Am J Physiol Renal Physiol 304: F103–F111, 2013.
First published November 7, 2012; doi:10.1152/ajprenal.00385.2012.
Urine concentration in the diabetic mouse requires both urea and water
transporters
Titilayo O. Ilori, Mitsi A. Blount, Christopher F. Martin, Jeff M. Sands, and Janet D. Klein
Renal Division, School of Medicine, Emory University, Atlanta, Georgia
Submitted 10 July 2012; accepted in final form 31 October 2012
urea transporter-A1/A3 knockout; diabetes; aquaporin-2; phosphorylated serine-256-aquaporin-2; phosphorylated serine-486-urea transporter-A1; vasopressin
are two main transporter families
that contribute to producing concentrated urine: aquaporins
(AQP) and urea transporters (UT). The collecting duct is unable
to reabsorb water in the absence of vasopressin (AVP), but the
permeability to water increases significantly in the presence of
AVP (14). AVP binds to V2 receptors in the basolateral membrane of collecting duct principal cells. Through a G proteincoupled pathway, adenylyl cyclase is stimulated, producing an
increase in cAMP. The cAMP-dependent protein kinase A
(PKA) phosphorylates AQP2, leading to its insertion in the
apical plasma membrane of collecting duct principal cells. This
leads to water reabsorption from the tubular lumen into the
inner medullary tissue. Therefore, AVP regulates water reabsorption by regulating trafficking of AQP2 between subapical
vesicles and the apical plasma membrane (16).
In the inner medullary collecting duct (IMCD), UT-A1
moves to the apical plasma membrane in response to AVP
IN THE COLLECTING DUCT, THERE
Address for reprint requests and other correspondence: T. Ilori, Emory Univ.
Renal Division, 338 Woodruff Memorial Bldg., 1639 Pierce Drive, Atlanta,
GA 30322 (e-mail: [email protected]).
http://www.ajprenal.org
(18). Similar to the mechanism by which AQP2 is activated,
AVP phosphorylates UT-A1 at serines-486 and -499 through
PKA (2). Phosphorylated UT-A1 moves to the apical plasma
membrane and causes reabsorption of urea from the tubular
lumen and, in conjunction with UT-A3, moves it into the
medullary interstitium. This results in an increase in the concentration of the medullary interstitium, ultimately resulting in
reabsorption of water (11). Therefore, the ability to concentrate
urine requires coordinated actions by AQP and UT. Both
mechanisms are AVP and phosphorylation dependent. AVP
increases the phosphorylation of AQP2, promoting apical
membrane insertion and increased water reabsorption. AVP
also promotes UT-A1 phosphorylation, membrane accumulation, and increased urea reabsorption.
Regulation of AQP2 is believed to be controlled by membrane trafficking, which involves phosphorylation at various
serines. There are four serines in the carboxyl terminal chain
that have been identified: serines-256, -261, -264, and -269 (6,
7, 9). Although they are all AVP sensitive, they have different
modes of action. Phosphorylation at serines-256, -264, and
-269 is increased in response to AVP. whereas phosphorylation
at serine-261 decreases in response to AVP (7–9). While we
know that phosphorylation of UT-A1 promotes its membrane
insertion, we do not know if diabetes affects phosphorylation
or membrane insertion of UT-A1. It is also unclear whether the
level of activity of AQP2 affects UT-A1 phosphorylation in the
diabetic animal.
Diabetes is a global health problem that affects multiple
organ systems. In the kidney, diabetes is known to cause
proteinuria, nephrotic syndrome, and may ultimately result in
renal failure. Diabetic patients also present with polyuria and a
reduced urine concentrating ability. Patients with poorly controlled diabetes are polyuric and develop a concentrating defect, but, with strict glycemic control, urine concentration
improves with better salt and water conservation. However,
under strenuous conditions such as extremes of temperature or
lack of access to hydration, diabetics become more susceptible
to developing a concentrating defect. It seems unlikely that
deficiencies in AVP are responsible for the concentrating
defect in diabetes since AVP levels are not suppressed in the
diabetic animal (10). However, AVP does increase urea permeability in diabetic rats (18). Diabetic rats are able to maintain urine osmolality (850 –900 mosmol/kgH2O) above plasma
levels despite polyuria. This ability has been attributed, at least
in part, to the upregulation of both UT-A1 and AQP2 protein
abundances (1, 10, 15). However, the degree to which each of
these proteins contributes to the maintenance of urine osmolality is unknown.
Our objective in this study was initially to verify that both
AQP and UT are needed to produce concentrated urine using
UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice. We further
1931-857X/13 Copyright © 2013 the American Physiological Society
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Ilori TO, Blount MA, Martin CF, Sands JM, Klein JD. Urine
concentration in the diabetic mouse requires both urea and water
transporters. Am J Physiol Renal Physiol 304: F103–F111, 2013. First
published November 7, 2012; doi:10.1152/ajprenal.00385.2012.—
The regulation of the inner medullary collecting duct (IMCD) urea
transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their
interactions in diabetic animals is unknown. We investigated whether
the urine concentrating defect in diabetic animals was a function of
AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout
(UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT)
and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT
mice, vasopressin increased urine osmolality from 3,000 to 4,550
mosmol/kgH2O. In contrast, urine osmolality was low (800 mosmol/
kgH2O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3
KO (114%) and WT (92%) mice. To define the role of UT-A1 and
UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were
injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only
40% survival at 7 days post-STZ injection compared with 70% in WT.
AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared
with a 133% increase in WT diabetic mice. Biotinylation studies in rat
IMCDs showed that membrane accumulation of UT-A1 increased by
68% in response to vasopressin in control rats but was unchanged by
vasopressin in diabetic rat IMCDs. We conclude that, even with
increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated
in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.
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UT-A1 AND AQP2 IN DIABETIC RATS
examined the interdependence of AQP2 and its phosphorylated
forms and the UT-A proteins in the urine concentration response in the diabetic kidney. Finally, we determined if the
AVP-stimulated membrane insertion of UT-A1 is altered in
response to diabetes mellitus (DM).
MATERIALS AND METHODS
RESULTS
Characteristics of the UT-A1/A3 KO. We assessed the various tissues in this strain of KO mice and compared our results
with the original strain reported by Fenton et al. (4). The only
difference in organ weights between our KO strain and the
Fenton KO strain was in kidney weight. The Fenton KO strain
had reduced weights compared with the WT while our KO
strain showed no difference in size compared with the WT
mice. Table 1 shows the average weights of different organs in
our UT-A1/A3 KO strain compared with C57BL6 WT mice.
The testes were significantly larger in the KO mice compared
with the WT, even when this was normalized by brain weight.
The kidney weights showed no difference between KO and
WT strains.
AVP increases stimulation of AQP2 only in the WT mice.
Following the 7-day treatment with AVP in WT and UTA1/A3 KO mice, we found that urine osmolality of the WT
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Experimental animals. All animal protocols were approved by the
Emory University Institutional Animal Care and Use Committee.
UT-A1/A3 KO mice were established by Fenton et al. (3, 5). Male
UT-A1/A3 KO mice were a generous gift from Dr. Mark Knepper,
National Institutes of Health. These mice were mated with C57BL6
female wild-type (WT) mice to produce a heterozygous litter. The
heterozygous litter was mated, and the offspring were tested to select
those that had complete absence of the UT-A1 and UT-A3 transporters. The homozygous offspring were mated repeatedly until we had
pure progeny of UT-A1/A3 KO mice. The absence of UT-A1 and
UT-A3 in the inner medulla (IM) was confirmed by Western blot of
tissue lysates.
Characterization of UT-A1/A3 KO mice. The mice were placed in
metabolic cages for 24 h of acclimatization, followed by a 24-h
collection period. Urine was collected for volume and osmolality
measurement using a Wescor vapor pressure osmometer. After the
24-h collection period, animals were killed, and various organs were
dissected and weighed.
AVP stimulation of AQP2 in UT-A1/A3 KO mice. WT and UTA1/A3 KO mice were given free access to food and water. The mice
were placed in metabolic cages, and urine was collected from the two
groups for baseline levels. To administer AVP, minipumps (Alzet
model 2002; Durect, Cupertino, CA) delivering AVP at 5 ng/h
(Sigma, St. Louis, MO) were implanted subcutaneously under light
ketamine anesthesia. After the 7-day treatment with AVP, the mice
were again placed in metabolic cages for 24-h urine collection.
Subsequently, the animals were killed, kidneys were harvested, IM
were dissected, and tissue lysates were prepared. After protein determination by BCA colorimetric protein assay (Bio-Rad, Hercules, CA),
the proteins were size separated by SDS-PAGE and electroblotted to
polyvinylidene difluoride membranes (Millipore, Bedford, MA).
Membranes were probed with primary antibody to AQP2 overnight at
4°C. The secondary antibody used for detection was Alexa Fluor
680-linked anti-rabbit IgG (Invitrogen, Chicago, IL). Proteins were
visualized using infrared detection with the LI-COR Odyssey protein
analysis system (LI-COR, Lincoln, NE). Quantification of the resulting bands was accomplished using the LICOR internal densitometry
program.
Response of UT-A1/A3 KO mice to diabetes. WT and UT-A1/A3
KO mice were individually housed in metabolic cages for 24 h for
baseline urine collection. After 24 h, they were injected intraperitoneally with streptozotocin (STZ), 175 mg/kg body wt in 0.1 M citrate
buffer (pH 4.0), to induce type 1 diabetes. Elevated blood glucose was
confirmed by tail blood after 24 – 48 h (One Touch Profile Diabetes
Tracking Kit; Lifescan, Milpitas, CA). Twenty-four hours before
death and 6 days after STZ injection, the animals were placed in
metabolic cages, and urine was collected for volume and osmolality
determination. The animals were killed after 7 days or when humane
end points were reached so survival was monitored for only 1 wk.
Diabetes was confirmed by checking blood glucose at the time of
death. The IMs were dissected, and tissue lysates were prepared for
Western blot analysis. Western blots were probed for AQP2, phosphorylated (p) serine (ser) 256-AQP2 (a generous gift from Dr. Rikke
Nørregaard, University of Aarhus), pser261-AQP2, pser264-AQP2,
and pser269-AQP2 (PhosphoSolutions, Aurora, CO).
Animal preparation for membrane accumulation experiments. Sprague
Dawley rats were injected intravenously with STZ (65 mg/kg) in fresh
citrate buffer via tail vein. Blood glucose was checked at 24 – 48 h to
confirm diabetes. Animals found not to be diabetic were reinjected
with half the prior dose by tail vein or the full dose via intraperitoneal injection, and elevated blood glucose was verified after 24
h. After 3 wk, the animals were killed, and the kidneys were
harvested. IMCD suspensions were prepared by enzyme digest as
previously described (19).
Biotinylation. IMCD suspensions from control and diabetic rat
kidneys were biotinylated as previously described (12). Briefly, suspended tubules were treated for 20 min at 37°C with 100 nM AVP.
Next, samples were washed free of excess solution two times with
PBS and three times with biotinylation buffer without biotin (mM:
215 NaCl, 4 KCl, 1.2 MgSO4, 2 CaCl2, 5.5 glucose, 10 triethanolamine, and 2.5 Na2HPO4) and then incubated with biotinylation
buffer containing 3 mg/ml biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (Sigma-Aldrich, St. Louis, MO) and 100 nM
AVP for 60 min at 4°C. The samples were then washed free of
unattached biotin with biotin quenching buffer, washed with lysis
buffer without detergent, and solubilized for 1 h in lysis buffer
containing 1% Nonidet P-40. After centrifugation (14,000 g, 10 min,
4°C) to remove insoluble particulates, streptavidin beads were added
and allowed to absorb biotinylated proteins overnight at 4°C. After
washing with high-salt and no-salt buffers, Laemmli SDS-PAGE sample
buffer was added directly to the pellets, samples were boiled for 1 min,
and the pool of biotinylated proteins was analyzed by Western blot.
Western blots of the biotinylated proteins were probed with antibodies to
total UT-A1 and UT-A1 phosphorylated on serine-486.
Immunohistochemistry. Sprague-Dawley rats were injected intravenously with STZ (65 mg/kg) in fresh citrate buffer via tail vein.
Blood glucose was checked at 24 – 48 h to confirm diabetes. Control
and diabetic Sprague-Dawley rats were injected with AVP subcutaneously 45 min before perfusion and paraformaldehyde fixation. The
animals were killed, and their kidneys were perfusion fixed with
paraformaldehyde. Paraffin sections of 4 ␮m thickness were sliced.
Sections were dewaxed and hydrated in preparation for immunostaining as previously described (13). Sections were incubated overnight at
4°C with the following primary antibodies: UT-A1 and pser486-UT-A1.
Slides were washed free of primary antibody and then incubated for
2 h in peroxidase-conjugated secondary antibody (donkey anti-rabbit
IgG). Diaminobenzidine and 35% H2O2 were added to detect peroxidase activity identifying the primary antibodies. Slides were also
stained with Mayers hematoxylin to visualize nuclei. Stained sections
were visualized using a bright field on an Olympus inverted microscope IX71 and a SPOT camera at ⫻400 magnification.
Statistical analysis. All data are presented as means ⫾ SE, and n is
the number of animals. To test for the statistical significance between
the results from two groups, Student’s t-test was used. The criterion
for statistical significance was P ⬍ 0.05.
UT-A1 AND AQP2 IN DIABETIC RATS
Table 1. Organ weight: UT-A1/A3 knockout mice vs.
wild-type mice
Body wt, g
Kidney, g
Liver, g
Spleen, g
Testes, g
Heart, g
Brain, g
Thymus, g
Gastronemius, g
Wild Type
UT-A1/A3
Knockout
22.7 ⫾ 0.8
0.32 ⫾ 0.02
1.00 ⫾ 0.05
0.062 ⫾ 0.004
0.016 ⫾ 0.005
0.13 ⫾ 0.01
0.32 ⫾ 0.006
0.05 ⫾ 0.004
0.13 ⫾ 0.003
23.25 ⫾ 0.28
0.34 ⫾ 0.04
1.1 ⫾ 0.03
0.06 ⫾ 0.003
0.28 ⫾ 0.004*
0.15 ⫾ 0.005
0.324 ⫾ 0.006
0.05 ⫾ 0.004
0.13 ⫾ 0.006
Values are means ⫾ SE; n ⫽ 6 mice/group. UT, urea transporter. *Statistically significant difference with P ⬍ 0.0001.
Diabetes reduces survival of UT-A1/A3 KO mice. The average blood glucose level of the mice 48 h after injection of STZ
were as follows: control WT, 114.5 mg/dl; diabetic WT, 299.8
mg/dl; UT-A1/A3 KO, 99.3 mg/dl; diabetic UT-A1/A3 KO,
294.5 mg/dl, n ⫽ 4 – 8. The apparently high dose of STZ, 175
mg/kg, was used in the C57BL6 strain of WT and KO because
lower doses were ineffective in inducing diabetes. At day 7,
100% of the control WT and UT-A1/A3 KO mice were still
alive. However, induction of diabetes reduced survival of the
mice. UT- A1/A3 KO mice showed 40% survival at 7 days
post-STZ injection compared with 70% survival in WT (Fig. 2).
Diabetes resulted in reduced survival of both WT and KO
mice, but its effect was more pronounced in the KO group. In
observing the mice, the WT mice with diabetes appeared to
stabilize over time after STZ injection while the KO animals
appeared to get progressively worse. We previously showed
that an upregulation of UT-A1 is part of the compensatory
response to DM (10). The present findings in the UT-A1/A3
KO suggest that, without this compensatory response, the
animal becomes sicker, rather than stabilizing at 7 days. An
alternative explanation, which seems less likely, is that UTA1/A3 KO mice are more sensitive to STZ toxicity than WT
mice through an unknown, non-DM mechanism. We chose to
study mice at 7 days post-STZ to avoid mice that were too
sickly. To verify that the mice were not experiencing overriding impaired renal function, we measured serum creatinines
and calculated creatinine clearances in an attempt to assess
their renal function. Serum creatinines in the mice were very
similar with no statistically significant difference between the
groups. The average serum creatinine was of 0.1 mg/dl in the
control WT, control UT-A1/A3 KO, and the diabetic UT-
Fig. 1. The effect of vasopressin (AVP) on the urea transporter (UT)-A1/A3 knockout (KO) mice. A: graph showing urine osmolality in C57BL6 wild-type (WT)
and UT-A1/A3 KO mice ⫾ a 7-day AVP treatment. Urine osmolality in the UT-A1/A3 KO mice remained low (800 mosmol/kgH2O) following AVP treatment,
whereas in WT mice, urine osmolality increased from 3,000 to 4,550 mosmol/kgH2O with AVP treatment. B: top, Western blot of tissue lysate from WT and
UT-A1/A3 KOs, with and without AVP treatment probed for aquaporin (AQP) 2. Each lane contains a sample from a different animal. Immediately below is
the loading control (LC) showing total protein (Ponceau) stained on the same membrane. Bottom, bar graph showing combined mean densities ⫾ SE from 2
experiments; n ⫽ 4/condition. *P ⬍ 0.05. AQP2 abundance increased in both WT and UT-A1/A3 KO mice following 7-day AVP treatment.
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mice increased by 51%, whereas that of the KO mice showed
no change (Fig. 1A). The WT mice were able to increase their
urine osmolality from 3,000 to 4,550 mosmol/kgH2O, whereas
the KO remained low at 800 mosmol/kgH2O. We measured the
baseline AVP levels and found that UT-A1/A3 KO mice do
have significantly higher AVP levels than WT mice (AVP:
WT ⫽ 14.96 ⫾ 2.19 pg/ml, n ⫽ 4; UT-A1/A3 KO ⫽ 25 ⫾
3.09 pg/ml, n ⫽ 4, P ⫽ 0.03). However, when we compared
AQP2 protein abundance between these two groups, we found
that AVP significantly increased AQP2 protein abundance
(densitometry in arbitrary units) in both WT and UT-A1/A3
KO mice (Fig. 1B). Statistical evaluation reveals that the 92%
increase in response to AVP in the KO animals is significantly
less than the 114% increase in the control animals (n ⫽
4/condition, P ⬍ 0.01).
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UT-A1 AND AQP2 IN DIABETIC RATS
A1/A3 mice (n ⫽ 3/group, data not shown). The calculated
creatinine clearances of the mice were as follows: control
WT ⫽ 0.48 ⫾ 0.13 ml/min, control UT-A1/A3 KO ⫽ 0.53 ⫾
0.11 ml/min, diabetic UT-A1/A3 KO ⫽ 0.82 ⫾ 0.23 ml/min,
n ⫽ 2– 4/group. The creatinine clearance for the diabetic WT
mice was not available for analysis. There were no significant
differences between the creatinine clearance of the KO diabetic
and control mice and the WT control mice.
Fig. 3. A: diabetes (DM) decreases urine osmolality in both WT and UT-A1/A3 KO mice. The bar graph shows the urine osmolality of WT and UT-A1/A3 KO
control and DM mice. Diabetes significantly decreased the urine osmolality of the WT and UT-A1/A3 KO mice (bar graph showing means ⫾ SE, n ⱖ 23/group,
*P ⬍ 0.03). B: diabetes increases AQP2 protein levels in WT but not in UT-A1/A3 KO mice. Top, Western blot of tissue lysate from WT and UT-A1/A3 KO
mice, with and without diabetes, probed for AQP2. Each lane is loaded with a sample from a different animal. Immediately below is the loading control (LC).
Bottom, bar graph showing combined mean densities ⫾ SE from 2 experiments; n ⫽ 4/condition. *P ⬍ 0.05.
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Fig. 2. The effect of diabetes on mouse survival. This shows a line graph
depicting the survival of WT and UT-A1/A3 KO mice over 7 days after
streptozotocin (STZ) injection to induce diabetes mellitus (DM). WT and
UT-A1/A3 KO control mice showed 100% survival. Induction of diabetes
reduced survival of UT-A1/A3 KO mice to 40% compared with 70% in WT
diabetics, i.e., of a group of 18 in the diabetic UT-A1/A3 KO mouse group,
only 7 remained by day 7 (n ⫽ 16 –18/group).
Diabetes decreases urine osmolality in both UT-A1/A3 KO
and WT but does not change AQP2 abundance in the KO. The
urine osmolality of WT and diabetic mice was compared following the induction of diabetes. Diabetes significantly decreased the
urine osmolality of the UT-A1/A3 KO mice (control UT-A1/A3
KO ⫽ 899 ⫾ 59 mosmol/kgH2O, UT-A1/A3 diabetic mice ⫽
715 ⫾ 11 mosmol/kgH2O, n ⱖ 23/group, P ⬍ 0.03). The WT
mice also showed a decrease in urine osmolality of 19% when
they were treated with STZ (WT ⫽ 2,407 ⫾ 97 mosmol/kgH2O,
diabetic WT ⫽ 2,053 ⫾ 91 mosmol/kgH2O, n ⫽ 17–22/group,
P ⬍ 0.01). The diabetes-induced decrease in urine osmolality
was more pronounced in the UT-A1/A3 KO mice compared
with the WT mice (Fig. 3A). We further compared AQP2
abundance in UT-A1/A3 KO mice and WT mice, with and
without diabetes. There was a greater than twofold increase in
AQP2 in the WT diabetic mice compared with the nondiabetic
WT group, but there was no change in AQP2 abundance in the
UT-A1/A3 KO diabetic mice compared with the nondiabetic
UT-A1/A3 KO mice (Fig. 3B).
pser256-AQP2 abundance was lower in the UT-A1/A3 KO
mice compared with the WT mice, and the KO mice were
unable to increase pser256-AQP2 in response to diabetes
unlike the WT mice. In the nondiabetic animals, pser256-AQP2
abundance was ⬃50% lower in the UT-A1/A3 KO compared
with the WT mice (P ⬍ 0.05, Fig. 4A). In the diabetic animals,
pser256-AQP2 abundance was lower in the UT-A1/A3 KO by
68% compared with the WT mice (P ⬍ 0.001). pser256-AQP2
was lower in the UT-A1/A3 KO vs. WT mice and did not
increase in the KO mice with diabetes. However, in the WT
mice, the amount of pser256-AQP2 was increased 2.3-times in
response to diabetes.
UT-A1 AND AQP2 IN DIABETIC RATS
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pser261-AQP2 abundance is decreased in response to diabetes in the UT-A1/A3 KO mice compared with the WT diabetic
mice. In the nondiabetic animals, pser261-AQP2 abundance
showed no significant difference [25 ⫾ 4.7 and 46.2 ⫾ 11.3 in
the KO and WT, respectively, P ⫽ not significant (NS)].
pser261-AQP2 did not increase in the UT-A1/A3 KO in
response to diabetes; however, in the WT animals, the amount
of pser261-AQP2 was increased fourfold in response to diabetes (P ⫽ 0.01, Fig. 4B).
pser264-AQP2 and pser269-AQP2 abundances in response
to diabetes. We also tested phosphorylation of pser264-AQP2
and pser269-AQP2 in WT and KO mice in response to diabetes. There was no difference observed in the abundance of
pser264-AQP2 or pser269-AQP2 in either WT or UT-A1/A3
KO mice, with or without diabetes, as assessed by Western blot
analysis in IMCD tissue samples (data not shown).
Plasma membrane accumulation. AVP increases apical
plasma membrane accumulation of UT-A1 in control IMCDs.
IMCD suspensions were biotinylated with and without exogenous
AVP to determine membrane accumulation of UT-A1. Figure 5A
provides a Western blot showing the membrane-bound (biotinylated) UT-A1 in control IMCD suspensions, with and without
AVP (100 nM), and the analogous Western blot of the IMCD
tissue lysates probed for total UT-A1. These protein values were
used to normalize the UT-A1 in the membrane (biotinylated). The
bar graph shows the amount of biotinylated UT-A1 per unit of
total UT-A1 protein, combining the results from four different
experiments (n ⫽ 8 –13, P ⬍ 0.05). AVP increases the membrane
expression of UT-A1 in the control rats treated with AVP but not
in the diabetic rats treated with AVP (combining four different
experiments, n ⫽ 12–16, P ⫽ NS, Fig 5B).
Immunohistochemistry. We had earlier shown that AVP increases urea permeability in the initial IMCD from the diabetic rat
(18). Therefore, we used immunohistochemistry to determine if
the cellular localization of UT-A1 was changed by AVP. We
looked at the membrane distribution of total UT-A1 and UT-A1
phosphorylated at serine-486 in control and diabetic rats that were
injected with AVP before perfusion and immunohistochemistry.
Figure 6, A and B, shows the membrane association of total
UT-A1 in the control animals, with and without AVP. Addition of
AVP increases membrane association of total UT-A1 in the
control IMCD. In the diabetic animal, total UT-A1 was seen to be
maximally expressed both at the membrane and in the cytoplasm.
This distribution did not change in the presence of AVP (Fig. 6, C and
D). When we studied serine-486 phosphorylated UT-A1, we
found an increased membrane association in control animals with
AVP treatment (Fig. 7, A and B); however, in the diabetic rats,
pser486-UT-A1 was already maximally expressed at the apical
membrane, and this did not change with AVP treatment (Fig. 7, C
and D). Our results also showed a higher level of pser486-UT-A1
at the apical surface with a greater proportion of phosphorylated
UT-A1 associated with the membrane than with the cytosol in the
diabetic animal.
DISCUSSION
The UT-A1/A3 KO mouse was established by Fenton et al.
in 2004 (3). They deleted 3 kb of the UT-A gene, resulting in
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Fig. 4. The effect of diabetes (DM) on the abundance of phosphorylated AQP2. A: phosphorylated (p) serine (ser) 256-AQP2 abundance is decreased in response
to DM in the UT-A1/A3 KO mice compared with the WT diabetic mice. Top, Western blot of tissue lysate from WT and UT-A1/A3 KO mice, with and without
diabetes, probed for pser256-AQP2. Bottom, bar graph showing combined mean densities ⫾ SE from 2 experiments; n ⫽ 4/condition. *P ⱕ 0.05. NS, not
significant. B: pser261-AQP2 abundance is decreased in response to diabetes in the UT-A1/A3 KO mice compared with the WT diabetic mice. Top, Western
blot of tissue lysate from WT and UT-A1/A3 KO, with and without diabetes, probed with pser261-AQP2. Each lane is loaded with a sample from a different
rat. Immediately below is the loading control (LC). Bottom, bar graph showing combined mean densities ⫾ SE from 2 experiments; n ⫽ 4/condition. *P ⱕ 0.01.
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UT-A1 AND AQP2 IN DIABETIC RATS
disruption of the region that encodes UT-A1 and UT-A3 (3).
This was pivotal in describing the functions of these UT in the
kidney. The IMCDs from these KO mice are unable to carry
out AVP-dependent urea transport and so cannot concentrate
their urine (3). We received fertile male UT-A1/A3 KO mice
from the Knepper laboratory, bred them back to C57BL/6
females, and then reestablished the homozygous line. Our KO
mice showed physiological characteristics that were comparable to the initial stock, including their inability to concentrate
urine.
AVP regulates AQP2 by binding to V2 receptors on the
basolateral membrane of the principal and IMCD cells and
promoting increased cAMP production. An increase in plasma
AVP levels results in increased abundance of AQP2. In the
present study, we show that, even when we maximally stimulate AQP2 with AVP in control UT-A1/A3 KO mice, the
protein abundance of AQP2 is increased, but there is no
improvement in the urine concentrating ability of the mice.
Although baseline AVP levels are higher in the KO mice and
this may have blunted the response to exogenous AVP, we do
not think the increase was sufficient to mask a AVP response
if it was present. Although the basal level of 25 pg/ml is
increased in the KO compared with the WT level of 15 pg/ml,
the maximal effect of AVP is not reached as mice are able to
achieve AVP levels close to 421 pg/ml (17) and the KO mice
had levels of 25 pg/ml. In addition, urine concentration was
still impaired in the KO mice treated with AVP. This implies
that urine concentration in the IMCD may involve an interaction between AQP2 and the urea transporters UT-A1 and
UT-A3, or at least a coordination of the actions of both water
and urea movement to effect a successful concentrating response. This may explain the observed inability of AVP to
increase urine osmolality in the UT-A1/A3 KO mice where,
even in the presence of maximum stimulation with AVP,
AQP2 cannot create a concentrating environment in the absence of UT-A1 and UT-A3.
We also found that survival of the UT-A1/A3 KO mice was
impacted by the presence of diabetes. The diabetic animals
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Fig. 5. Vasopressin (AVP) increases apical membrane accumulation of UT-A1 in inner medullary collecting ducts (IMCDs) from nondiabetic control rats (A)
but not diabetic (DM) rats (B). A: top, Western blot showing the membrane-bound UT-A1 (biotinylated) in nondiabetic control (CTRL) rat IMCD suspensions,
with and without AVP treatment. Immediately below is the Western blot showing total UT-A1. B: top, Western blot showing the membrane-bound UT-A1 in
diabetic rat IMCD suspensions, with and without AVP treatment. Immediately below is the Western blot showing total UT-A1 in diabetic rats. Western blots
include representative Western blot protein bands from three of the four experiments in which we looked at the biotinylation of UT-A1 under these conditions.
C: left, bar graph showing the amount of biotinylated UT-A1 per unit of total protein in control animals with and without AVP, combining the results from four
different experiments (n ⫽ 8 –13, P ⬍ 0.05). Right, bar graph showing biotinylated UT-A1 per unit of total protein in diabetic animals treated with and without
AVP, combining the results from four different experiments [n ⫽ 12–16, P ⫽ not significant (NS)]. *P ⬍ 0.05.
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UT-A1 AND AQP2 IN DIABETIC RATS
showed a 40% survival compared with controls, which showed
a 70% survival. The possible explanation for this is that the
absence of UT-A1 and UT-A3 leads to hypotonic urine, which
would cause dehydration and may result in volume depletion.
In an animal that is polyuric at baseline, these characteristics
may result in profound morbidity and mortality. This is reflected in the urine concentration of the diabetic animals. The
control diabetic mice were able to concentrate urine to a
reasonable degree. Urine concentration decreased by 19% in
the WT diabetic mice, and this was statistically significant; the
UT-A1/A3 KO had a decrease of 25% in urine concentration.
The dramatic reduction in urine osmolality in the KO animals
can be explained by diabetes. Diabetes results in polydipsia
and polyuria, which cause a free water excretion and reduced
urine osmolality. AQP2 is upregulated in abundance in the
UT-A1/A3 KO mice and in the diabetic WT. This finding
agrees with previous work done by Nejsum et al. and supports
this being a compensatory mechanism to conserve water in
diabetes (15). However, in the UT-A1/A3 KO, diabetes is
unable to increase AQP2 abundance. This suggests that, in
diabetes, there is interdependence between UT-A1 and AQP2,
which results in a compensatory mechanism for the polyuric
state. The absence of UT-A1 and UT-A3 in the diabetic mouse
results in a failure of this compensatory mechanism to be
activated, and this may be the cause of the increased mortality
seen in the UT-A1/A3-deficient animals. Nejsum et al. have
previously shown that the abundance of AQP2, AQP3, and
phosphorylated AQP2 increases in response to diabetes (15).
AVP appears to be responsible for the upregulation of
phosphorylated AQP2 in response to the polyuria. Our study
also looked at the levels of phosphorylation of aquaporins
(AQP) in the WT and KO animals. We saw that phosphorylation at serine-256, which is AVP sensitive, was more increased
in the WT diabetic mice compared with the UT-A1/A3 KO
diabetic mice. In addition, pser256-AQP2 was increased in
nondiabetic WT mice compared with pser256-AQP2 levels in
the nondiabetic UT-A1/A3 KO. There was a significant upregulation of pser256-AQP2 in the WT mice with induction of
diabetes, but in the UT-A1/A3 KO mice diabetes did not
significantly change the abundance of pser256-AQP2. This
suggests that the inability to phosphorylate AQP2 at serine-256
may be, at least in part, a contributor to the poor urine
concentration in these mice. It also suggests that serine-256
may be affected by the absence of UT-A1/A3 although the
mechanism is yet to be understood. pser261-AQP2 was also
downregulated in the diabetic UT-A1/A3 KO mice compared with the diabetic WT mice but may not be a large
contributor to the reduced urine concentrating ability, since
it is not upregulated by AVP (8) and diabetes is an AVPsensitive state. There was no significant difference in phosphorylation of AQP2 at serines-264 and -269 in the WT and
UT-A1/A3 KO, implying that these sites may not directly be
implicated in the urine concentrating ability of the diabetic
UT-A1/A3 KO mice.
Along with its role in promoting water movement with AQP,
AVP acts on UT-A1 to promote phosphorylation and mem-
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Fig. 6. Immunohistochemistry showing the
membrane distribution of UT-A1 in control and
diabetic rats ⫾ vasopressin (AVP). A: membrane
association of total UT-A1 in the control
animal. B: membrane association of total
UT-A1 in the control animal with AVP.
Membrane association of total UT-A1 is
increased in the control IMCD treated with
AVP. C: total UT-A1 in the diabetic rat.
D: total UT-A1 in the diabetic rat treated
with AVP. Total UT-A1 was seen to be
already maximally expressed both at the
membrane and in the cytoplasm of control
diabetic rats, and this did not change in the
presence of AVP. Insets in each panel have
been arbitrarily increased in size to allow
clearer visualization of the membrane distribution. L, lumen of the tubule in the inset.
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UT-A1 AND AQP2 IN DIABETIC RATS
brane accumulation of this transporter. When we studied the
membrane accumulation of UT-A1 in control and diabetic rats
treated exogenously with AVP, we found that control rats
increased membrane expression of UT-A1, but this was not the
case in the diabetic rats. This result was not expected as
previous studies had shown an increase in urea permeability in
isolated perfused IMCDs in the diabetic animals compared
with the WT (18). Immunohistochemistry helped to explain
our results. Our observations were that UT-A1 is maximally
membrane associated in the diabetic animal, and further stimulation by exogenous AVP does not increase the membrane
association. Diabetes also causes UT-A1 that is phosphorylated
at serine-486 to be localized predominantly to the apical
plasma membrane. This phosphorylated form is one of the
active forms of UT-A1 that moves to the membrane (2), causes
increased urea reabsorption from the urine, and ultimately
results in water reabsorption, which would compensate for the
polyuria seen in the diabetic state.
Results from these studies provide new evidence about the
relationship between UT-A and AQP2 in the urine concentrating mechanism. We conclude that AQP2 alone, even when
stimulated by AVP, is not sufficient to achieve optimal urine
concentration. This may explain, in part at least, why the
absence of UT-A1 and UT-A3 increases mortality in the
diabetic mice. The ability of diabetic animals to cope with
osmotic diuresis by increasing AQP2 does not occur in the
absence of UT-A1 and UT-A3. For maximum urine concentration and compensation in diabetes, both AQP and UT must
be present and functional. It appears that UT-A1 is already
maximally stimulated in diabetic IMCDs. Acute administration
of AVP, therefore, does not change UT-A1 membrane accumulation and cannot further stimulate urea movement since the
more active pser486-UT-A1 is already located almost exclusively at the apical plasma membrane.
ACKNOWLEDGMENTS
We thank Dr. Mark Knepper (National Institutes of Health) for the gift of
a breeder stock of the UT-A1/A3 knockout mouse. We are also grateful to Dr.
Rikke Nørregaard (University of Aarhus) for the gift of the phosphoantibody
to pser256-AQP2.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants R01-DK-41707, R01-DK-89828, R21-DK-91147,
and T32-DK-07656.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: T.O.I., J.M.S., and J.D.K. conception and design of
research; T.O.I. and C.F.M. performed experiments; T.O.I., C.F.M., and J.D.K.
analyzed data; T.O.I., J.M.S., and J.D.K. interpreted results of experiments;
T.O.I., C.F.M., and J.D.K. prepared figures; T.O.I. drafted manuscript; T.O.I.,
M.A.B., J.M.S., and J.D.K. edited and revised manuscript; T.O.I., M.A.B.,
J.M.S., and J.D.K. approved final version of manuscript.
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Fig. 7. Immunohistochemistry showing the
membrane distribution of pser486-UT-A1 in
diabetic rats ⫾ vasopressin (AVP). pser486UT-A1 in control rats (A), control rats ⫹
AVP (B), diabetic rats (C), and diabetic rats
treated with AVP (D). AVP increased membrane association of pser486-UT-A in control but not diabetic rats. pser486-UT-A1 in
diabetic rats was already maximally expressed at the membrane, and this did not
change with AVP. Insets in each panel have
been arbitrarily increased in size to allow
clearer visualization of the membrane
distribution.
UT-A1 AND AQP2 IN DIABETIC RATS
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