Am J Physiol Renal Physiol 299: F1401F1406, 2010.

First published September 22, 2010; doi:10.1152/ajprenal.00322.2010.

Protein kinase C regulates urea permeability in the rat inner medullary

collecting duct
Yanhua Wang,1 Janet D. Klein,1 Carole M. Liedtke,2 and Jeff M. Sands1
1

Department of Medicine, Renal Division, Emory University, Atlanta, Georgia; and 2Departments of Pediatrics
and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio
Submitted 3 June 2010; accepted in final form 19 September 2010

Wang Y, Klein JD, Liedtke CM, Sands JM. Protein kinase C

regulates urea permeability in the rat inner medullary collecting duct.
Am J Physiol Renal Physiol 299: F1401F1406, 2010. First published
September 22, 2010; doi:10.1152/ajprenal.00322.2010.Hypertonicity increases urea transport independently of, as well as synergistically
with, vasopressin in the inner medullary collect duct (IMCD). We
previously showed that hypertonicity does not increase the level of
cAMP in the IMCD, but it does increase the level of intracellular
calcium. Since we also showed that hypertonicity increases both the
phosphorylation and biotinylation of the urea transporters UT-A1 and
UT-A3, this would suggest involvement of a calcium-dependent
protein kinase in the regulation of urea transport in the inner medulla.
In this study, we investigated whether protein kinase C (PKC), which
is present in the IMCD, is a regulator of urea permeability. We tested
the effect of PKC inhibitors and activators on urea permeability in the
isolated, perfused rat terminal IMCD. Increasing osmolality from 290
to 690 mosmol/kgH2O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either
10 M chelerythrine or 50 M rottlerin. To determine the potential
synergy between vasopressin and PKC, phorbol dibutyrate (PDBu)
was used to stimulate PKC. Vasopressin stimulated urea permeability
247%. Although PDBu alone did not change basal urea permeability,
in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea
permeability was reduced to AVP alone levels by inhibition of PKC.
We conclude that hypertonicity stimulates urea transport through a
PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of
a cascade of activations remains to be determined.
tubule perfusion; calcium signaling; urine concentration; vasopressin;
hypertonicity
UREA AND UREA TRANSPORTERS (UT) are crucial for the urinary
concentrating mechanism. Since the first observation that urea
is important in the control of water homeostasis by Gamble
et al. in 1934 (6), many studies have reported the integral role
of urea in the production of a concentrated urine. Early studies
looked at the effect of varying the protein level in the diets of
animals and people (5, 6, 12). The most rigorous method of
assessing urea transport is to perform ex vivo perfusion studies
to directly measure the movement of urea across the inner
medullary collecting duct (IMCD). Tubule perfusion studies
established that there are UT involved in the movement of urea
through the IMCD cells (20). The transporters perform at both
basal and stimulated levels. The regulation of these transporters is an important determinant in the permeability of the
IMCD to urea. Studies show that urea permeability can be

Address for reprint requests and other correspondence: Y. Wang, Emory

Univ. School of Medicine, Renal Div., 1639 Pierce Dr., NE, WMB Rm. 3304,
Atlanta, GA 30322 (e-mail: ywang68@emory.edu).
http://www.ajprenal.org

affected by vasopressin, angiotensin II, calcium, steroid hormones, and changes in tonicity (8, 14, 20, 21).
One of the earliest observations made using tubule perfusion
studies was that vasopressin is the primary hormone that
regulates urea permeability in the IMCD (20, 24). Vasopressin
binds to the V2 receptors on the basolateral plasma membrane
of the IMCD, which results in stimulation of adenylyl cyclase
and generation of cAMP. cAMP stimulates two downstream
signals: protein kinase A and exchange protein activated by
cAMP (25), thereby resulting in an increase in urea permeability.
Vasopressin stimulation of urea permeability is due, in large
part, to the stimulation of the UT-A1 and UT-A3 urea transporters in the IMCD (14). These urea transporters are members
of the SLC14A2 family. UT-A1 is the largest of the urea
transporters and is located at the apical plasma membrane
of the IMCD. UT-A3 is essentially the NH2-terminal half of
UT-A1 and is located at the basolateral plasma membrane of
the IMCD (18). UT-A3 is also present in the apical plasma
membrane when the animal has been treated with vasopressin,
suggesting that UT-A3, as well as UT-A1, is regulated by
vasopressin (3). The mechanism for vasopressins regulation
involves vasopressin-stimulated increases in both the phosphorylation and apical plasma membrane accumulation of the
UT-A1 urea transporter (3, 4, 27).
Vasopressin is not the only regulatory stimulus for urea
transport, however. Several studies show that urea permeability
in isolated, perfused IMCDs is also activated by hypertonicity
(adding NaCl or mannitol) in the absence of vasopressin (7, 15,
21). Increasing osmolality in the presence of vasopressin produces an additive effect on urea permeability (7, 15, 21).
Unlike vasopressin, hypertonicity increases intracellular calcium, but does not change cAMP accumulation (8). These
findings indicate that hypertonicity is an independent activator
of urea transport, and it acts through intracellular calcium
instead of cAMP. Hypertonicity increases the phosphorylation
and plasma membrane accumulation of UT-A1 and UT-A3,
which may result in an increase in urea permeability (2). The
manner by which hypertonicity stimulates the urea transporters
or urea permeability is not yet understood.
The present study extends information about PKC signaling
on urea permeability. We first determined whether PKC is
involved in hypertonicity-stimulated urea permeability. The
primary rationale for performing this aspect of our study is that
hypertonicity increases urea permeability via changes in intracellular calcium, suggesting involvement of a calcium-dependent protein kinase in the urea transport response. Next, we
explored the mechanisms for PKC regulation and tested the
hypothesis that PKC synergistically works with cAMP to
stimulate urea permeability.

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PKC REGULATES UREA PERMEABILITY

METHODS

Animals. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male SpragueDawley rats (Charles River Laboratories, Wilmington, MA), weighing
50 75 g, were injected with furosemide (5 mg/kg body wt) 30 min
before death. Kidneys were dissected to remove inner medullas.
Single tubules from the terminal IMCD were microdissected at 17C.
Tubule perfusion. Single IMCDs were dissected, mounted on glass
pipettes, and perfused as described previously (19, 20). In general, 45
min after the tubules were warmed to 37C in 1 ml of bath solution,
three 1-min collections of 30 50 nl/min were made.
To measure the response to treatments, buffers were changed or
treatments were added to the bath, tubules were allowed to equilibrate
for 15 min, then three further collections were made, and urea was
assessed using ultramicrofluorometry. Urea permeability was calculated as described previously (19, 20).
To subject the tubules to hypertonic conditions, the osmolalities of
both the perfusate and the bath were increased from 290 to 690
mosmol/kgH2O by adding 200 mM NaCl. To assess the contribution
of PKC, cell-permeable PKC inhibitors (10 M chelerythrine or 50
M rottlerin) were added to the bath. Alternatively, the PKC stimulator phorbol-dibutyrate (PDBu; 200 nM) was added to the bath.
To test for synergistic effects of cAMP and PKC on urea permeability, basal urea permeability was measured followed by sequential
addition of vasopressin and PDBu. First, 200 pM vasopressin was
added to the bath and urea permeability was measured. Next, 200 nM
PDBu was added to the bath in the presence of vasopressin, and urea
permeability was measured. In an additional approach, both 200 pM
vasopressin and 200 nM PDBu were added simultaneously to the bath
45 min after the tubule was warmed to 37C. After 15 min, urea
permeability was measured. To verify that the effects observed with
simultaneous PDBu/vasopressin treatment were due to PKC, 10 M
chelerythrine was added to the bath, and urea permeability was
measured.
Western blot analysis. Identical samples of whole inner medullary
protein lysate (20 g/lane) were size separated by SDS-PAGE on
10% gels and then electroblotted to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA). Blots were blocked
with 5% nonfat dry milk in Tris-buffered saline (20 mM TrisHCl, 0.5
M NaCl, pH 7.5) and then incubated with primary antibodies overnight at 4C. Attached primary antibodies were identified using Alexa
Fluor 680-linked anti-rabbit IgG (Molecular Probes, Eugene, OR) and
visualized using infrared detection with the LICOR Odyssey protein
analysis system (Lincoln, NE). Antibodies to PKC , , , , , , and
were purchased from Cell Signaling Technology (Danvers, MA).

Statistics. All data are presented as means SE. Data from tubule
perfusion studies were analyzed using a paired Students t-test or
repeated-measures ANOVA, depending on whether two conditions or
more than two conditions were assessed in each tubule (22). Each
tubule serves as its own control, allowing a paired analysis of the data.
The criterion for statistical significance is P 0.05.
RESULTS

Urea permeability in hypertonic conditions. Increasing the

osmolality of the perfusate and bath solutions from 290 to 690
mosmol/kgH2O resulted in a significant increase in urea permeability from 24 2 to 49 4 105 cm/s (n 4, P
0.01; Fig. 1A). The increased urea permeability was significantly reduced to 30 3 105 cm/s by addition of the PKC
inhibitor chelerythrine (10 M) to the hypertonic bath solution
(n 4, P 0.01; Fig. 1A). The bar graph in Fig. 1B shows the
urea permeabilities as a percent of control levels. Hypertonicity
increased urea permeability to 208% of the level in isotonic
conditions. Urea permeability was returned to nearly control
levels (129%) by inhibition of PKC with chelerythrine.
To further confirm the role of PKC in the regulation of urea
permeability, 50 M rottlerin (a second chemical inhibitor of
PKC that inhibits a wide spectrum of PKCs at 50 M) was
added to the hypertonic bath. As before, hypertonicity increased urea permeability from 23 2 to 43 3 105 cm/s
(n 4, P 0.01; Fig. 2A). Addition of rottlerin significantly
decreased urea permeability to 21 2 105 cm/s (n 4,
P 0.01; Fig. 2A). The changes in permeability as percent of
control are presented in bar graph form in Fig. 2B. Hypertonicity increased urea permeability by 191%. Urea permeability
was returned to nearly control levels (90%) by inhibition of
PKC with rottlerin.
Urea permeability stimulated by PDBu. To verify the effects
of the PKC inhibitors, we measured urea permeability when
PKC was stimulated using a phorbol ester, PDBu (200 nM),
which is known to stimulate the regulatory domain of PKC
(16). The urea permeability after PDBu treatment (32 3
105 cm/s) was not different from basal urea permeability (29
1 105 cm/s, n 4).
Three approaches were designed to test for synergistic effects of PDBu and vasopressin. First, urea permeabilities were

Fig. 1. Urea permeability in terminal inner medullary collecting ducts (IMCDs) was increased
by hypertonicity. A: terminal IMCDs were perfused with isotonic buffer (290 mosmol/kgH2O),
then switched to hypertonic buffer (690 mosmol/
kgH2O), and then supplemented with 10 M
chelerythrine (Chel). Each line represents a separate IMCD from a different rat. B: bar graph
shows the percent change from isotonic control
levels. Bars means SE. *P 0.01 vs. 290
mosmol/kgH2O; #P 0.01 vs. 690 mosmol/
kgH2O without inhibitor; n 4 rats/condition.

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Fig. 2. Hypertonicity-induced increase in urea

permeability was reversed by inhibition of protein kinase C (PKC) with rottlerin (Rott).
A: terminal IMCDs were perfused with isotonic buffer (290 mosmol/kgH 2 O), then
switched to hypertonic buffer (690 mosmol/
kgH2O), and then supplemented with 50 M
Rott. Each line represents a separate IMCD
from a different rat. B: bar graph showing
the percent change from isotonic control
levels. Bars means SE. *P 0.01 vs.
290 mosmol/kgH2O; #P 0.01 vs. 690
mosmol/kgH2O without inhibitor; n 4
rats/condition.

assessed under conditions where vasopressin-stimulated

IMCDs were then treated with a PKC stimulant. Figure 3
shows this approach. Vasopressin significantly increased urea
permeability from 21 2 to 74 16 105 cm/s (n 6,
P 0.05; Fig. 3A). Subsequent addition of 200 nM PDBu to
the bath (to stimulate PKC with vasopressin present) further
increased urea permeability to 94 18 105 cm/s (n 5,
P 0.05; Fig. 3A). The changes in permeability as percent of
control are presented in bar graph form in Fig. 3B. Vasopressin
increased urea permeability from 100% (basal control) to
347%. Urea permeability was further increased to 439% by
stimulation of PKC with PDBu. We also performed the time
control experiment where vasopressin was added after the
initial 45-min warming period (basal readings), and urea permeability was monitored periodically over an additional 45
min. Within 15 min following addition of vasopressin to the
bath, urea permeability increased from 15 1 to 60 7
105 cm/s, where it was maintained for an additional 30 min
(data not shown).
The second approach was to stimulate urea permeability by
adding vasopressin and PDBu together, and then assess
whether inhibition of PKC removed the stimulation of urea
permeability. The tubules were treated initially with both 200
pM vasopressin and 200 nM PDBu and urea permeability was
measured (80 8 105 cm/s, n 4; Fig. 4A). To verify

that the effects observed with simultaneous PDBu/

vasopressin treatment include a PKC-mediated stimulation,
10 M chelerythrine was added to the bath. Urea permeability significantly decreased to 58 6 105 cm/s (n
4, P 0.01 by paired t-test; Fig. 4A). The changes in
permeability as percent of control are presented in bar graph
form in Fig. 4B. Chelerythrine decreased urea permeability
from 100% (vasopressinPDBu control) to 72%. To ensure
that the changes observed with chelerythrine were not due to
the prolonged exposure to PDBu, we performed a time
control in which PDBu and vasopressin were both present in
both periods. Urea permeability was 72 7 105 cm/s in
period 1 and showed a slight increase to 84 4 105
cm/s in period 2 (n 3, P NS).
The third approach to determining the potential synergy
between vasopressin and PKC was to determine whether inhibition of PKC would block the action of PDBu on urea
permeability. To ensure that chelerythrine was inhibiting by
blocking PKC and not directly affecting vasopressin-mediated
urea permeability, tubules were first treated with 200 pM
vasopressin, then incubated with 10 M chelerythrine, and
finally treated with 200 nM PDBu. Chelerythrine significantly
decreased urea permeability from 61 6 to 53 7 105
cm/s in the IMCDs initially treated with 200 pM vasopressin
alone (n 4, P 0.05; Fig. 5A). Subsequent addition of 200

Fig. 3. PKC stimulates urea permeability

above cAMP-stimulated levels. A: perfused
IMCDs after 45 min with buffer without
stimulants (basal), then with 200 pM vasopressin (AVP) added to the bath for 15 min,
and finally with 200 nM phorbol dibutyrate
(PDBu) added to the bath for a further 15
min. Each line represents a separate IMCD
from a different rat. B: bar graph showing
the percent change from basal control levels.
Bars means SE. *P 0.05 vs. basal
(unstimulated) conditions; #P 0.05 vs.
AVP-stimulated levels; n 5 separate rat
terminal IMCDs.

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Fig. 4. Stimulation of urea permeability by AVP

and PDBu is decreased on inhibition of PKC with
Chel. A: IMCDs were perfused with buffer containing both 200 pM AVP and 200 nM PDBu for
15 min and then treated for an additional 15 min
with 10 M Chel. Each line represents a separate
IMCD from a different rat. B: bar graph showing
the percent change in urea permeability on inhibition of PKC. Bars means SE. *P 0.01 vs.
AVP PDBu, n 4 rat terminal IMCDs.

nM PDBu (with vasopressin and chelerythrine present) to the

bath did not increase the urea permeability. Instead, urea
permeability continued to decrease to 43 6 105 cm/s
(n 4, P 0.01; Fig. 5A). The changes in urea permeability
as percent of control are presented in bar graph form in Fig. 5B.
Urea permeability was decreased from 100% (vasopressin
control) to 87% by addition of chelerythrine, and continued to
decrease to 71% during the 15 min after addition of PDBu.
To ensure that the decrease in urea permeability was not due
to an effect of the PKC inhibitor that was greater during the
third compared with the second set of collections, we performed a time control in which PDBu and vasopressin were
both present in periods 2 and 3. Urea permeability was 84
4 105 cm/s in period 2 and 90 2 105 cm/s in period
3 (n 3, P NS).
Effect of PKC inhibition under isotonic conditions. To examine the effect of PKC antagonists on urea permeability in the
absence of hypertonicity, we perfused tubules using isotonic
(290 mosmol/kgH2O) solutions. The PKC inhibitor chelerythrine (10 M) did not change urea permeability (basal: 15 1,
chelerythrine: 12 1 105 cm/s, n 3, P NS; Fig. 6A).
Subsequent addition of vasopressin (200 pM) with chelerythrine still present did not change urea permeability (13 1
105 cm/s, n 3, P NS; Fig. 6B).

PKC isoforms in kidney inner medulla. The identity of the

PKC isoform that is involved in the increase of urea permeability in the inner medulla is unknown. There are several PKC
isoforms that are expressed in the inner medulla (Fig. 7)
including conventional PKC isoforms that are responsive to
calcium and diacylglycerol (PKC, PKC, and PKC), as well
as novel isoforms (PKC, PKC, PKC, and PKC) which are
calcium independent, and the atypical isoform, PKC. Of
these, both conventional and novel PKC isoforms are responsive to phorbol ester activation and thus would be potential
activators of urea permeability in the inner medulla.
DISCUSSION

It is well-established that urea permeability is regulated by

vasopressin via cAMP-dependent signaling pathways. This
study provides evidence that hypertonicity increases urea permeability through a PKC-signaling pathway. In addition, direct
activation of PKC by a phorbol ester, PDBu, requires the
synergy of vasopressin to stimulate urea permeability. Together with findings from previous studies (14, 27), the present
study suggests that maximal increases in urea transport in rat
terminal IMCDs require stimulation of both cAMP and PKCsignaling pathways.

Fig. 5. Inhibition of PKC blocks stimulation of

urea permeability by phorbol esters. A: IMCDs
were stimulated with 200 pM AVP for 15 min,
then 10 M Chel was added for 15 min, and
finally 200 nM PDBu was added for 15 min.
Each line represents a separate IMCD from a
different rat. B: bar graph showing the percent
change in AVP-stimulated urea permeability on
inhibition of PKC with Chel, and then stimulation of PKC with PDBu. Bars means SE.
*P 0.05 vs. AVP; #P 0.01 vs. AVP Chel;
n 4 rat terminal IMCDs.

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Fig. 6. Chel does not inhibit urea permeability

under isotonic conditions and AVP fails to increase urea permeability in the presence of Chel.
A: IMCDs were equilibrated at 290 mosmol/
kgH2O, then 10 M Chel was added for 15 min,
and finally 200 pM AVP was added for 15 min.
Each line represents a separate IMCD from a
different rat. B: bar graph showing the percent
change in urea permeability on inhibition of PKC
with Chel, and then stimulation of PKC with
AVP. Bars means SE. P NS; n 3 rat
terminal IMCDs.

Previous studies show that hypertonicity increases urea permeability (21) and plasma membrane accumulation of both
UT-A1 and UT-A3 in rat terminal IMCDs (2). The responses
to hypertonicity occur through increases in intracellular calcium and activation of PKC has been suggested to play a role
(8, 14). To determine the role of PKC in hypertonicity-stimulated urea permeability, hypertonicity (690 mosmol/kgH2O)
was used as a means to stimulate PKC in the present study. Our
data show that hypertonicity significantly increases urea permeability (Figs. 1 and 2), which is consistent with a previous
study (21). To verify that the increase in urea permeability is in
response to activation of PKC, two PKC inhibitors, chelerythrine and rottlerin, were used to decrease urea permeability.
Chelerythrine acts on the catalytic domain of PKC and is a
broad-type inhibitor of PKC. Rottlerin is a PKC-specific
inhibitor. However, at the higher concentration (50 M) used
in this study, rottlerin also inhibits a broad spectrum of PKC
isoforms, including PKC, PKC, PKC, and PKC, and
PKC, PKC, and PKC with a reduced inhibitory activity
(9 11, 23). The data in the present study show that both PKC
inhibitors reversed the increase by hypertonicity to nearly basal
levels of urea permeability (Figs. 1 and 2), which strongly
supports the concept that hypertonicity increases urea permeability by stimulating PKC.
To understand the mechanism by which the PKC pathway
regulates urea permeability, we also used the phorbol ester
PDBu to directly stimulate PKC. PDBu binds to the regulatory
domain of PKC and thereby activates it (16). The present study
shows that PDBu alone does not change urea permeability.
However, PDBu significantly increased vasopressin-stimulated

urea permeability (Fig. 3). These data suggest that an interaction between vasopressin and PKC is necessary for PKC to
stimulate urea permeability.
Angiotensin II has been shown to exert its effects through
PKC-mediated pathways. Liu and Cogan (17) showed that
PKC may be involved in as much as one-third of the angiotensin II-stimulated bicarbonate and water transport in the S1
and S2 segments of the proximal tubule. Amlal et al. (1)
reported that a high concentration (5 1011 M) of angiotensin II stimulates Na-K-NH4-2Cl cotransport via PKC
in the medullary thick ascending limb. In rat IMCDs, angiotensin II significantly increases urea permeability above vasopressin-stimulated levels (14), similar to the effects observed in
the present study for PDBu. Angiotensin II has also been
shown to increase UT-A1 phosphorylation in rat IMCD suspensions (14). The angiotensin II-mediated increases in function and phosphorylation can be blocked by a PKC inhibitor
(14), suggesting a role for PKC-mediated signaling in the
regulation of urea transport.
Interestingly, our previous data showed that angiotensin II
alone, which can activate PKC in several renal tubule segments
(1, 17), had no effect on basal (no added vasopressin) urea
permeability (14). However, angiotensin II significantly increased vasopressin-stimulated urea permeability (14). The
comparable pattern of regulation by PDBu and by angiotensin
II adds more credence to the hypothesis that PKC is involved
in regulation of urea permeability by supplementing or enhancing vasopressin-stimulated urea permeability. The increase in
urea permeability observed with simultaneous PDBu/vasopressin treatment was significantly reversed by chelerythrine, con-

Fig. 7. Seven PKC isoforms are present in rat

inner medulla. Lysate from whole rat inner
medulla was analyzed by Western blot using
PKC isoform-specific antibodies. An arrow
identifies the PKC protein bands.

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sistent with PKC-mediated stimulation (Fig. 4). Finally, after

PKC was blocked with chelerythrine, PDBu treatment did not
increase vasopressin-stimulated urea permeability (Fig. 5). The
agreement of the inhibitor and stimulator results strongly
supports the idea of an interaction between vasopressin and
PKC.
Recently, Yano et al. (26) demonstrated that glucagon reduces urea permeability and UT-A1 expression via a glucagon
receptor and by stimulating PKC with phorbol myristate. This
differs from our finding where stimulating PKC with PDBu
had no effect on basal urea permeability. Their finding also
differs from Isozaki et al. (13) who found no effect of glucacon
on urea permeability. We do not have an explanation for these
differences, but possibilities include the use of different rat
strains (Wistar by Yano et al. vs. Sprague-Dawley by Isozaki
et al. and present study) and different agonists (phorbol myristate vs. PDBu).
We do not know why hypertonicity independently initiates
PKC activation in the absence of overt vasopressin stimulation.
It is possible that it only requires the endogenous vasopressin
level to support the action of PKC in response to hypertonicity.
In light of this hypertonic response, it is unclear why stimulation of PKC with PDBu or angiotensin II (14) does not show
any stimulation of urea permeability in the absence of additional exogenous vasopressin. Perhaps hypertonicity activates
different PKC isoform(s) than PDBu or angiotensin II, is a
better activator, or stimulates additional signaling pathways?
Further studies are needed to test these possible mechanisms.
Clearly, however, in the presence of vasopressin, PDBu stimulation of PKC causes a significant increase in urea permeability.
In conclusion, the findings from the present study show that
PKC regulates urea permeability in conjunction with vasopressin. These findings are likely to be physiologically significant.
During antidiuresis, the inner medulla is often quite hypertonic.
Under antidiuretic conditions, therefore, the increase in tonicity
may be instrumental in increasing urea permeability through
PKC-signaling pathways, in addition to the actions of vasopressin. We conclude that maximal stimulation of urea permeability in rat terminal IMCDs requires stimulation by both
cAMP and PKC. Whether PKC directly phosphorylates UT-A1
and/or UT-A3, or phosphorylates as a consequence of a cascade of activations, remains to be determined.
GRANTS
This work was supported by National Institutes of Health Grants R01-DK41707, R01-DK-62081, and P01-DK-61521.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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