 BASIC RESEARCH www.jasn.org
reduced ECM proteins in PKD mouse kidneys, which could
not be reversed by dDAVP treatment. These results suggest
that (1) V2R and Hippo signaling pathways interact, (2) YAP
is a downstream effector of V2R signaling, and (3) cystic ep-
ithelial YAP is critical for V2R-stimulated fibrosis in PKD. The
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importance of V2R and YAP in myofibroblast activation and
fibrosis was also shown by reduced a-SMA expression and
fibrosis in Pkd1-YapKO mice and the inability of dDAVP to
induce significant a-SMA expression and fibrosis in these
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8K2+Ya6H515kE= on 04/27/2023
mice. Use of verteporfin, a YAP inhibitor and FDA-
approved drug for age-related neovascular macular degener-
ation, could be a potentially useful therapeutic strategy to
slow or inhibit renal fibrosis in PKD.
In this study, human ADPKD primary culture cells and
Pkd1KO mice showed increased YAP expression and tran-
scriptional activity when treated with forskolin or dDAVP.
This increase in YAP coincided with increased pERK1/2 levels
in Pkd1KO mice and was inhibited by ERK inhibition in
ADPKD cells. These findings in ADPKD are in contrast with
prior observations that cAMP-PKA signaling stimulated by
forskolin increases Hippo signaling, leading to LATS-
mediated phosphorylation and inhibition of YAP in breast
cancer cell lines, HEK293 cells,49 and NIH3T3L1 fibroblasts.50
This could be because cAMP-mediated signaling activates the
B-Raf/ERK pathway in cultured PKD cells but not in normal
cells.30 V2R-cAMP-PKA–mediated ERK1/2 activation pro-
motes cystic epithelial cell proliferation and cyst-filling fluid
secretion in PKD,9,10,30 and ERK1/2 is known to regulate YAP
expression in tumor cells.31,32
Our finding that epithelial cell V2R and YAP can stimulate
myofibroblast activation and fibrosis in ADPKD kidneys sug-
gests the involvement of secreted factors in mediating this
paracrine epithelial-myofibroblast interaction. Consistently,
examination of dDAVP-treated Pkd1KO mouse kidneys
showed increased mRNA levels of YAP-regulated secreted pro-
fibrotic factors, such as CCN2, AREG, PAI-1, and CCL2.
Moreover, these factors could be significantly reduced by ver-
teporfin treatment in ADPKD cells or Pkd1KO mice. CCN2
could be an important V2R-YAP–regulated secreted factor
that contributes to myofibroblast activation in ADPKD kid-
neys. The role of CCN2 in fibrosis19,20 has been conclusively
shown using CCN2 inactivation in a wide variety of systems,
including CKD models such as UUO, diabetic nephropathy,
GN, and severe AKI.21–26 Although high-throughput sequenc-
ing of human and mouse PKD kidneys has shown CCN2 to be
a major RNA transcript,41,51–53 it is unknown if CCN2 plays a
pathogenic role in PKD. We found that CCN2 gene deletion in
collecting ducts significantly reduced not just cyst growth but
also, the pericystic myofibroblast population in mouse
ADPKD kidneys. Although other experimental CKD models
studied autocrine regulation of CCN2 by fibroblasts,21–23,25,26
we found abundant CCN2 expression in the cystic epithelial
cells in human and mouse ADPKD kidneys. CCN2 was also
increased by dDAVP treatment in Pkd1KO mice and reduced
by YAP inactivation in Pkd1KO mice and ADPKD cells. This
1708 JASN
Fibroblast
CCN2
YAP ECM α-SMA
V2 R
AVP
Myofibroblast
Cystic epithelial cell
Figure 7. Cystic epithelial cells stimulate myofibroblast activa-
tion in the pericystic microenvironment leading to fibrosis in
ADPKD. In ADPKD kidneys, V2R mediated cell signaling activates
YAP in cystic epithelial cells leading to increased CCN2 expres-
sion, paracrine activation of interstitial myofibroblasts and ECM
production.
suggests that cystic epithelial CCN2 plays an important role in
V2R-YAP–mediated stimulation of renal fibrosis in PKD. Al-
though immune cell infiltrates were not examined in this
study, the observation that V2R and YAP can regulate multiple
proinflammatory factors, including CCN2, suggests that they
could be involved in attracting/activating macrophages,
T cells, and granulocytes in ADPKD kidneys and importantly,
could also be involved in fibrosis.
In conclusion, these findings provide new insights into a
novel pathogenic mechanism by which V2R, an epithelial-
specific hormone receptor, stimulates fibrosis in ADPKD.
We identify YAP as an important mediator of myofibroblast
activation and ECM production (Figure 7). Pharmacologic
approaches targeting the V2R-YAP-CCN2 molecular axis
may have strong implications for control of fibrosis in
ADPKD, suggesting new targets for therapy.
ACKNOWLEDGMENTS
We thank the University of Kansas PKD Biomarkers and Biomaterials
Core (PKDP30DK106912) for human specimens and primary cul-
ture cells, Dr. Igarashi for Pkhd1cre mice, and Dr. Somlo and the Yale
PKD Center (PKDP30DK090744) for Pkd1/f mice.
Dr. Reena Rao conceptualized and designed studies; Dr. Nidhi
Dwivedi, Dr. Christianna Howard, Dr. Abeda Jamadar, Dr. Sonali
Sinha, and Dr. Shixin Tao performed experiments; Dr. James P. Calvet,
Dr. Timothy Fields, Dr. Andrew Leask, and Dr. Darren Paul Wallace
provided reagents; Dr. Nidhi Dwivedi, Dr. Christianna Howard,
Dr. Abeda Jamadar, Dr. Reena Rao, Dr. Sonali Sinha, and Dr. Shixin
Tao analyzed results; Dr. Reena Rao wrote the manuscript; Dr. Nidhi
Dwivedi, Dr. Christianna Howard, Dr. Abeda Jamadar, Dr. Sonali Sinha,
JASN 31: 1697–1710, 2020

and Dr. Shixin Tao wrote parts of the manuscript; and Dr. James
P. Calvet, Dr. Timothy Fields, Dr. Andrew Leask, and Dr. Darren Paul
Wallace reviewed and edited the manuscript.
Downloaded from http://journals.lww.com/jasn by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
DISCLOSURES
A. Leask reports that he is a shareholder (,5%, ,$500,000) in FibroGen. D.
Wallace is a consultant for NovaTarg Therapeutics, Chinook Therapeutics,
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8K2+Ya6H515kE= on 04/27/2023
and Vertex Pharmaceuticals. All remaining authors have nothing to disclose.
FUNDING
This study was supported by National Institute of Diabetes and Digestive
and Kidney Diseases grant R01-DK083525 (to R. Rao). N. Dwivedi was sup-
ported by American Heart Association postdoctoral fellowship grant
19POST34380932 and a postdoctoral fellowship grant from the University
of Kansas Biomedical Research and Training Program.
SUPPLEMENTAL MATERIAL
This article contains the following supplemental material online at
http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2020020190/-/
DCSupplemental.
Supplemental Figure 1. Quantitation of band density of a-SMA
and collagen-1a relative to GAPDH for mice treated with dDAVP.
Supplemental Figure 2. Urinary osmolality in mice treated with
dDAVP.
Supplemental Figure 3. Effect of conditioned media from human
primary culture NHK or ADPKD renal cystic epithelial cells on hu-
man or rodent renal fibroblasts and mRNA expression of secreted
factors from ADPKD mouse kidneys.
Supplemental Figure 4. CCN2 induces myofibroblast activation
and migration in vitro.
Supplemental Figure 5. Immunostaining and cyst number in
Pkd1KO and Pkd1KO-CCN2KO mouse kidneys.
Supplemental Figure 6. Immunostaining for YAP, collagen-1a, and
a-SMA and mRNA levels.
Supplemental Figure 7. Quantitation of band density from im-
munoblot of kidney lysate from WT or Pkd1KO mice treated with
vehicle or verteporfin.
Supplemental Figure 8. Immunostaining, cyst number, and
cystic index of Pkd1YAPKO mice or Pkd1KO mice treated with
verteporfin.
Supplemental Figure 9. YAP expression in primary culture human
ADPKD cells treated with sorskolin, H-89, or UO126.
Supplemental Table 1. List of mouse primers used for quantitative
RT-PCR and list of human primers used for quantitative RT-PCR.
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