Am J Physiol Renal Physiol 310: F284–F293, 2016.
First published November 25, 2015; doi:10.1152/ajprenal.00360.2015.
Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct
Alexis A. Gonzalez,1 Flavia Cifuentes-Araneda,1 Cristobal Ibaceta-Gonzalez,1 Alex Gonzalez-Vergara,1
Leonardo Zamora,1 Ricardo Henriquez,1 Carla B. Rosales,2 L. Gabriel Navar,2,3 and Minolfa C. Prieto2,3
1
Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; 2Department of Physiology Tulane
University, School of Medicine, New Orleans, Louisiana; and 3Hypertension and Renal Center of Excellence, Tulane
University, School of Medicine, New Orleans, Louisiana
Submitted 7 August 2015; accepted in final form 18 November 2015
Gonzalez AA, Cifuentes-Araneda F, Ibaceta-Gonzalez C, Gonza-
lez-Vergara A, Zamora L, Henriquez R, Rosales CB, Navar LG,
Prieto MC. Vasopressin/V2 receptor stimulates renin synthesis in the
collecting duct. Am J Physiol Renal Physiol 310: F284 –F293, 2016. First
published November 25, 2015; doi:10.1152/ajprenal.00360.2015.—
Renin is synthesized in the principal cells of the collecting duct (CD),
and its production is increased via cAMP in angiotensin (ANG)
II-dependent hypertension, despite suppression of juxtaglomerular
(JG) renin. Vasopressin, one of the effector hormones of the renin-
angiotensin system (RAS) via the type 2-receptor (V2R), activates the
cAMP/PKA/cAMP response element-binding protein (CREB) path-
way and aquaporin-2 expression in principal cells of the CD. Accord-
ingly, we hypothesized that activation of V2R increases renin synthe-
sis via PKA/CREB, independently of ANG II type 1 (AT1) receptor
activation in CD cells. Desmopressin (DDAVP; 10⫺6 M), a selective
V2R agonist, increased renin mRNA (⬃3-fold), prorenin (⬃1.5-fold),
and renin (⬃2-fold) in cell lysates and cell culture media in the M-1
CD cell line. Cotreatment with DDAVP⫹H89 (PKA inhibitor) or
CREB short hairpin (sh) RNA prevented this response. H89 also
blunted DDAVP-induced CREB phosphorylation and nuclear local-
ization. In 48-h water-deprived (WD) mice, prorenin-renin protein
levels were increased in the renal inner medulla (⬃1.4- and 1.8-fold).
In WD mice treated with an ACE inhibitor plus AT1 receptor block-
ade, renin mRNA and prorenin protein levels were still higher than
controls, while renin protein content was not changed. In M-1 cells,
ANG II or DDAVP increased prorenin-renin protein levels; how-
ever, there were no further increases by combined treatment. These
results indicate that in the CD the activation of the V2R stimulates
renin synthesis via the PKA/CREB pathway independently of
RAS, suggesting a critical role for vasopressin in the regulation of
renin in the CD.
intrarenal renin-angiotensin system; collecting duct; water depriva-
tion; distal tubular renin; prorenin; PKA/CREB
THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays a pivotal role in the
regulation of blood pressure (BP) and in the maintenance of
body sodium and fluid balance. There is growing evidence
demonstrating the presence of the RAS in the tissues of renal
and cardiovascular systems (5–7, 35), supporting the impor-
tance of the tissue RAS in the accomplishment of its functions.
The kidneys generate angiotensin II (ANG II) de novo (46, 47).
The luminal presence of angiotensinogen (AGT) in the proxi-
mal tubules (PT) (24), angiotensin-converting enzyme (ACE)
in the collecting duct (CD) (3, 15, 25, 39), and prorenin and
renin in the distal nephron has been established (40, 41). These
findings further support the formation of intratubular ANG II
from sequential coordinated actions of tubular renin and ACE
Address for reprint requests and other correspondence: A. A. Gonzalez,
Institute of Chemistry, Pontificia Universidad Católica de Valparaíso, Val-
paraíso, Chile 2340000 (e-mail: alexis.gonzalez@pucv.cl).
F284 1931-857X/16 Copyright © 2016 the American Physiological Society
that act on AGT substrate delivered from the proximal seg-
ments. As evidence, ANG II, AGT, and renin contents are
increased in the kidneys from ANG II-infused hypertensive
rats (12), which contribute to sodium reabsorption (48). How-
ever, the mechanisms regulating renin in the CD and its
physiological implications are not well understood.
There is consensus evidence showing that the cAMP-PKA
and cAMP response element-binding protein (CREB) pathway
constitute the central pathway for renin regulation in JG cells
(17, 26). Klar et al. (23) showed that cAMP formation medi-
ated by forskolin, an activator of adenylate cyclase (AC),
increased the activity of renin promoter thus stimulating
renin gene transcription in juxtaglomerular (JG) cells. In the
distal nephron, renin synthesis and transcription are upregu-
lated by ANG II through the activation of the AT1 receptor
and PKC (8).
Vasopressin (AVP) is a potential effector of the RAS (16) in
CD cells. The activation of the AVP type 2 receptor (V2R)
stimulates the cAMP/PKA/CREB pathway and aquaporin-2
(AQP2) expression in the apical plasma membrane of principal
cells in the CD (28). AT1 receptor blockade in rats treated with
the specific V2R agonist desmopressin (DDAVP) and sub-
jected to dietary NaCl restriction decreased urine osmolality
and blunted the DDAVP-induced upregulation and phosphor-
ylation of AQP2 via PKA (27). This further supports a role for
ANG II in the regulation of AQP2. Nonetheless, it has not been
determined whether the V2R regulates CD renin abundance.
In the present study, we tested the hypothesis that V2R
activation in CD cells stimulates renin synthesis via the PKA/
CREB pathway independently of the AT1 receptor. We per-
formed in vitro studies using M-1 cells to determine the effects
of DDAVP treatment on renin and the signaling pathways
involved. To establish in vivo relevance, we evaluated the
renin and prorenin responses in renal inner medullary tissues,
devoid of renin from JG cells, from mice subjected to 48-h
water deprivation and RAS blockade.
MATERIALS AND METHODS
M-1 cell cultures. The M-1 cells were obtained from the American
Type Culture Collection (Manassas, VA). Cells were harvested after
6 h of treatments. DDAVP (catalog no. 16679-58-6, Sigma-Aldrich, St.
Louis, MO) was tested at doses of 10⫺10 to 10⫺6 M. Forskolin (catalog
no. 66575-29-9, Sigma-Aldrich) was used at 10⫺6 M. H89 (catalog no.
B1427, Sigma-Aldrich) was used at 10⫺6 M. ANG II (catalog no.
4474-91-3, Sigma-Aldrich), was used at 10⫺7 M, and tolvaptan (cata-
log no. 150683-30-0, Sigma-Aldrich) was used at 10⫺6 M. Calphostin
C (catalog no. 121263-19-2, Santa Cruz Biotechnology, Santa Cruz,
CA) was used at 10⫺7 and 10⫺6 M. Losartan (catalog no. 124750-
99-8, Sigma) was used at 10⫺6 M.
PKA assay and cAMP measurements. M-1 cells were seeded and
treated with DDAVP, and the reaction was stopped by removal of the
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 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN
medium and addition of a cocktail of protease inhibitors (catalog no.
P9599, Sigma-Aldrich). PKA activity was measured using a PKA
kinase activity ELISA kit from Enzo-Life Sciences (catalog no.
ADI-EKS-390A, Glasgow, UK) after 20-min treatment. The cAMP
levels of M-1 cells were determined with cAMP ELISA (Cayman,
Ann Arbor, MI) according to the manufacturer’s protocol. Briefly,
cells were stimulated with DDAVP or DDAVP plus inhibitors or
vehicle (PBS) for 20 min and harvested in 100 ␮l of 0.1 M HCl
containing 10⫺9 M IBMX to prevent the degradation of cAMP. The
ANG II⫹IBMX group (see Fig. 5B) was also harvested in the
presence of IBMX. After centrifugation at 1,000 g for 10 min, the
supernatants were decanted into clean new tubes to process immedi-
ately or store at ⫺80°C. For cAMP assay, the samples were diluted
two- to threefold using a kit provided for ELISA buffer. Fifty
microliters of the diluted samples and the cAMP standards were added
to each well of the plate precoated with mouse anti-rabbit IgG cAMP
ELISA (Cayman) using equal amounts of protein following the
manufacturer’s instructions.
RNA isolation and real-time quantitative PCR for renin mRNA.
Total RNA was extracted by using the RNeasy Mini Kit (Qiagen,
Valencia, CA). Primers and probes used to amplify renin mRNA
using the TaqMan System were forward: 5=-AGTACTATGGT-
GAGATCGGCATT-3=, reverse: 5=-AGATTCACAACCTCTAT-
GACTCCTC-3=, and a fluorogenic probe: 5=6=-FAM-TTCAAAGT-
CATCTTTGACACGGGTTCAG-BHQ1-3. A mouse ␤-actin gene
was used as an internal standard: forward: 5=-ATCATGAAGTGT-
GACGTTGA-3=, reverse: 5=-GATCTTCATGGTGCTAGGAGC-3=,
and a fluorogenic probe: 5=-6-HEX-TCTATGCCAACACAGTGCT-
GTCTGGT-BHQ2-3=. The qRT-PCR was performed in the Mx3000P
system (Stratagene, Santa Clara, CA).
Renin, prorenin, and CREB protein expression by western blot
analysis. Twenty micrograms of total protein were separated and
transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA).
Anti-phospho-CREB and total CREB were obtained from Cell Sig-
naling (catalog no. mAb 9198, Danvers, MA). For prorenin and renin
detection, polyclonal IgG B-12 antibody was used (Santa Cruz Bio-
technology). Results were expressed as the ratio between the abun-
dance of the protein of interest and ␤-actin. Recombinant mouse
prorenin (AnaSpec, Fremont, CA) and renin (Lee Biosolutions St.
Louis, MO) were used as standards. Cell culture media (1 ml) was
centrifuged at 3,000 rpm for 5 min, and the supernatant was concen-
trated (10⫻) using Amicon ultrafilter units (Millipore, Temecula, CA)
after 20 –30 min of centrifugation. Final recovery volume was 40 ␮l,
and 40 ␮g of total protein were used for Western blot analysis.
CREB knockdown in M-1 cells using short hairpin RNA. Plasmids
containing four different sequences [SureSilencing short hairpin (sh)
RNA Plasmids, catalog no. 336311, Qiagen] were purified (see Fig.
4A) and transfected (1 ␮g of DNA) using Lipofectamine LTX (Invit-
rogen, Carlsbad, CA) for 36 h. Efficiency of transfection was con-
firmed by green fluorescent protein (GFP) detection (see Fig. 4B).
Reduction of CREB protein expression reached 67 ⫾ 9% quantified
by the CREB vs. ␤- actin ratio (see Fig. 4C). A scrambled shRNA
sequence containing GFP was used as a negative control.
Renin and CREB immunofluorescence. M-1 cells were fixed in cold
methanol, blocked, stained with either rabbit anti-renin (B-12, Santa
Cruz Biotechnology) or total CREB (Cell Signaling, New England
Biolabs, Beverly, MA), and detected with Alexa Fluor 594-conjugated
to anti-rabbit IgG (Invitrogen). Samples were counterstained with
4=,6-diamidino-2-phenylindole (DAPI; Invitrogen). Negative controls
were obtained by omission of the specific primary antibody. NIS
Elements analysis software (Nikon) was used to quantify renin-
prorenin immunostaining (object count) and nuclei CREB localization
based on their DAPI (nuclei) colocalization by measuring the merging
area of CREB-DAPI in n ⫽ 6 fields (⫻100). Results are expressed as
the percentage of nuclei-CREB colocalization (see Fig. 3B).
AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org
F285
In vivo treatments, water restriction protocol, and animal tissue
sampling. The Institutional Animal Care and Use Committees
approved all animal protocols. Male CF-1 mice (18 –20 g, n ⫽ 5,
Charles River, Wilmington, MA) were subjected to 48-h water
deprivation or ad libitum tap water supplementation. Another set of
animals (n ⫽ 5) was subjected to a combined treatment with losartan
(30 mg·kg⫺1·day⫺1, Cozaar) and captopril (40 mg·kg⫺1·day⫺1, Fisher
Scientific, Houston, TX) supplemented in food, with free access to
water or water restriction for 48 h. To increase plasma osmolality, we
performed intraperitoneal injection of 20% mannitol (34) equivalent
to ⬃1.6 g/kg in saline solution in an additional set of mice (n ⫽ 5).
Mice were euthanized after 1 h, and prorenin and renin protein
abundance was compared with control animals receiving saline injec-
tion. Overall, all mice were euthanized using CO2 following treat-
ments. Renal tissues were stereo microdissected, washed in PBS three
times, and 15 mg of inner medullary tissues devoid of renin from JG
cells were used to measure renin mRNA or protein. As previously
demonstrated (37), no differences between the amount of prorenin or
renin protein levels were found in perfused kidneys vs. freshly
isolated dissected medullary tissues (data not shown).
Immunostaining. Renin immunostaining was performed in para-
formaldehyde-fixed kidney consecutive sections with the peroxidase
technique using renin antibody provided by Dr. Tadashi Inagami
(Vanderbilt University) as previously described (37). AQP2 immuno-
staining was performed with the peroxidase technique using an
anti-AQP2 antibody (sc-28829, Santa Cruz Biotechnology). Periodic
acid-Schiff (PAS) staining was performed for better characterization
of the structures.
Plasma osmolality measurements. Plasma osmolality was mea-
sured using a vapor pressure osmometer (model 5520; Wescor,
Logan, UT). Blood samples were centrifuged at 14,000 rpm, and 10
␮l were used. Measurements were made from filter disks absorbed
with plasma fluid. Appropriate osmometer calibration was verified by
measurement of standards before the sampling.
Statistical analyses. The mRNA measurements were done in trip-
licate. For Western blotting, an average number of five to six inde-
pendent observations were performed for each treatment. Data were
evaluated by the Grubb test followed by paired and unpaired Student
t-tests or by one-way ANOVA with Tukey’s posttest. For mRNA and
protein data, control levels were defined as 100%. Significance was
defined as P ⬍ 0.05. Results are expressed as means ⫾ SE.
RESULTS
DDAVP increases renin synthesis in M-1 cells. The immu-
noblotting data revealed two bands, one at ⬃45 and the other
at 38 kDa (Fig. 1A), consistent with previous reports (12, 19,
32). DDAVP elicited dose-related increases in renin mRNA
(306 ⫾ 30 vs. 100 ⫾ 12%, P ⬍ 0.001), prorenin (165 ⫾ 23 vs.
100 ⫾ 10%, P ⬍ 0.05), and renin (200 ⫾ 13 vs. 100 ⫾ 15%,
P ⬍ 0.05) in cell lysates (Fig. 1, B and C). At 10⫺8 M, we
observed slight increases in prorenin protein levels but signif-
icant increases in renin protein (142 ⫾ 15%, P ⬍ 0.05) and
renin mRNA (135 ⫾ 19%, P ⬍ 0.05) levels. PKA activity was
increased at a dose of 10⫺8 M of DDAVP (Abs-450: 0.67 ⫾
0.11 vs. vehicle: 0.45 ⫾ 0.12, n ⫽ 4, P ⬍ 0.05) and 10⫺6 M
(Abs 450: 0.95 ⫾ 0.14, n ⫽ 4, P ⬍ 0.05 vs. vehicle). These
results agree with the increased phosphorylation of CREB at
the same doses (p-CREB/T-CREB ratio: 135 ⫾ 13 and 150 ⫾
15%, P ⬍ 0.05, Fig. 1D). Immunofluorescence in M-1 cells
(Fig. 1E) showed a marked increase in prorenin-renin immu-
nostaining after DDAVP treatment (object count: 435 ⫾ 13 vs.
control: 27 ⫾ 8). DDAVP significantly increased the prorenin
band, starting at 10⫺10 M [arbitrary units (AU)/mg protein:
28 ⫾ 4 vs. 12 ⫾ 3, P ⬍ 0.05] in the cell culture media. We did
F286 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN

C 400

Renin mRNA / β actin

**
A 300

(% Control)
Human Human
kDa MW r-Prorenin r-Renin M-1 200
50 -Prorenin
*
100
40 -Renin
0 E
Veh 10-10 10-8 10-6
Veh
B kDa DDAVP (Mol/L)
Prorenin-
Renin-
-45 D
-40
P-CREB -
β actin- -40 Total-CREB -
250 Prorenin band * P-CREB vs. Total CREB
200
*
*
Prorenin or renin vs. β actin

Renin band
200 *
* 150
(% Control)
(% Control)

150 DDAVP
100
100

50
50

0 0
Veh 10-10 10-8 10-6 Veh 10-10 10-8 10-6
DDAVP (Mol/L) DDAVP (Mol/L)

DDAVP
Prorenin-
F G Renin- + Tolvaptan
Prorenin-
β actin-
Renin-
Prorenin or renin vs. β actin

Prorenin band
Prorenin or renin vs. total protein

250
Renin band
40 Prorenin band * *
Renin band
* 200 *
(% Control)

30
(arbitrary units)

* 150

20
100

10 50

0 0
Veh 10-10 10-8 10-6 Veh DDAVP DDAVP+Tolv
DDAVP (Mol/L)
Fig. 1. Renin expression in M-1 collecting duct cells is increased by desmopressin (DDAVP) treatment. A: renin and prorenin protein bands were visualized by
Western blotting using 40 ␮g of protein extracts. The identity of renin (⬃38 kDa) and prorenin (⬃45 kDa) was based on the comparison with positive controls
using human recombinant renin and prorenin proteins. B: dose-response curve for DDAVP (10⫺10, 10⫺8, and 10⫺6 M) showed prorenin and renin protein
expression levels in cell lysates. C: renin mRNA expression at 3 different doses of DDAVP. D: cAMP response element-binding protein (CREB) phosphorylation
at 3 different doses of DDAVP showed a significant increase in phospho-CREB at 10⫺8 and 10⫺6 M DDAVP. E: immunofluorescence (⫻100) using a
prorenin/renin antibody showing the increased punctuated red staining (anti-mouse IgG secondary antibody Alexa Fluor 594) in response to DDAVP (10⫺7 M).
F: dose-response curve for DDAVP showed prorenin and renin expression levels in cell culture media (⫻10) concentrated. G: tolvaptan, a V2 receptor antagonist,
blunted the DDAVP-mediated increase in prorenin and renin protein abundances. *P ⬍ 0.05 vs. vehicle-treated group. **P ⬍ 0.01 vs. vehicle-treated group;
n ⫽ 6.

not detect a renin band at basal conditions; however, the band we examined the effects of H89, a PKA inhibitor, in the
was present at 10⫺10 M DDAVP (Fig. 1F). Tolvaptan, a V2R presence of DDAVP treatment. As shown in Fig. 2, pretreat-
antagonist, blunted DDAVP-mediated inductions on prorenin ment (20 min) with H89 completely prevented the DDAVP-
and renin protein abundances (Fig. 1, E and G) and prorenin- induced expression of prorenin [DDAVP⫹H89: 95 ⫾ 20% vs.
renin immunofluorescence (object count: 15 ⫾ 4 vs. control: control: 100 ⫾ 10%, P ⫽ not significant (NS)] and renin
27 ⫾ 8), confirming the receptor specificity of DDAVP. (DDAVP⫹H89: 98 ⫾ 18 vs. control: 100 ⫾ 13%, P ⫽ NS).
DDAVP increases renin in M-1 cells through the PKA/ No effects were observed with H89 alone (prorenin: 89 ⫾
CREB pathway. To investigate the intracellular pathways in- 20%, renin 82 ⫾ 15%, P ⫽ NS). Forskolin, an enhancer of
volved in the V2R-dependent upregulation of renin in the CD, intracellular cAMP formation, greatly increased prorenin

AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org

 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN
Prorenin-
Renin-
β actin-
Prorenin band
**
Prorenin or renin vs. β actin
300 Renin band
250
*
* with ANG II plus IBMX.
(% Control)
200
150
100
50
0
Veh DDAVP DDAVP H89 Forskolin
+ pared with controls (300 ⫾ 56 vs. 100 ⫾ 15%, P ⬍ 0.05).
H89
Fig. 2. Activation of V2 receptor (V2R) increases prorenin and renin protein
levels via PKA pathway in M-1 cells. Augmentation of prorenin and renin
protein was blunted by H89 a PKA inhibitor, while forskolin treatment
increased prorenin and renin expression. *P ⬍ 0.05 vs. vehicle-treated group;
n ⫽ 6.
(230 ⫾ 23%, P ⬍ 0.05) and renin (255 ⫾ 17%, P ⬍ 0.05)
protein abundance. Because cAMP-dependent activation of
PKA leads to CREB phosphorylation and CREB localization in
the nucleus, we further investigated the effects of H89 on the
CREB pathway. DDAVP increased the phosphorylation levels
of CREB after 20 min of treatment, while H89 pretreatment
prevented this effect (Fig. 3A). Nuclear localization of CREB
protein was analyzed by immunofluorescence after 20 min of
DDAVP treatment. As shown in Fig. 3, B and C, the increased
nuclear localization of CREB immunostaining was blunted by
H89 treatment. To further confirm the role of CREB in this
pathway, we used a CREB shRNA to knock down CREB
expression. As shown in Fig. 4D, CREB knockdown (67 ⫾
9%) prevented the DDAVP-mediated increase in prorenin
(119 ⫾ 9%, P ⫽ NS vs. control) and renin protein bands
(121 ⫾ 16%, P ⫽ NS vs. control).
ANG II and DDAVP independently increase prorenin and
renin in M-1 cells. We next examined the effects of ANG II
treatment plus DDAVP in M-1 cells. ANG II mediated renin
upregulation in CD cells at submicromolar concentrations in
vitro (19). M-1 cells were incubated with DDAVP (10⫺6
M), ANG II (10⫺7 M), or both for 4 h. As shown in Fig. 5A,
DDAVP treatment increased prorenin and renin protein
levels to the same extent observed with ANG II incubations
compared with controls (prorenin: 195 ⫾ 23%, P ⬍ 0.05
and renin: 200 ⫾ 20% P ⬍ 0.05). Treatment of cells with
ANG II plus DDAVP caused a similar induction of (pro-
renin: 172 ⫾ 13% P ⬍ 0.05 and renin: 189 ⫾ 15%, P ⬍ 0.05
vs. control). Figure 5B shows the cAMP levels in M-1 cells
subjected to various treatments. DDAVP increased cAMP
levels to a similar extent as observed by ANG II treatment
alone. ANG II plus DDAVP increased cAMP levels to the
same extent, and AT1 receptor blockade does not affect the
DDAVP/ANG II-mediated increases in cAMP levels, dem-
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F287
onstrating that there is not an additive effect. Calphostin C
(PKC inhibitor) partially prevented the cAMP formation in
the ANG II⫹DDAVP group at 10⫺7 M. This effect was even
greater at a higher calphostin C concentration (10⫺6 M),
indicating that the stimulation of cAMP formation by
DDAVP and ANG II is dependent on PKC. As a positive
control, we tested whether phosphodiesterase inhibition may
show further increases in measurable cAMP. As shown in
Fig. 5B, cAMP levels were even greater in M-1 cells treated
Water deprivation stimulates renin expression in medullary
CDs. To examine the in vivo effects of V2R activation on renin
in the CD from the renal inner medulla, we used 48-h water-
deprived mice. To avoid contamination of the JG renin com-
ponent, we exclusively used microdissected renal inner med-
ullary tissues to measure renin mRNA and prorenin and renin
protein abundance. As shown in Fig. 6A, renin mRNA levels
were augmented in the tissues of water-deprived mice com-
Similarly, the prorenin (165 ⫾ 23 vs. 100 ⫾ 13%, P ⬍ 0.05)
and renin (254 ⫾ 18 vs. 100 ⫾ 11%, P ⬍ 0.05) protein levels
(Fig. 6B) were augmented, and there was increased specific
positive immunostaining in the renal cortical and medullary
CD cells (Fig. 6C). To confirm the physiological effect of
water deprivation, we performed AQP2 immunostaining in
consecutive kidney sections. As expected, water deprivation
increased apical AQP2 abundance. Figure 6 also shows PAS
staining to better appreciate the structures.
Increased renin mRNA and prorenin protein expression in
renal medullary tissues following water deprivation is in-
dependent of AT1 receptor activation. Figure 7A shows the
expression of medullary prorenin and renin in mice sub-
jected to 48 h of water deprivation with and without losartan
plus captopril in the food. Despite RAS blockade, renin
mRNA was significantly higher than in control mice (285 ⫾
56%, P ⬍ 0.05). Prorenin protein levels were significantly
higher than controls (171 ⫾ 10 vs. 100 ⫾ 23%, P ⬍ 0.05)
but not those from renin (113 ⫾ 13%). As previously
demonstrated, ACE inhibition or AT1 receptor blockade
does not affect prorenin renin expression at basal conditions
(14, 37). These data indicate that renin mRNA and prorenin
protein levels are augmented by water deprivation indepen-
dently of AT1 receptor activation. The posttranslated un-
changed abundance of renin also suggests that the AT1
receptor might mediate processing and cleavage of prorenin
in CD cells since losartan plus captopril treatment prevented
the increase in renin protein.
Intraperitoneal injection of mannitol induces upregulation
of renin in the renal medulla. To assess the acute AVP effect
on CD renin, we used mice subjected to a single intraperitoneal
injection of hypertonic mannitol to increase osmotic-mediated
vasopressin release. Mice were euthanized after 1 h, and
medullary tissues and plasma osmolality were analyzed.
Plasma osmolality was slightly but significantly increased in
the mannitol-treated group (mannitol: 315 ⫾ 4 vs. control:
298 ⫾ 6 mosmol/l, P ⬍ 0.05, n ⫽ 5). As shown in Fig. 7B,
prorenin and renin protein levels were significantly higher than
in controls (prorenin: 182 ⫾ 13 vs. 100 ⫾ 15%, P ⬍ 0.05;
renin: 172 ⫾ 12 vs. 100 ⫾ 9%, P ⬍ 0.05).
F288 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN

A
P-CREB -

C
Total-CREB - 100 *

% CREB –DAPI colocalization

200 * 80

P-CREB vs. Total CREB

150 60

(% Control)
100 40

50 20

0 0
Veh DDAVP DDAVP
+
Fig. 3. Inhibition of PKA activity diminished the H89
DDAVP-induced CREB phosphorylation and
nuclear localization in M-1 cells. A: levels of the
phosphorylated form of CREB normalized by B
total CREB after immunoblotting. B: CREB-
4=,6-diamidino-2-phenylindole (DAPI) colocal-
No antibody Veh
ization by immunofluorescence showing that
CREB is undergoing nuclear destination after
DDAVP treatment; however, H89 pretreatment
blunted this effect as judged by immunofluores-
cence colocalization (red) with DAPI (blue)
staining. C: percentage of CREB-DAPI colocal-
ization in each group *P ⬍ 0.05 vs. vehicle-
treated group; n ⫽ 6.

DDAVP DDAVP + H89

DISCUSSION medullary CD by water deprivation even in the presence of

The present study demonstrates that 1) V2R activation
stimulates renin synthesis and secretion in the CD via the
PKA/CREB pathway; 2) renin and prorenin are secreted by
M-1 cells in response to DDAVP stimulation; 3) renin is
augmented in the renal inner medullary CD of mice after 48 h
of water deprivation; 4) renin is stimulated in the renal inner
AT1 receptor blockade; and 5) rats with mannitol-induced
increases in plasma osmolality exhibit rapid increases in pro-
renin and renin synthesis in the renal medulla.
Although the mechanisms of renin regulation in JG cells are
well established, the regulation of distal tubular renin remains
unclear. It is known that the cAMP/PKA/CREB pathway

AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org


A B
D (sh)RNA plasmids (1– 4) and scrambled control
Prorenin-
Renin-
β actin-
Prorenin band
Prorenin or renin vs. β actin
300
Renin band
250
(% Control)
200
150
100
50
0
Scr
shRNA
constitutes the central pathway for the stimulation of renin
expression in JG cells (17, 26). However, ANG II suppresses
renin synthesis through PKC and Ca2⫹ in JG cells, as part of
the physiological negative feedback regulation of renin release
(36). In the CD, increases in intracellular Ca2⫹ and in cAMP
levels are required to target AQP2 to increase osmotic water
permeability (4). However, the lack of a potentiation effect of
thapsigargin on AVP-dependent cAMP accumulation indicates
that the increases in intracellular Ca2⫹ may not play a major
role on the responses elicited by AVP (20). Previous studies
demonstrated that CD renin is upregulated by PKC activation
and that the ANG II-mediated upregulation of renin is sup-
pressed by PKC inhibitors (12). Studies in rat inner medullary
CD cells showed that PKC inhibitors decrease cAMP accumu-
lation (29). These findings suggest that the activation of PKC
plays a role in stimulation of AC, cAMP accumulation, and
subsequent renin upregulation in the CD. In the present study,
we demonstrated that cAMP accumulation in M-1 cells also
depends on intact PKC activity, since PKC inhibition partially
suppressed cAMP accumulation in response to ANG II plus
DDAVP treatment.
Kang et al. (15) showed that M-1 cells treated with ANG II
showed an increase in renin mRNA levels. They also demon-
strated that the CD is the main source of prorenin in diabetic
rats (15). Our data agree with these results; however, we
detected the presence of two protein bands (renin and prorenin)
in cell lysates and cell culture media of M-1 cells, both
augmented by DDAVP and ANG II treatments (Fig. 4). We
previously showed that freshly isolated rat inner medullary CD
cells express mainly prorenin and that its protein levels are
stimulated by ANG II (12). In the present study using M-1 cells
AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org
VASOPRESSIN STIMULATES COLLECTING DUCT RENIN F289
C
Scr-shRNA shRNA-CREB
-Total CREB
- β actin
Fig. 4. A: integrity of the CREB-short hairpin
(Scr) after purification assessed by agarose gels.
MW, molecular weight standard. B: green fluo-
rescent protein (GFP)-containing shRNA plasmid
(green) showing transfection efficiency. C: repre-
sentative Western blot showing the knockdown of
CREB protein (67 ⫾ 9%). D: CREB-shRNA
prevented the DDAVP-mediated increase in pro-
renin and renin upregulation in M-1 cells. *P ⬍
0.05 vs. control group (Scr shRNA).
*
*
Scr shRNA shRNA shRNA CREB
+ DDAVP CREB + DDAVP
treated with either DDAVP or ANG II, we showed increases in
prorenin and renin to the same extent, but not in response to a
combined treatment, which only caused further increases in
prorenin. This suggests that lower concentrations of both
hormones may interact in vivo to enhance renin production in
the CD. Interestingly, increases in PKA activity were only
observed in response to a micromolar dose of DDAVP, con-
sistent with previous reports using cultured rat inner medullary
CD cells (9) (28). Lee et al. (28) demonstrated that ANG II
plays a role in the regulation of AQP2 targeting to the plasma
membrane in CD cells. This effect is mediated by the activa-
tion of the AT1 receptor (28). The effect of activation of the
AT1 receptor is probably mediated by the activation of PKC
(12). Rozengurt et al. (43) showed that PKC activation en-
hanced the cAMP accumulation induced by forskolin and that
this effect can be prevented by downregulation of PKC. This
modulatory effect of ANG II may be mediated by direct
activation of AC (1, 2).
Previous studies have ruled out the involvement of AC3 and
AC4 in the vasopressin-dependent stimulation of cAMP (21,
22) and suggested that the most likely AC involved is AC6
(42), which is the most abundant AC in the CD. However, the
present study did not allow defining which of the ACs is
involved in the V2R-dependent stimulation of renin in the CD.
Future studies are needed to address this question.
Interestingly, we observed granule-like patches with positive
immunofluorescence for prorenin/renin in M-1 cells treated
with DDAVP (Fig. 1). This observation along with the pres-
ence of prorenin and, to a lesser extent of renin in the cell
culture media, strongly suggests that prorenin and renin are
secreted in response to V2R stimulation. Secretion of renin
F290 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN

A
Prorenin-
Renin-
B
500
β actin-
**

cAMP accumulation
400

(pmol/mg protein)
*
*
Prorenin band 300
* *
Prorenin or renin vs. β actin

250 Renin band

** *#
200 ** *
* 200
#
(% Control)

150
100

100
0
50

0
Veh DDAVP Ang II Ang II
+
DDAVP

Fig. 5. A: DDAVP- and ANG II-independent upregulation of prorenin and renin in vitro. M-1 cells were treated with DDAVP (10⫺6 M), ANG II (10⫺7 M), or
DDAVP⫹ANG II for 4 h. Combined treatment did not enhance prorenin and renin protein levels. B: cAMP levels in M-1 cells subjected to various treatments.
DDAVP increased cAMP levels, as well as was observed by ANG II treatment alone. ANG II⫹DDAVP increased cAMP levels to the same extent in M-1 cells
without an additive effect. AT1 receptor blockade with losartan (Los) did not affect the increases in cAMP. Calphostin C (PKC inhibitor, 10⫺7 M) in the presence
of ANG II and DDAVP partially reduced cAMP formation. Higher dose of calphostin C blunted this effect, indicating that the stimulation of cAMP formation
by DDAVP and ANG II is dependent of PKC. *P ⬍ 0.05. **P ⬍ 0.01 vs. vehicle-treated group. #P ⬍ 0.05 vs. ANG II⫹DDAVP and DDAVP groups.

from the CD of rats has been previously reported (31). The
apical secretion of renin by the CD may favor the luminal
interaction with AGT delivered from proximal segments to
contribute to intratubular ANG II de novo generation during
water deprivation.
ANG II increases AVP release from the supraoptic nucleus
via the pituitary gland into the blood (38). It is possible that
during the water deprivation period of 48 h, intrarenal RAS
activation may also exert stimulatory effects on CD renin. In
the microdissected inner medullary tissues, renin transcript was
markedly augmented by water deprivation. RAS activation due
to volume depletion may also contribute to renin upregulation
in the renal medulla (12). In this regard, the role of the AT1
receptor was clearly demonstrated in the AT1aR knockout
mice, which exhibit increased 24-h urine excretion (30). This
effect is not suppressed by exogenous AVP or water restric-
tion, and it was associated with reductions in AC protein
expression (44), suggesting the importance of cross talk be-
tween AVP and ANG II in the mechanisms of urinary concen-
tration. Our current findings demonstrate that both mechanisms
also regulate renin in the CD. Indeed, renin and prorenin in the
CD of the inner medulla were stimulated by water deprivation
in mice; however, pharmacological blockade of RAS during
water deprivation did not change renin protein. These results
suggest that intrarenal RAS stimulation by water deprivation
may further enhance prorenin cleavage, thus leading to in-
creases in renin formation and release from the CD cells.
The physiological significance of these studies is highlighted
by the fact that volume depletion and augmented plasma
osmolality can act synergistically through the stimulation of
intrarenal RAS, contributing to sodium and water retention.
Indeed, the demonstrations that ANG II and AVP can directly
stimulate epithelial sodium channels in the CD (10, 33) and
suggest a parallel pathway for the stimulation of synthesis and
secretion of prorenin-renin, as well as of AQP-2 expression, in
the principal cells. Furthermore, the binding of renin and
prorenin to the (pro)renin receptor on the apical side of the CD
may help prevent the losses of these proteins in the urine (11)
and may also contribute to increase their catalytic activity to
further increase intratubular ANG II generation (11, 13). Im-
portantly, it has been shown that plasma AVP concentrations
are increased in some patients with heart failure, despite
hyponatremia and low plasma osmolality (45). Thus the in-
creases in plasma AVP levels may result from the activation of
pressure mechanoreceptors due to low cardiac output (18).
However, it is likely that the increases in CD renin may help to
restore water and salt equilibrium in volume-depletion condi-
tions or during low cardiac output through the enhancement of
intratubular ANG II formation.
In summary, the present study provides clear evidence for
the V2R-mediated regulation of CD renin. V2R activation in
the M-1 cells increases renin synthesis and secretion. Further-
more, the demonstration of increased medullary renin/prorenin
formation following 48 h of water deprivation suggests that
V2R activation also regulates CD renin in vivo; thus AVP may
contribute to intratubular ANG II formation, leading to further
stimulation of water and sodium reabsorption in the distal
nephron segments.

AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org

 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN F291

B Control WD
Prorenin-
Renin-
β acn-
A
*

Prorenin or renin vs. β actin

400 Prorenin band
*
Medullary renin mRNA

200 Renin band

vs. β actin RNA

300 *
(% Control)

(% Control)
150

200
100 Fig. 6. Renin expression is increased in the
medullary collecting ducts following water
deprivation. A: mice subjected to water de-
100 50 privation (WD) showed increased renin
mRNA levels in inner medullary tissues,
0 0 which are not of juxtaglomerular origin.
Control WD Control WD *P ⬍ 0.05 vs. control group; n ⫽ 4. B:
prorenin and renin protein levels in inner
medullary tissues were augmented by WD.
*P ⬍ 0.05 vs. control group; n ⫽ 4. C:
C AQP2 RENIN PAS consecutive kidney sections (3 ␮m) from a
control mouse (top) and a WD mouse (bot-
IM tom) showing aquaporin-2 (AQP2), renin,
CD CD and periodic acid-Schiff (PAS) staining. Kid-
ney sections are from cortical regions to show
juxtaglomerular (JG) renin as positive control
JG
Control

(renin, top middle, arrow). Middle: renin

staining in collecting ducts (CD) from cortex
and inner medulla (IM; insets). Pictures were
captured using a digital camera and an
Eclipse Nikon microscope. Bar ⫽ 400 ␮m.

CD CD
WD

IM

ACKNOWLEDGMENTS GRANTS
We thank the Molecular Core of the Tulane Renal Hypertension Center of Funds were received from FONDECYT-Chile 11121217 and 79112017
Excellence for providing the physical infrastructure to perform some of the grants to A. A. Gonzalez; from a National Institute of General Medical
experiments of this study. Sciences National Institutes of Health (NIH: CoBRE P30GM-103337) grant to

A B
Prorenin- Prorenin- Fig. 7. WD increases prorenin and renin expres-
Renin-
Renin- sion independently of renin-angiotensin system
β actin-
(RAS) activation. A: in an additional experi-
β actin- ment, mice were subjected to WD or WD plus
losartan (Los) and captopril (Capt) treatment in
Prorenin band food for 48 h. Prorenin and renin were aug-
250 Prorenin band 250 Renin band mented in medullary tissues of WD mice. WD
Prorenin or renin vs. β actin
Prorenin or renin vs. β actin

Renin band
200
* 200 * * plus RAS blockade caused increased expression
of prorenin, but renin was not different from
* *
(% Control)

control, suggesting posttranscriptional process-

(% Control)

150 150
ing mediated by AT1 receptor activation. No
100
effects were observed in mice treated with lo-
100
sartan plus captopril alone. B: mice subjected to
50 50 intraperitoneal injection of mannitol (20%, 1 h)
showed increased expression of prorenin and
0 0 renin in the renal medulla. *P ⬍ 0.05.
Veh WD WD+Los Los Saline Mannitol
+Capt +Capt

AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org

F292 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN
L. G. Navar; and from NIH Grants DK104375-01 and LA-CaTS (U54-
GM104940) to M. C. Prieto.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: A.A.G., L.G.N., and M.C.P. provided conception and
design of research; A.A.G., F.C.-A., C.I.-G., A.G.-V., L.Z., R.H., and C.B.R.
performed experiments; A.A.G. and M.C.P. analyzed data; A.A.G., L.G.N.,
and M.C.P. interpreted results of experiments; A.A.G. prepared figures;
A.A.G. and M.C.P. drafted manuscript; A.A.G., L.G.N., and M.C.P. edited and
revised manuscript; A.A.G., L.G.N., and M.C.P. approved final version of
manuscript.
REFERENCES
1. Beazely MA, Alan JK, Watts VJ. Protein kinase C and epidermal growth
factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in
intact cells. Mol Pharmacol 67: 250 –259, 2005.
2. Beazely MA, Watts VJ. Galphaq-coupled receptor signaling enhances
adenylate cyclase type 6 activation. Biochem Pharmacol 70: 113–120,
2005.
3. Casarini DE, Boim MA, Stella RCR, Krieger-Azzolini MH, Krieger
JE, Schor N. Angiotensin I-converting enzyme activity in tubular fluid
along the rat nephron. Am J Physiol Renal Physiol 272: F405–F409, 1997.
4. Chou CL, Yip KP, Michea L, Kador K, Ferraris JD, Wade JB,
Knepper MA. Regulation of aquaporin-2 trafficking by vasopressin in the
renal collecting duct—roles of ryanodine-sensitive Ca2⫹ stores and cal-
modulin. J Biol Chem 275: 36839 –36846, 2000.
5. Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga
SE, Oliveira-dos-Santos AJ, da Costa J, Zhang L, Pei Y, Scholey J,
Ferrario CM, Manoukian AS, Chappell MC, Backx PH, Yagil Y,
Penninger JM. Angiotensin-converting enzyme 2 is an essential regulator
of heart function. Nature 417: 822–828, 2002.
6. Danser AHJ, Saris JJ. Prorenin uptake in the heart: a prerequisite for
local angiotensin generation? J Mol Cell Cardiol 34: 1463–1472, 2002.
7. Danser AH, van Kats JP, Admiraal PJ, Derkx FH, Lamers JM,
Verdouw PD, Saxena PR, Schalekamp MA. Cardiac renin and angio-
tensins. Uptake from plasma versus in situ synthesis. Hypertension 24:
37–48, 1994.
8. Dellabruna R, Pinet F, Corvol P, Kurtz A. Opposite regulation of renin
gene-expression by cyclic-amp and calcium in isolated mouse juxtaglo-
merular cells. Kidney Int 47: 1266 –1273, 1995.
9. Ecelbarger CA, Chou CL, Lolait SJ, Knepper MA, DiGiovanni SR.
Evidence for dual signaling pathways for V-2 vasopressin receptor in rat
inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte
Physiol 270: F623–F633, 1996.
10. Ecelbarger CA, Kim GH, Terris J, Masilamani S, Mitchell C, Reyes I,
Verbalis JG, Knepper MA. Vasopressin-mediated regulation of epithe-
lial sodium channel abundance in rat kidney. Am J Physiol Renal Physiol
279: F46 –F53, 2000.
11. Gonzalez AA, Lara LS, Luffman C, Seth DM, Prieto MC. Soluble form
of the (pro) renin receptor is augmented in the collecting duct and urine of
chronic angiotensin II-dependent hypertensive rats. Hypertension 57:
859 –864, 2011.
12. Gonzalez AA, Liu L, Lara LS, Seth DM, Navar LG, Prieto MC.
Angiotensin II stimulates renin in inner medullary collecting duct cells via
protein kinase C and independent of epithelial sodium channel and
mineralocorticoid receptor activity. Hypertension 57: 594 –599, 2011.
13. Gonzalez AA, Prieto MC. Renin and the (pro)renin receptor in the renal
collecting duct: role in the pathogenesis of hypertension. Clin Exp Phar-
macol Physiol 42: 14 –21, 2015.
14. Gonzalez-Villalobos RA, Satou R, Seth DM, Semprun-Prieto LC,
Katsurada A, Kobori H, Navar LG. Angiotensin-converting enzyme-
derived angiotensin II formation during angiotensin II-induced hyperten-
sion. Hypertension 53: 351–355, 2009.
15. Harrison-Bernard LM, Zhuo J, Kobori H, Ohishi M, Navar LG.
Intrarenal AT1 receptor and ACE binding in angiotensin II-induced hy-
pertensive rats. Am J Physiol Renal Physiol 281: F19 –F25, 2001.
16. Hogarty DC, Tran DN, Phillips MI. Involvement of angiotensin receptor
subtypes in osmotically induced release of vasopressin. Brain Res 637:
126 –132, 1994.
AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org
17. Jensen BL, Schmid C, Kurtz A. Prostaglandins stimulate renin secretion
and renin mRNA in mouse renal juxtaglomerular cells. Am J Physiol
Renal Fluid Electrolyte Physiol 271: F659 –F669, 1996.
18. Kalra PR, Anker SD, Coats AJS. Water and sodium regulation in
chronic heart failure: the role of natriuretic peptides and vasopressin.
Cardiovasc Res 51: 495–509, 2001.
19. Kang JJ, Toma I, Sipos A, Meer EJ, Vargas SL, Peti-Peterdi J. The
collecting duct is the major source of prorenin in diabetes. Hypertension
51: 1597–1604, 2008.
20. Katsura T, Verbavatz JM, Farinas J, Ma TH, Ausiello DA, Verkman
AS, Brown D. Constitutive and regulated membrane expression of aqua-
porin-1 and aquaporin-2 water channels in stably transfected LLC-PK1
epithelial cells. Proc Natl Acad Sci USA 92: 7212–7216, 1995.
21. Kittikulsuth W, Stuart D, Kohan DE. Adenylyl cyclase 4 does not
regulate collecting duct water and sodium handling. Physiol Rep 2:
e00277, 2014.
22. Kittikulsuth W, Stuart D, Van Hoek AN, Stockand JD, Bugaj V,
Mironova E, Blount MA, Kohan DE. Lack of an effect of collecting
duct-specific deletion of adenylyl cyclase 3 on renal Na⫹ and water
excretion or arterial pressure. Am J Physiol Renal Physiol 306: F597–
F607, 2014.
23. Klar J, Sandner P, Muller MW, Kurtz A. Cyclic AMP stimulates renin
gene transcription in juxtaglomerular cells. Pflügers Arch 444: 335–344,
2002.
24. Kobori H, Harrison-Bernard LM, Navar LG. Expression of angio-
tensinogen mRNA and protein in angiotensin II-dependent hypertension. J
Am Soc Nephrol 12: 431–439, 2001.
25. Komlosi P, Fuson AL, Fintha A, Peti-Peterdi J, Rosivall L, Warnock DG,
Bell PD. Angiotensin I conversion to angiotensin II stimulates cortical collecting
duct sodium transport. Hypertension 42: 195–199, 2003.
26. Kurtz A, Wagner C. Cellular control of renin secretion. J Exp Biol 202:
219 –225, 1999.
27. Kwon TH, Nielsen J, Knepper MA, Frøkiær J, Nielsen S. Angiotensin
II AT1 receptor blockade decreases vasopressin-induced water reabsorp-
tion and AQP2 levels in NaCl-restricted rats. Am J Physiol Renal Physiol
288: F673–F684, 2005.
28. Lee YJ, Song IK, Jang KJ, Nielsen J, Frøkiær J, Nielsen S, Kwon TH.
Increased AQP2 targeting in primary cultured IMCD cells in response to
angiotensin II through AT1 receptor. Am J Physiol Renal Physiol 292:
F340 –F350, 2007.
29. Lee YJ, Shin SJ, Tan MS, Hsieh TJ, Tsai JH. Increassed renal atrial natriuretic
peptide synthesis in rats with deoxycorticosterone acetate-salt treatment. Am J
Physiol Renal Fluid Electrolyte Physiol 271: F779–F789, 1996.
30. Li XC, Shao Y, Zhuo JL. AT1a receptor knockout in mice impairs urine
concentration by reducing basal vasopressin levels and its receptor sig-
naling proteins in the inner medulla. Kidney Int 76: 169 –177, 2009.
31. Liu L, Gonzalez AA, McCormack M, Seth DM, Kobori H, Navar LG,
Prieto MC. Increased renin excretion is associated with augmented
urinary angiotensin II levels in chronic angiotensin II-infused hypertensive
rats. Am J Physiol Renal Physiol 301: F1195–F1201, 2011.
32. Liu L, Lara LS, Gonzalez AA, Bourgeois CR, Seth DM, Prieto MC.
Angiotensin II stimulates renin synthesis and secretion via protein kinase
C activation and cAMP accumulation in collecting duct M-1 cells. J Invest
Med 60: 456 –457, 2012.
33. Mamenko M, Zaika O, Pochynyuk O. Direct regulation of ENaC by
bradykinin in the distal nephron. Implications for renal sodium handling.
Curr Opin Nephrol Hypertens 23: 122–129, 2014.
34. Manninen PH, Lam AM, Gelb AW, Brown SC. The effect of high-dose
mannitol on serum and urine electrolytes and osmolality in neurosurgical
patients. Can J Anaesth 34: 442–446, 1987.
35. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological
and pathological effects in the cardiovascular system. Am J Physiol Cell
Physiol 292: C82–C97, 2007.
36. Peti-Peterdi J, Harris RC. Macula densa sensing and signaling mecha-
nisms of renin release. J Am Soc Nephrol 21: 1093–1096, 2010.
37. Prieto-Carrasquero MC, Kobori H, Ozawa Y, Gutierrez A, Seth D,
Navar LG. AT1 receptor-mediated enhancement of collecting duct renin
in angiotensin II-dependent hypertensive rats. Am J Physiol Renal Physiol
289: F632–F637, 2005.
38. Qadri F, Culman J, Veltmar A, Maas K, Rascher W, Unger T.
Angiotensin-II-induced vasopressin release is mediated through alpha-1-
adrenoceptors and angiotensin-II AT1 receptors in the supraoptic nucleus.
J Pharmacol Exp Ther 267: 567–574, 1993.
 VASOPRESSIN STIMULATES COLLECTING DUCT RENIN
39. Redublo Quinto BM, Camargo de Andrade MC, Ronchi FA, Santos
EL, ves Correa SA, Shimuta SI, Pesquero JB, Mortara RA, Casarini
DE. Expression of angiotensin I-converting enzymes and bradykinin B-2
receptors in mouse inner medullary-collecting duct cells. Int Immunop-
harmacol 8: 254 –260, 2008.
40. Rohrwasser A, Ishigami T, Gociman B, Lantelme P, Morgan T,
Cheng T, Hillas E, Zhang S, Ward K, Bloch-Faure M, Meneton P,
Lalouel JM. Renin and kallikrein in connecting tubule of mouse. Kidney
Int 64: 2155–2162, 2003.
41. Rohrwasser A, Morgan T, Dillon HF, Zhao L, Callaway CW, Hillas E,
Zhang S, Cheng T, Inagami T, Ward K, Terreros DA, Lalouel JM.
Elements of a paracrine tubular renin-angiotensin system along the entire
nephron. Hypertension 34: 1265–1274, 1999.
42. Roos KP, Bugaj V, Mironova E, Stockand JD, Ramkumar N, Rees S,
Kohan DE. Adenylyl cyclase VI mediates vasopressin-stimulated ENaC
activity. J Am Soc Nephrol 24: 218 –227, 2013.
F293
43. Rozengurt E, Murray M, Zachary I, Collins M. Protein kinase C
activation enhances cAMP accumulation in Swiss 3T3 cells: inhibition by
pertussis toxin. Proc Natl Acad Sci USA 84: 2282–2286, 1987.
44. Schrier RW. Interactions between angiotensin II and arginine vasopressin
in water homeostasis. Kidney Int 76: 137–139, 2009.
45. Schrier RW. Use of diuretics in heart failure and cirrhosis. Semin Nephrol
31: 503–512, 2011.
46. Shao W, Seth DM, Navar LG. Augmentation of endogenous intrarenal
angiotensin II levels in Val5-ANG II-infused rats. Am J Physiol Renal
Physiol 296: F1067–F1071, 2009.
47. Shao W, Seth DM, Navar LG. Angiotensin II type 1 receptor-mediated
augmentation of urinary excretion of endogenous angiotensin II in Val5-
angiotensin II-infused rats. Hypertension 56: 378 –383, 2010.
48. Zhao D, Seth DM, Navar LG. Enhanced distal nephron sodium reab-
sorption in chronic angiotensin II-infused mice. Hypertension 54: 120 –
126, 2009.

AJP-Renal Physiol • doi:10.1152/ajprenal.00360.2015 • www.ajprenal.org