Molecular Mechanisms of
Antidiuretic Effect of Oxytocin
Li, C et al. J Am Soc Nephrol 19: 225-232, 2008.
Alicia Notkin
March 31, 2008
Outline
• Background
• Purpose
• Methods
• Results
• Conclusion
Background
Location of aquaporins in the
kidney (Nielsen 2002)
Vasopressin & aquaporins
• Vasopressin  increased AQP2 expression long•
•
•
term
Vasopressin is involved in trafficking of AQP2 to
the apical membrane of the principal cells shortterm
Vasopressin  increased AQP3 expression on
the basolateral membrane of the principal cells
Vasopressin  upregulation of the NKCC2
Signal Transduction Via the V2
Receptor (Brenner & Rector 2004)
Brattleboro rats
• Have a single base deletion in the vasopressin
•
•
•
•
gene
Lack endogenous vasopressin (behave like
central DI)
Are profoundly polyuric
Have lower AQP2 & AQP3 expression
The fact that there is even 1/3-1/2 of normal
AQP expression suggests a probable
vasopressin-independent effect
Brattleboro rats
• Vasopressin administration in these
animals 
- significant increases in AQP2 & AQP3
expression
- reverses polyuria
Brattleboro rats
• Two V2R antagonists (SR121463A &
OPC31260) have previously been shown
to increase polyuria & decrease AQP2
expression by ~ 50% in Brattleboro rats
 again suggests that another ligand has
effects at the V2R (since these rats are
already vasopressin-deficient)
Background: oxytocin
• 9 amino acid peptide hormone secreted by
the posterior pituitary (like vasopressin)
• 7 out of 9 AAs are identical between
oxytocin & vasopressin
• Both peptides form a cyclic structure with
disulfide bonds between cysteines at
similar positions
Amino acid sequence in oxytocin &
vasopressin
http://www.neurosci.pharm.utoledo.edu/MBC3320/vasopressin.htm
Background: oxytocin
• Used in pregnancy to induce labor (previously
•
•
•
administered in 5% dextrose solution, though
now recommended in LR or NS)
Has been associated with water retention &
hyponatremia
In vitro: increases osmotic water permeability in
microdissected renal inner medullary collecting
ducts (reversible by a V2 receptor antagonist)
In vivo: causes an antidiuresis in vasopressindeficient Brattleboro rats (reversible by a V2
receptor antagonist)
Purpose
Purpose of this paper
• To understand the molecular mechanisms
behind the antidiuretic effect of oxytocin
• Specifically, to study the effects of OT on
urine concentration, water channels, & ion
transporters in the presence or absence of
a V2 receptor antagonist or an OT
receptor antagonist
Methods
Methods
• Animals given 1 week to acclimate to high
altitude of Denver
• 12 hour light-dark cycle
• Standard diet
• Free access to water
Methods: protocol 1
• 8 Brattleboro rats: 4 in 1st group (control
group) & 4 in 2nd group (experimental
group – given SR121463B, a V2-selective
nonpeptide vasopressin receptor
antagonist, at a dose of 1mg/kg/d sc 2x/d
x 3d)
• 4 Sprague-Dawley rats matched as normal
controls (to measure plasma OT levels)
Methods: protocol 1
• Urine was collected & osmolality &
creatinine were measured
• Trunk blood was collected for serum
osmolality, sodium, & creatinine
Methods: protocol 2
• 4 groups, each with 6 Brattleboro rats
• 1st group: sham-operated controls – had
an osmotic minipump delivering saline
(instead of OT) to the peritoneum
• Other 3 groups: had osmotic minipumps
delivering 3 µg/kg/h OT x 5d (a dose
previously shown to have an antidiuretic
effect)
Methods: protocol 2
3 OT treatment groups:
given OT x 5d
OT control group:
given OT only
OT + SR group:
given SR121463B 2x/d
sc at a dose of 1mg/kg
on day 5
OT + GW group:
given GW796679X
(a selective OT
receptor antagonist)
2x/d by gavage
at a dose of 5mg/kg
on day 5
Methods: protocol 2
• 24 hours later the rats were decapitated
• Urine was collected & osmolality & creatinine were
•
•
•
•
measured
Trunk blood was collected for serum osmolality, sodium,
& creatinine
Kidneys were removed, dissected into OM + C & IM
regions, homogenized, & centrifuged
Protein concentration was determined & immunoblotting
for AQP channels & Na & urea transporters was
performed
Immunolabeling was performed on paraffin-embedded
sections
Results
Results: protocol 1
• Brattleboro rats had marked polyuria &
•
•
•
decreased Uosm
Plasma OT concentrations were increased in
Brattleboro rats compared to Sprague-Dawley
rats (statistically significant)
V2 receptor antagonist administration 
increased urine flow rate & decreased Uosm
V2 receptor antagonist administration 
decreased AQP2 in the IM & OM + C & the
NKCC2 in the OM + C
Urine flow rate (µl/min/kg BW) in
control (CTR) v. V2 receptor antagonist
(SR) treated Brattleboro rats (fig 1A)
Urine osmolality (mOsm/kg H20) in
control (CTR) v. V2 receptor antagonist
(SR) treated Brattleboro rats (fig 1B)
Densitometric analysis of immunoblots of the IM
& OM + C in control (CTR) & V2 receptor
antagonist (SR) treated Brattleboro rats (table 1)
Immunoblots of membrane fractions of IM in
kidneys from control (CTR) & V2 receptor
antagonist (SR) treated Brattleboro rats using
anti-AQP2 antibodies (fig 2A)
Immunoblots of membrane fractions of OM +
C in kidneys from control (CTR) & V2 receptor
antagonist (SR) treated Brattleboro rats using
anti-AQP2 antibodies (fig 2B)
Immunoblots of membrane fractions of OM +
C in kidneys from control (CTR) & V2 receptor
antagonist (SR) treated Brattleboro rats using
anti-NKCC2 antibodies (fig 2C)
Results: protocol 2
• During OT infusion, urine flow rate
decreased, Uosm increased, & solute-free
water reabsorption (TcH20) increased
• These antidiuretic effects were reversed
by the V2 receptor antagonist, but not the
OT receptor antagonist
Urine flow rate (µl/min/kg BW) in Brattleboro rats: controls
(CTL), oxytocin infused (OT), OT plus V2 receptor antagonist
administration (OT + SR), OT plus OT receptor antagonist
administration (OT + GW) (fig 3A)
Urine osmolality (mosm/kg H2O) in Brattleboro rats: controls
(CTL), oxytocin infused (OT), OT plus V2 receptor antagonist
administration (OT + SR), OT plus OT receptor antagonist
administration (OT + GW) (fig 3B)
Characteristics of the 4 different
groups of Brattleboro rats (table 2)
Results: protocol 2
• OT infusion for 5d 
•
- increased AQP2 protein levels in the IM
& OM + C
- increased p-AQP2 in the IM & OM + C
- increased AQP3 expression in the IM & OM + C
These effects were reversed with the V2
receptor antagonist, but not the OT receptor
antagonist
Immunoblot & densitometric analysis
of AQP2 expression in the IM (fig 4A)
Immunoblot & densitometric analysis of
AQP2 expression in the OM + C (fig 4B)
Immunoblot & densitometric analysis
of AQP2 expression in the IM (fig 4C)
Immunoblot & densitometric analysis of
AQP2 expression in the OM + C (fig 4D)
Immunoblot & densitometric analysis of
p-AQP2 expression in the IM (fig 5A)
Immunoblot & densitometric analysis of
p-AQP2 expression in the IM (fig 5B)
Immunoblot & densitometric analysis of
AQP3 expression in the IM (fig 5C)
Immunoblot & densitometric analysis of
AQP3 expression in the IM (fig 5D)
Summary of densitometric analysis of immunoblots in
the IM & OM + C of kidneys of control, OT infused, &
OT infused plus V2 receptor antagonist administered
Brattleboro rats (table 3)
Summary of densitometric analysis of immunoblots
in the IM & OM + C of kidneys of control, OT
infused, & OT infused plus OT receptor antagonist
administered Brattleboro rats (table 4)
Results: protocol 2
• OT 
•
•
- increased apical plasma membrane
labeling density of AQP2 & p-AQP2 in the
principal cells of the IMCD
- increased basolateral staining intensity of AQP3
in the principal cells of the IMCD
The V2 receptor antagonist reversed the above
The OT receptor antagonist did not reverse the
above
Immunoperoxidase microscopy of AQP2 &
p-AQP2 in the IM in the 4 groups of
Brattleboro rats
Immunoperoxidase microscopy of AQP3
in the IM in the 4 groups of Brattleboro
rats
Conclusion
Conclusion
• The molecular mechanisms of the antidiuretic effect of
•
•
oxytocin involve:
- increased AQP2 expression
- increased p-AQP2 expression & trafficking
- increased AQP3 expression
These changes are mediated via the V2 receptor on the
basolateral membrane of the collecting duct
V2 receptor antagonists can potentially be used to treat
symptomatic hyponatremia in pregnant women receiving
oxytocin for labor induction, without affecting uterine
contractions
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