Ablation of Transient Receptor Potential Vanilloid 1
Abolishes Endothelin-Induced Increases in Afferent
Renal Nerve Activity
Mechanisms and Functional Significance
Chaoqin Xie, Donna H. Wang
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Abstract—Endothelin 1 (ET-1) and its receptors, ETA and ETB, play important roles in regulating renal function and blood
pressure, and these components are expressed in sensory nerves. Activation of transient receptor potential vanilloid
(TRPV) 1 channels expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity
(ARNA), diuresis, and natriuresis. We tested the hypothesis that ET-1 increases ARNA via activation of ETB, whereas
ETA counterbalances ETB in wild-type (WT) but not TRPV1–null mutant mice. ET-1 alone or with BQ123, an ETA
antagonist, perfused into the left renal pelvis increased ipsilateral ARNA in WT but not in TRPV1–null mutant mice,
and ARNA increases were greater in the latter. [Ala1, 3,11,15]-endothelin 1, an ETB agonist, increased ARNA that was
greater than that induced by ET-1 in WT mice only. [Ala1, 3,11,15]-endothelin 1–induced increases in ARNA were
abolished by chelerythrine, a protein kinase C inhibitor, but not by H89, a protein kinase A inhibitor. Chelerythrine, H89,
and BQ788, an ETB antagonist, did not affect ARNA triggered by capsaicin in WT mice. Substance P release from the
renal pelvis was increased by [Ala1, 3,11,15]-endothelin 1 in WT mice only, and the increase was abolished by
chelerythrine but not by H89. Chelerythrine, H89, and BQ788 did not affect capsaicin-induced substance P release. Our
data show that ET1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in WT but not in
TRPV1-null mutant mice, suggesting that TRPV1 mediates ETB-dependent increases in ARNA, diuresis, and natriuresis
possibly via the protein kinase C pathway. (Hypertension. 2009;54:1298-1305.)
Key Words: TRPV1 䡲 ET-1 䡲 ETB receptors 䡲 PKC 䡲 afferent renal nerve activity
T
nerves.6 – 8 Given the important role of TRPV1 in mediating
renal function, deletion of TRPV1 results in the loss of
protection against renal injury.9 Indeed, ablation of TRPV1
exaggerates renal functional and tissue damage induced by
deoxycorticosterone acetate-salt hypertension.9
Endothelin 1 (ET-1), a potent vasoconstrictor, is found as
a neurotransmitter in primary afferent neurons and their nerve
terminals.10 Immunocytochemistry results show that its receptor subtypes, endothelin A (ETA) and endothelin B (ETB)
receptors, are present in medium- and large-sized cell bodies
of human trigeminal ganglia.11 In rats, ET-1 perfusion into
the renal pelvis increases ARNA via activation of ETB when
a high-salt diet is given and decreases ARNA via activation
of ETA in the face of salt deprivation.12 Colocalization of
TRPV1 and ETA has been found in a subpopulation of
primary sensory neurons, whereas ET-1 sensitizes capsaicin
(CAP)-induced TRPV1 current in this population of neurons.13 In HEK293 cells, ET-1–induced potentiation of
TRPV1 action depends on activation of ETA but not ETB via
a protein kinase C (PKC)– dependent pathway.13 Moreover,
he transient receptor potential vanilloid type 1 (TRPV1)
channel is mainly expressed in sensory nerves of unmyelinated C-fibers or thinly myelinated A␦-fibers that innervate the cardiovascular and kidney tissues.1 Activation of
TRPV1 causes release of a variety of sensory neuropeptides,
including substance P (SP) and calcitonin gene-related peptide, which have profound effects on the modulation of
cardiovascular and renal function (Figure 1).1–3 For example,
the renal pelvis is densely innervated by TRPV1-positive
sensory nerves.4 Agonist-induced activation of TRPV1 expressed in the unilateral renal pelvis leads to increases in
ipsilateral afferent renal nerve activity (ARNA) and contralateral urinary sodium and water excretion via the renorenal
reflex, which can be abolished by renal denervation (Figure
1).5,6 Hypertonic saline perfusion of the renal pelvis or
increased renal pelvis pressure as a mean of mechanostimulation may activate TRPV1, leading to increased ARNA and
diuresis and natriuresis, a sequence of events that depends on
TRPV1-mediated SP release and subsequent SP activation of
the neurokinin 1 (NK1) receptors expressed in sensory
Received March 4, 2009; first decision March 23, 2009; revision accepted October 2, 2009.
From the Department of Medicine (C.X., D.H.W.), Neuroscience Program (D.H.W.) and Cell and Molecular Biology Program (D.H.W.), Michigan
State University, East Lansing, Mich.
Correspondence to Donna H. Wang, Division of Nanomedicine and Molecular Intervention, Department of Medicine, Michigan State University, B338
Clinical Center, East Lansing, MI 48823. E-mail [email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.132167
1298
Xie and Wang
NK1
CGRP
SP
TRPV1
ETA
Reno
renal
reflex
RSNA
X
PKA
ARNA
PKC
ETB
Diuresis
Natriuresis
ET-1
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Figure 1. Illustration depicting possible molecular pathways
mediating ET-1–induced regulation of renal sensory nerve function. Solid lines, Enhancement; dash line, suppression. Activation of the ETB receptors by ET-1 causes activation of the
TRPV1 channel through a PKC- but not a PKA-dependent pathway, resulting in the release of SP and/or CGRP, the subsequent activation of the NK1 receptors by SP, and the enhancement of the ipsilateral ARNA. Activation of the ETA receptors
appears to inhibit this process. Increased ARNA would inhibit
RSNA via renorenal reflex and would cause diuresis and
natriuresis.
activation of TRPV1 via the ETA-PKC pathway contributes
to ET-1–induced thermal hyperalgesia.14
Despite the fact that TRPV1 and the components of the
ET-1 system are coexpressed in primary afferent nerves and
the fact that activation of ETB expressed in renal tubules
mediates ET-1–induced diuresis and natriuresis,15–18 it is
unknown whether TRPV1 plays a role in ET-1–induced
changes in renal function. Understanding the interaction
between TRPV1 and the ET1 system may provide insight into
the mechanism underlying ET-1–mediated pathological
changes in diseases and may identify downstream targets for
drug development. The present study tests the following
hypotheses: (1) ET-1 perfusion into the renal pelvis increases
ARNA in wild-type (WT) but not TRPV1-null mutant
(TRPV⫺/⫺) mice; (2) ET1-induced increases in ARNA are
mediated by activation of ETB, whereas ETA plays a counterbalance role in WT but not in TRPV⫺/⫺ mice; and (3)
ETB-mediated increases in ARNA are via activation of the
PKC but not the protein kinase A pathway in WT but not
TRPV⫺/⫺ mice (Figure 1).
Methods
In Vivo Study
All of the experimental protocols were approved by the institutional
animal care and use committee of Michigan State University.
Ten-week– old male TRPV1⫺/⫺ strain B6.129S4-TRPV1tm1Jul and
C57BL/6 mice (WT; Jackson Laboratories, Bar Harbor, ME) were
used in the experiments (total 170 mice). Mice were anesthetized by
IP administration of pentobarbital sodium at 50 mg/kg. A phycoerythrin 10 catheter was inserted into the left carotid artery for
monitoring mean arterial pressure with a Statham 231D pressure
transducer coupled to a Gould 2400s recorder (Gould Instrument
Systems). Two MD-2000 microdialysis tubes (ID 0.18/OD 0.22 mm;
BASi) were bonded together and placed inside the left ureter via a
TRPV1 and ETB-PKC Pathway
1299
midline incision. One of the tubes, of which the tip extended 1 to 2 mm
into the renal pelvis compared with the other, was used for drug
perfusion, whereas the other was used for urine draining. The perfusion
was performed at a rate of 20 ␮L/min, at which time the pelvis pressure
did not change, and the drugs were perfused into the renal pelvis for
3 minutes for ARNA measurement.6 Mice were given the following
treatments (n⫽5 to 6 in each group): (1) 10⫺8 M or 10⫺7 M ET-1
(Sigma-Aldrich) perfused into the left renal pelvis of WT mice; (2)
10⫺7 M ET-1 with or without 5⫻10⫺6 M BQ123 (Sigma-Aldrich),
a selective ETA antagonist, given into the renal pelvis in WT and
TRPV1⫺/⫺ mice; (3) 10⫺7 M [Ala1, 3,11,15]-ET-1 (4 Ala-ET-1;
Sigma-Aldrich), a selective ETB agonist, given into the renal pelvis
of WT and TRPV1⫺/⫺ mice; (4) 4⫻10⫺6 M BQ788 (Sigma-Aldrich), a selective ETB antagonist, with 4⫻10⫺6 M CAP administrated into the renal pelvis of WT mice; (5) 10⫺5 M chelerythrine
(CHE; Tocris Bioscience), a PKC inhibitor, given with 10⫺7 M 4
Ala-ET-1 or 4⫻10⫺6 M CAP into the renal pelvis of WT mice; and (6)
2⫻10⫺5 M H89 (Sigma-Aldrich), a protein kinase A (PKA) inhibitor,
given with 10⫺7 M 4 Ala-ET-1 or 4⫻10⫺6 M CAP into the renal pelvis
of WT mice.
The renal nerves were isolated at the angle between the abdominal
aorta and the renal artery via a left flank incision with the use of a
stereoscopic dissecting microscope. The nerves were placed on the
bipolar platinum electrodes to record multifiber nerve activity. The
electrode was connected to a high-impedance probe (HIP-511; Grass
Instruments). The signals were amplified ⫻20 000, filtered with a
high-frequency cutoff at 1000 Hz and a low-frequency cutoff at 100
Hz by a Grass model P511 AC amplifier, and recorded by a Gould
2400s recorder (Grould Instrument System). After the renal nerve
activity was verified using its pulse synchronous rhythmicity with
the heartbeat, the nerves were sectioned, and the distal part was
placed on the electrode for ARNA recording. The electrode was
fixed to the renal nerve with Kwik-Cast and Kwik-Sil (World
Precision Instruments). The renal nerve activity was transformed into
voltage integration. The experiment started 30 minutes after the
surgery. The basal value of ARNA was recorded 10 minutes before
the treatment, and the recovery value of ARNA was recorded 10
minutes after the treatment. The postmortem renal nerve activity
recorded as the background of renal nerve activity was subtracted
from all of the values. Average responses of ARNA were used for
analysis and ARNA was expressed in the percent of its basal
value.6,19
In Vitro Study
SP Release From the Renal Pelvis
The renal pelvis wall was removed from anesthetized mice and
incubated in 37°C HEPES buffer (HEPES, 25.0 mmol/L; NaCl,
135.0 mmol/L; KCl, 3.5 mmol/L; CaCl2, 2.5 mmol/L; MgCl2,
1.0 mmol/L; d-glucose, 3.3 mmol/L; and ascorbic acid, 0.1 mmol/L;
pH 7.45) with 95% O2/5% CO2. The pelvis was incubated with drugs
for 1 hour after it was equilibrated in the HEPES buffer for 30
minutes. The incubation solution was collected and measured by
radioimmunoassay (rat radioimmunoassay kits; Peninsula Laboratories Inc), as described previously, and the SP concentration was
normalized by kidney weight.20
Immunofluorescence Staining
Frozen kidney sections obtained from WT and TRPV⫺/⫺ mice were
fixed with formalin for 15 minutes and washed with PBS-0.01%
Tween 20 for 5 minutes. After blocking nonspecific binding sites
with 5% normal donkey serum for 30 minutes, tissues were incubated with goat anti-TRPV1 (1:100; Santa Cruz), rabbit anti-ETB
receptor (1:200; Santa Cruz), or rabbit anti-ETA receptor (1:200;
Santa Cruz) diluted with 5% normal donkey serum at 4°C overnight,
whereas negative controls were incubated with serum overnight
only. The sections were rinsed with PBS-0.01% Tween 20 and
incubated with donkey-antigoat fluorescein isothiocyanate–labeled
IgG or donkey-antirabbit Cy3-labeled IgG for 1 hour at room
temperature. The sections were washed, dehydrated with 95% and
100% ethanol, and covered with antifade mounting medium and
1300
ARNA (%)
300
Hypertension
A
WT
WT
200
TRPV1-/-
**
*
100
0
December 2009
basal 10-8 MET-1 recovery basal 10-7MET-1 recovery basal 10-7 MET-1
recovery
B
-8
-7
10 M ET-1
-7
10 M ET-1
Figure 2. Ipsilateral ARNA induced by
ET-1 perfused into the left renal pelvis in
WT or the TRPV1⫺/⫺ mice. A, ARNA at the
basal, response to ET1, or recovery in WT
or TRPV1⫺/⫺ mice. B, Representative recording of ARNA. n⫽5 to 6 in each group.
*P⬍0.05 or **P⬍0.01 vs basal values of
each group.
10 M ET-1
50uv
-50uv
coverslips.21 In the double immunofluorescence staining study, the
sections were incubated with the mixture of primary antibodies
overnight at 4°C and then incubated with the mixture of secondary
antibodies after rinse.21,22
Statistical Analysis
All of the values were expressed as mean⫾SE. The differences of
ARNA among groups were analyzed using 1-way ANOVA, followed by the Tukey-Kramer multiple comparison tests. The unpaired
Student t test was used to determine the difference of SP levels
between groups. Differences were considered statistically significant
at P⬍0.05.
Results
There was no difference in the body weight between WT
(29.4⫾0.5 g) and TRPV⫺/⫺ (28.8⫾0.6 g) mice in all of the
groups. The mean arterial pressure between WT
(95⫾6 mm Hg) and TRPV1⫺/⫺ (97⫾4 mm Hg) mice was not
300
ARNA(%)
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5min
A
WT
200
WT
statistically different, and it maintained at these levels before,
during, and after the treatments.
In Vivo Results
To examine the role of ET-1 in the regulation of ARNA in
WT and TRPV1⫺/⫺ mice, ET-1 was perfused into the left
renal pelvis. Ipsilateral ARNA was increased by ET-1 perfusion at the concentrations of 10⫺8 M (119⫾9%; P⬍0.05) or
10⫺7 M (136⫾11%; P⬍0.01) in WT mice (Figure 2). In
contrast, ARNA was not altered (Figure 2; 99⫾11%;
P⬎0.05), even when the higher dose of ET-1 (10⫺7 M) was
perfused into the left renal pelvis in TRPV1⫺/⫺ mice.
To examine the role of the ETA receptor in ET-1–induced
increases in ARNA, an ETA receptor antagonist, BQ123, was
perfused into the left renal pelvis with or without 10⫺7 M
ET-1. BQ123 alone did not change ARNA in WT (Figure 3;
TRPV1-/-
TRPV1-/-
**
100
0
basal
5x10-6 M recovery basal BQ123 + recovery basal 5x10-6 M recovery
BQ123
BQ123
10 -7 M ET-1
basal
BQ123+ recovery
10 -7M ET-1
B
BQ123
BQ123 +
ET-1
BQ123
BQ123 +
ET1
50uv
-50uv
5min
Figure 3. Ipsilateral ARNA response to BQ123, a selective ETA receptor antagonist, with or without ET-1 perfused into the left renal
pelvis in WT or the TRPV1⫺/⫺ mice. A, ARNA at the basal response to BQ123 with or without ET1 or recovery in WT or TRPV1⫺/⫺
mice. B, Representative recording of ARNA. n⫽5 to 6 in each group. **P⬍0.01 vs basal values of each group.
Xie and Wang
ARNA (%)
300
A
WT
TRPV1-/-
WT
**
**
200
TRPV1 and ETB-PKC Pathway
1301
WT
**
100
0
basal
-6
10-7 M recovery basal 10-7 M recovery basal 4x10-6M recovery basal 4x10 BQ788 recovery
CAP
4 Ala ET-1
4 Ala ET-1
+4X10-6 CAP
B
4 Ala ET-1
CAP
4 Ala ET-1
BQ788
+CAP
50uv
-50uv
5min
106⫾7%; P⬎0.05) or TRPV1⫺/⫺ (98⫾8%; P⬎0.05) mice.
BQ123 combined with ET-1 perfusion into the left renal
pelvis increased ARNA to 166⫾18% in WT mice (Figure 3;
P⬍0.01), but it had no effect on ARNA in TRPV1⫺/⫺ mice
(105⫾13%; P⬎0.05). Furthermore, the increase in ARNA
induced by BQ123 plus 10⫺7 M ET-1 (Figure 3; 166⫾18%)
was higher than that induced by 10⫺7 M ET-1 alone (Figure
2; 136⫾11%) in WT mice (P⬍0.05).
To examine the role of the ETB receptor in the regulation
of ARNA in WT and TRPV1⫺/⫺ mice, an ETB receptor
agonist, 4 Ala-ET-1, was perfused into the left renal pelvis. 4
Ala-ET-1 increased ARNA in WT mice (Figure 4;
177⫾35%; P⬍0.01) but not in TRPV1⫺/⫺ mice (106⫾18%;
P⬎0.05). Moreover, the increase in ARNA induced by 10⫺7
M 4 Ala-ET-1 (Figure 4; 177⫾35%) was higher than that
induced by 10⫺7 M ET-1 (Figure 2; 136⫾11%) in WT mice
300
ARNA (%)
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Figure 4. Ipsilateral ARNA response to 4 Ala-ET-1, an ETB receptor agonist, perfused into the left renal pelvis in WT or TRPV1⫺/⫺ mice
or response to CAP, a TRPV1 receptor agonist, with or without BQ788, a selective ETB receptor antagonist, perfused into the left renal
pelvis in WT mice. A, ARNA at the basal, response to 4 Ala-ET-1 or CAP with or without BQ788 or recovery in WT or TRPV1⫺/⫺ mice.
B, Representative recording of ARNA. n⫽5 to 6 in each group. **P⬍0.01 vs basal values of each group.
A
WT
WT
200
(P⬍0.05). To determine whether the ETB receptor mediates
CAP-induced increases in ARNA, an ETB receptor antagonist, BQ788, was perfused into the left renal pelvis with or
without CAP. CAP increased ARNA (Figure 4; 201⫾21%;
P⬍0.01) in WT mice. CAP combined with BQ788 also
increased ARNA (202⫾23%; P⬍0.01) in WT mice, but the
magnitude of the increases in ARNA was the same between
CAP alone and CAP plus BQ788 (P⬎0.05).
To examine the role of PKC and PKA in ETB-induced
increases in ARNA in WT mice, PKC or PKA inhibitors were
perfused into the left renal pelvis with or without the ETB
receptor agonist. The PKC inhibitor, CHE, perfused alone did
not alter ARNA (Figure 5; 102⫾9%; P⬎0.05) in WT mice.
However, CHE abolished 4 Ala-ET-1–induced increases in
ARNA (Figure 5; 109⫾4%; P⬎0.05) in WT mice. In
contrast, CHE had no effect on CAP-induced increases
WT
**
100
0
CHE+
basal 10-5M CHE recovery basal CHE+10-7M recoverybasal
recovery
4x10-6M CAP
4 Ala ET-1
B
CHE
50uv
-50uv
5min
CHE +
4 Ala ET-1
CHE+CAP
Figure 5. Ipsilateral ARNA response to
CHE, a PKC inhibitor, with or without 4
Ala-ET-1, an ETB receptor agonist, or
CAP, a TRPV1 receptor agonist, perfused
into the left renal pelvis in WT mice. A,
ARNA at the basal, response to CHE with
or without 4 Ala-ET-1 or CAP, or recovery
in WT mice. B, Representative recording of
ARNA. n⫽5 to 6 in each group. **P⬍0.01
vs basal values of each group.
1302
Hypertension
ARNA (%)
300
A
December 2009
WT
WT
WT
**
**
200
100
0
-7
basal 2x10-5M H89 recovery basal H89+10 M recovery
4 Ala ET-1
basal
H89+
recovery
4x10-6M CAP
B
H89 +
4 Ala ET-1
H89
H89+CAP
Figure 6. Ipsilateral ARNA response to
H89, a PKA inhibitor, with or without 4
Ala-ET-1, an ETB receptor agonist, or
CAP, a TRPV1 receptor agonist, perfused
into the left renal pelvis in WT mice. A,
ARNA at the basal value, response to H89
with or without 4 Ala-ET-1 or CAP, or
recovery in WT mice. B, Representative
recording of ARNA. n⫽5 to 6 in each
group. **P⬍0.01 vs basal values of each
group.
50uv
-50uv
in ARNA (200⫾23%; P⬍0.01). The PKA inhibitor H89
perfused alone did not alter ARNA (Figure 6; 105⫾11%;
P⬎0.05) in WT mice. Neither 4 Ala-ET-1- nor CAP-induced
increases in ARNA were affected by H89 (Figure 6;
173⫾19% and 199⫾14%, respectively; P⬍0.01).
In Vitro Results
Radioimmunoassay was used to determine the level of SP
released from the renal pelvis incubated in vitro (Figure 7).
The SP levels were not different between WT and TRPV1⫺/⫺
mice at the baseline (0.57⫾0.15 versus 0.57⫾0.18 pg/g per
minute, respectively; P⬎0.05), treated with ET-1 alone
(0.59⫾0.13 versus 0.66⫾0.10 pg/g per minute, respectively;
P⬎0.05), or treated with BQ123 combined with ET-1
(0.72⫾0.23 versus 0.67⫾0.11 pg/g per minute, respectively;
P⬎0.05). 4 Ala-ET-1 alone increased SP release in WT
(0.83⫾0.10 versus 0.57⫾0.15 pg/g per minute; P⬍0.05) but
not in TRPV1⫺/⫺ (0.58⫾0.13 versus 0.57⫾0.18 pg/g per
minute; P⬎0.05) mice compared with their respective base1.5
SP (pg/g/min)
1.0
WT
TRPV-/-
* *
** *
*
#
0.5
C
on
4
BQ A E T
l
a
12 E - 1
3+ T1
ET
1
0.0
C
o
C B 4A E n
H Q la T1
E
H + 123 -ET
89 4 + 1
+ Ala ET
4 E 1
Al T
a 1
BQ
ET
78 C 1
8
C + AP
H C
E
H +CAP
89 A
+C P
AP
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5min
Figure 7. Levels of SP released into the incubation buffer from
the isolated renal pelvis in WT or TRPV1⫺/⫺ mice. n⫽7 to 8 in
each group. *P⬍0.05 vs control groups. #P⬍0.05 vs 4 Ala-ET1–treated group.
lines. 4 Ala-ET-1-induced SP release in WT mice was
abolished by CHE (0.83⫾0.10 versus 0.58⫾0.07 pg/g per
minute; P⬍0.05) but not by H89 (0.83⫾0.10 versus
0.81⫾0.08 pg/g per minute; P⬎0.05). CAP increased SP
release (0.84⫾0.26 versus 0.57⫾0.15 pg/g per minute;
P⬍0.05) compared with the baseline in WT mice, and
CAP-induced increases in SP release in WT mice were not
affected by BQ788 (0.85⫾0.16 pg/g per minute), CHE
(0.92⫾0.11 pg/g per minute), or H89 (0.90⫾0.21 pg/g per
minute).
Immunofluorescence staining was performed to determine
the expression and coexpression of TRPV1, ETA, and ETB
receptors in the renal pelvis of WT and TRPV⫺/⫺ mice.
TRPV1-positive nerve fibers were detected in the epithelial
layer in the pelvis wall of WT but not TRPV1⫺/⫺ mice
(Figure 8). ETA staining was not observed in the renal pelvis
wall in either WT or TRPV1⫺/⫺ mice. In contrast, ETB was
expressed in nerve fibers innervating the epithelial layer of
the renal pelvis in both WT and TRPV⫺/⫺ mice (Figure 8).
Moreover, ETB colocalized with TRPV1 in the nerve fibers,
innervating the pelvis wall in WT mice.
Discussion
It has been reported that urine ET-1 levels are much higher
than that of plasma.23 In normal rats, the plasma ET-1 level is
at 28⫾3 fmol/mL, whereas the ET-1 concentration in the
urine is ⬇4.7⫾0.3 pmol/24 hours and in the kidney tissue, 2.6
fmol/mg of protein.24,25 Evidence shows that little circulating
ET-1 is excreted into the urine, and the most urinary ET is
renal in origin.26 The preparation used in the present study,
namely, renal pelvis perfusion, allows for renal afferent nerve
exposure to perfused drugs, similar to urine. It has been
shown that affinity of 4 Ala-ET-1 binding to ETB is 1700
times higher than that binding to ETA.27 ET-1 induces
vasoconstriction with EC50 10⫺9 M, which is abolished by the
ETA antagonist, whereas 4 Ala-ET-1 with a concentration
ⱕ10⫺6 M has no effect.28 These data indicate that 4 Ala-ET-1
is unlikely to activate the ETA receptor at a concentration
⬍10⫺6 M.27,28 Furthermore, ET-1 incubated with cultured
Xie and Wang
TRPV1 and ETB-PKC Pathway
1303
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Figure 8. Immunofluorescence single or
double labeling of TRPV1 and ETA
receptors (A) or TRPV1 and ETB receptors (B) in the renal pelvis of WT or
TRPV1⫺/⫺ mice. TRPV1 was labeled
with fluorescein isothiocyanate (green
fluorescence, arrows) in the left column,
whereas the ETA or ETB receptors were
labeled with Cy3 (red fluorescence,
arrows) in the middle column. The overlay of TRPV1 and ETA or ETB is shown
in the right column (yellow staining,
arrows). The controls by preabsorption
of primary antibodies are negative (not
shown). The bar in A represents 100 ␮m
for all of the panels.
juxtaglomerular cells (IC50 3⫻10⫺9 M) inhibits renin release,
an effect that is mimicked by 10⫺6 M 4 Ala-ET-1 but not
affected by BQ123.29 Taken together, these results provide
rationales for the selection of doses of ET-1 and 4 Ala-ET-1
used for renal pelvis perfusion that ensures effectiveness and
avoids nonspecific binding.
All of the components of the ET-1 system, including ET-1,
ETA, and ETB, have been found to express in the sensory
nervous system.11,30 Ablation of ET-1 leads to an elevation in
the resting renal sympathetic nerve activity (RSNA), and an
attenuation in hypercapnia-induced increases in RSNA,31
suggesting that endogenous ET-1 governs the basal and reflex
controls of RSNA. Moreover, ET-1 injected into the hind paw
of rats induces pain that is transmitted by sensory nerve fibers
expressing ETA and ETB, which play distinct roles in
mediating the pain pathway.32,33 The ETA-PKC pathway
contributes to ET-1–induced thermal hyperalgesia.14 In contrast, the ETB-PKC pathway contributes to ET-1–mediated,
mechanical-induced hypernociception.33 It has been shown
that activation of ETA increases RSNA, whereas activation of
ETB inhibits RSNA.34,35 In contrast, activation of ETA
expressed in the renal pelvis suppresses ARNA in low-salt–
treated rats, whereas activation of ETB enhances ARNA in
high-salt–fed rats.12,36 These studies indicate that the ET
system also plays a key role in the control of sensory nerve
function and function of organs/tissues innervated by sensory
nerves. Our data show that ET-1 perfused into the renal pelvis
increases ARNA in WT but not in TRPV1⫺/⫺ mice, indicating that ET-1–induced increases in ARNA require the pres-
ence or activation of TRPV1. In addition, activation of ETB
increases ARNA, and inhibition of ETA potentiates ET-1–
induced increases in ARNA in WT but not in TRPV1⫺/⫺
mice. These results indicate that TRPV1 mediates ETBinduced increases and ETA-induced suppression of ARNA.
ET-1 may modulate renal sodium and urine excretion,17,37,38 and this effect may be mediated by interaction
with renal nerves.37 An ETA and ETB antagonist, bosentan,
given into the kidney causes a reduction in urine flow in both
normal and hypertensive rats, whereas bosentan-mediated
decreases in renal excretory function are abolished after renal
denervation,37 indicating a role for ET-1 receptors expressed
in renal nerves in the regulation of renal function. Furthermore, decreased expressions of ET-1, ETA, and ETB have
been found in the kidney of spontaneously hypertensive rats,
where downregulation of ETB may contribute to excessive
sodium retention in spontaneously hypertensive rats.37 Indeed, ETB in the kidney is involved in ET-1–induced
inhibitory effects on antidiuresis.39 The ETB agonist, sarafotoxin, given into the kidney causes enhanced diuresis in
anesthetized dogs, whereas excretion of sodium and the
glomerular filtration rate remain unchanged.38 Activation of
ARNA also results in diuresis and natriuresis.40 It has been
shown that increased renal pelvis perfusion pressure leads to
activation of ipsilateral ARNA, which causes an inhibitory
renorenal reflex and leads to diuresis and natriuresis via suppression of contralateral renal sympathetic nerve activity.40,41 Our
previous data show that activation of TRPV1 by CAP
perfused into the unilateral renal pelvis leads to activation of
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December 2009
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ipsilateral ARNA and bilateral diuresis and natriuresis via the
renorenal reflex.5,6 Our data in the present study show that
activation of ETB increases ARNA in WT but not in
TRPV1⫺/⫺ mice, whereas blockade of ETB has no effect on
CAP-induced increases in ARNA in WT mice. Taken together, these data indicate that TRPV1 mediates ETBdependent increases in ARNA induced by ET-1 and thereby
contributes to ETB-induced increases in sodium and water
excretion.
The primary sequence of TRPV1 contains many putative
phosphorylation sites, and PKC- and PKA-mediated phosphorylation of TRPV1 is critical for its functions.14,42– 44
PKC-mediated phosphorylation of TRPV1 has been shown to
increase TRPV1-mediated effects.14,42– 44 Activation of PKC
potentiates or sensitizes TRPV1 responses to heat, protons, or
its agonists and increases TRPV1-mediated SP and calcitonin
gene-related peptide release.43,44 PKC also participates in
ET-1–induced pain sensation.14,33 Previous data show that
activation of ETB by ET-1 leads to hypernociception induced
by mechanical stimulation via activation of the PKC pathway.33 Our data in the present study show that the PKC
inhibitor but not the PKA inhibitor perfused into the renal
pelvis abolishes ETB-induced increases in ARNA, whereas
CAP-induced increases in ARNA are not affected by the PKC
or PKA inhibitors. These data indicate that the ETB-PKC
pathway mediates the ET-1 effect on activation of TRPV1
and ARNA.
SP release has been shown to be regulated by several
factors,42,45 and mechanoinduced increases in ARNA and SP
release are abolished when NK1 receptors45 or TRPV1
channels8 are blocked. Our previous data show that TRPV1induced increases in ARNA and renal excretory function
depend on NK1 receptor activation by SP on its release.6 Data
in the present study show that SP release is elevated when
ETB is activated in WT but not in TRPV1⫺/⫺ mice, which is
abolished by the PKC but not PKA inhibitors, whereas
neither the PKC nor PKA inhibitors affect CAP-induced
increases in SP release. Taken together, these data indicate
that TRPV1 mediates ETB-induced increases in SP release
that are PKC dependent.
TRPV1 is mainly expressed in small- and medium-sized
neurons in dorsal root and trigeminal ganglia and is transported to both the central and peripheral terminals of these
primary afferent neurons.1 TRPV1 has been found primarily
in unmyelinated C-fibers or thinly myelinated A␦-fibers in
the periphery.1 TRPV1-containing sensory nerves heavily
innervate the upper ureter, the pelvis wall presenting in
between uroepithelial and smooth muscle layer, and in the
tubular cells of distal tubules and collecting ducts in the
cortex and medulla.4,6,8 The data in the present study show
that TRPV1 expresses in nerve fibers innervating the epithelial layer of the renal pelvis wall in WT but not in TRPV1⫺/⫺
mice. Similarly, both ETA and ETB have been found in
medium- to large-sized neurons in the trigeminal ganglia.11 In
the kidney, ETA that mainly locates in glomeruli and renal
vasculature15,46,47 mediates the cortical and medullary vasoconstriction.15,46 ETB that is mainly distributed in the renal
tubules contributes to ET-1–induced water and sodium excretion.15,46 Our immunofluorescence data show that ETB,
colocalized with TRPV1, expresses in nerve fibers innervating the renal pelvis wall in both WT and TRPV1⫺/⫺ mice.
The reason that ETA staining was undetectable is unknown at
the present time. However, it is likely that the staining
method was not sensitive enough for detecting low abundance of ETA expressed in the renal pelvis wall in WT and
TRPV1⫺/⫺ mice.15,46,47
In conclusion, the data in the present study show that
deletion of TRPV1 abolishes ET-1–induced increases in
ARNA and SP release that is via activation of ETB, whereas
activation of ETA plays a counterbalancing role. Moreover,
ETB-induced activation of TRPV1 is mediated through a
PKC but not a PKA pathway. These findings indicate for the
first time that TRPV1 may govern or contribute to ETBmediated control of water and sodium excretion in the kidney.
Perspectives
TRPV1-containing sensory nerves, when activated, regulate
diuresis and natriuresis of the kidney.5 Neonatal degeneration
of this population of sensory nerves leads to increased salt
sensitivity and arterial pressure.48 Similarly, ET-1 also plays
an important role in regulating renal excretory function
through activation of the ETB receptors.49 Collecting ductspecific knockout of the ETB receptors causes sodium retention and hypertension.18 The data in the present study indicate
that TRPV1 colocalizes with ETB in sensory nerve fibers
innervating the renal pelvis, mediates ETB-induced increases
in ARNA, and, therefore, may contribute to ETB-governed
renal excretory function that is sensory nerve dependent. The
molecular interaction of ETB and TRPV1 depends on activation of the PKC pathway. On the other hand, activation of
ETA conveys an inhibitory role, which counterbalances ETB
stimulatory action, in the regulation of ET-1–induced increases in ARNA. Thus, it is conceivable that, in the cases
when single or multiple dysfunction(s) of components in the
ETA- or ETB-PKC-TRPV1 pathway in sensory nerves innervating the kidney occur, they may lead to impaired renal
sensory nerve function and disturbed sodium and water
homeostasis, as depicted in Figure 1.
Sources of Funding
This study was supported by the National Institutes of Health (grants
HL-57853, HL-73287, and DK67620) and a grant from the Michigan
Economic Development Corporation.
Disclosures
None.
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Ablation of Transient Receptor Potential Vanilloid 1 Abolishes Endothelin-Induced
Increases in Afferent Renal Nerve Activity: Mechanisms and Functional Significance
Chaoqin Xie and Donna H. Wang
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Hypertension. 2009;54:1298-1305; originally published online October 26, 2009;
doi: 10.1161/HYPERTENSIONAHA.109.132167
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