Molecular Mechanisms of Antidiuretic Effect of Oxytocin Li, C et al. J Am Soc Nephrol 19: 225-232, 2008. Alicia Notkin March 31, 2008

Molecular Mechanisms of Molecular Mechanisms of Antidiuretic Effect of Antidiuretic Effect of

OxytocinOxytocinLi, C et al. J Am Soc Nephrol 19: 225-232, 2008. Li, C et al. J Am Soc Nephrol 19: 225-232, 2008.

Alicia NotkinAlicia Notkin

March 31, 2008March 31, 2008

OutlineOutline

• BackgroundBackground

• PurposePurpose

• MethodsMethods

• ResultsResults

• ConclusionConclusion

BackgroundBackground

Location of aquaporins in the Location of aquaporins in the kidney (Nielsen 2002)kidney (Nielsen 2002)

Vasopressin & aquaporinsVasopressin & aquaporins

• Vasopressin Vasopressin increased AQP2 expression increased AQP2 expression long-termlong-term

• Vasopressin is involved in trafficking of Vasopressin is involved in trafficking of AQP2 to the apical membrane of the AQP2 to the apical membrane of the principal cells short-termprincipal cells short-term

• Vasopressin Vasopressin increased AQP3 expression increased AQP3 expression on the basolateral membrane of the on the basolateral membrane of the principal cellsprincipal cells

• Vasopressin Vasopressin upregulation of the NKCC2 upregulation of the NKCC2

Signal Transduction Via the V2 Signal Transduction Via the V2 Receptor (Brenner & Rector Receptor (Brenner & Rector

2004)2004)

Brattleboro ratsBrattleboro rats

• Have a single base deletion in the Have a single base deletion in the vasopressin genevasopressin gene

• Lack endogenous vasopressin (behave like Lack endogenous vasopressin (behave like central DI)central DI)

• Are profoundly polyuricAre profoundly polyuric• Have lower AQP2 & AQP3 expressionHave lower AQP2 & AQP3 expression• The fact that there is even 1/3-1/2 of The fact that there is even 1/3-1/2 of

normal AQP expression suggests a normal AQP expression suggests a probable vasopressin-independent effectprobable vasopressin-independent effect


• Vasopressin administration in these Vasopressin administration in these animals animals

- significant increases in AQP2 & - significant increases in AQP2 & AQP3AQP3 expression expression- reverses polyuria- reverses polyuria


• Two V2R antagonists (SR121463A & Two V2R antagonists (SR121463A & OPC31260) have previously been OPC31260) have previously been shown to increase polyuria & shown to increase polyuria & decrease AQP2 expression by ~ 50% decrease AQP2 expression by ~ 50% in Brattleboro rats in Brattleboro rats again suggests again suggests that another ligand has effects at the that another ligand has effects at the V2R (since these rats are already V2R (since these rats are already vasopressin-deficient)vasopressin-deficient)

Background: oxytocinBackground: oxytocin

• 9 amino acid peptide hormone 9 amino acid peptide hormone secreted by the posterior pituitary secreted by the posterior pituitary (like vasopressin)(like vasopressin)

• 7 out of 9 AAs are identical between 7 out of 9 AAs are identical between oxytocin & vasopressinoxytocin & vasopressin

• Both peptides form a cyclic structure Both peptides form a cyclic structure with disulfide bonds between with disulfide bonds between cysteines at similar positionscysteines at similar positions

Amino acid sequence in Amino acid sequence in oxytocin & vasopressinoxytocin & vasopressin

http://www.neurosci.pharm.utoledo.edu/MBC3320/vasopressin.htm

Background: oxytocinBackground: oxytocin

• Used in pregnancy to induce labor (previously Used in pregnancy to induce labor (previously administered in 5% dextrose solution, though administered in 5% dextrose solution, though now recommended in LR or NS)now recommended in LR or NS)

• Has been associated with water retention & Has been associated with water retention & hyponatremiahyponatremia

• In vitro: increases osmotic water permeability In vitro: increases osmotic water permeability in microdissected renal inner medullary in microdissected renal inner medullary collecting ducts (reversible by a V2 receptor collecting ducts (reversible by a V2 receptor antagonist)antagonist)

• In vivo: causes an antidiuresis in vasopressin-In vivo: causes an antidiuresis in vasopressin-deficient Brattleboro rats (reversible by a V2 deficient Brattleboro rats (reversible by a V2 receptor antagonist)receptor antagonist)

PurposePurpose

Purpose of this paperPurpose of this paper

• To understand the molecular To understand the molecular mechanisms behind the antidiuretic mechanisms behind the antidiuretic effect of oxytocineffect of oxytocin

• Specifically, to study the effects of OT Specifically, to study the effects of OT on urine concentration, water channels, on urine concentration, water channels, & ion transporters in the presence or & ion transporters in the presence or absence of a V2 receptor antagonist or absence of a V2 receptor antagonist or an OT receptor antagonistan OT receptor antagonist

MethodsMethods

MethodsMethods

• Animals given 1 week to acclimate to Animals given 1 week to acclimate to high altitude of Denverhigh altitude of Denver

• 12 hour light-dark cycle12 hour light-dark cycle

• Standard dietStandard diet

• Free access to waterFree access to water

Methods: protocol 1Methods: protocol 1

• 8 Brattleboro rats: 4 in 18 Brattleboro rats: 4 in 1stst group (control group (control group) & 4 in 2group) & 4 in 2ndnd group (experimental group (experimental group – given SR121463B, a V2-selective group – given SR121463B, a V2-selective nonpeptide vasopressin receptor nonpeptide vasopressin receptor antagonist, at a dose of 1mg/kg/d sc 2x/d antagonist, at a dose of 1mg/kg/d sc 2x/d x 3d)x 3d)

• 4 Sprague-Dawley rats matched as 4 Sprague-Dawley rats matched as normal controls (to measure plasma OT normal controls (to measure plasma OT levels)levels)


• Urine was collected & osmolality & Urine was collected & osmolality & creatinine were measuredcreatinine were measured

• Trunk blood was collected for serum Trunk blood was collected for serum osmolality, sodium, & creatinineosmolality, sodium, & creatinine


• 4 groups, each with 6 Brattleboro rats4 groups, each with 6 Brattleboro rats• 11stst group: sham-operated controls – had group: sham-operated controls – had

an osmotic minipump delivering saline an osmotic minipump delivering saline (instead of OT) to the peritoneum(instead of OT) to the peritoneum

• Other 3 groups: had osmotic minipumps Other 3 groups: had osmotic minipumps delivering 3 µg/kg/h OT x 5d (a dose delivering 3 µg/kg/h OT x 5d (a dose previously shown to have an previously shown to have an antidiuretic effect)antidiuretic effect)


3 OT treatment groups:given OT x 5d

OT control group:given OT only

OT + SR group:given SR121463B 2x/dsc at a dose of 1mg/kg

on day 5

OT + GW group:given GW796679X

(a selective OTreceptor antagonist)

2x/d by gavageat a dose of 5mg/kg

on day 5


• 24 hours later the rats were decapitated24 hours later the rats were decapitated• Urine was collected & osmolality & creatinine Urine was collected & osmolality & creatinine

were measuredwere measured• Trunk blood was collected for serum osmolality, Trunk blood was collected for serum osmolality,

sodium, & creatininesodium, & creatinine• Kidneys were removed, dissected into OM + C & Kidneys were removed, dissected into OM + C &

IM regions, homogenized, & centrifugedIM regions, homogenized, & centrifuged• Protein concentration was determined & Protein concentration was determined &

immunoblotting for AQP channels & Na & urea immunoblotting for AQP channels & Na & urea transporters was performedtransporters was performed

• Immunolabeling was performed on paraffin-Immunolabeling was performed on paraffin-embedded sectionsembedded sections

ResultsResults

Results: protocol 1Results: protocol 1

• Brattleboro rats had marked polyuria & Brattleboro rats had marked polyuria & decreased Uosmdecreased Uosm

• Plasma OT concentrations were increased Plasma OT concentrations were increased in Brattleboro rats compared to Sprague-in Brattleboro rats compared to Sprague-Dawley rats (statistically significant)Dawley rats (statistically significant)

• V2 receptor antagonist administration V2 receptor antagonist administration increased urine flow rate & decreased increased urine flow rate & decreased UosmUosm

• V2 receptor antagonist administration V2 receptor antagonist administration decreased AQP2 in the IM & OM + C & the decreased AQP2 in the IM & OM + C & the NKCC2 in the OM + CNKCC2 in the OM + C

Urine flow rate (µl/min/kg BW) in Urine flow rate (µl/min/kg BW) in control (CTR) v. V2 receptor control (CTR) v. V2 receptor

antagonist (SR) treated antagonist (SR) treated Brattleboro rats (fig 1A)Brattleboro rats (fig 1A)

Urine osmolality (mOsm/kg H20) in Urine osmolality (mOsm/kg H20) in control (CTR) v. V2 receptor control (CTR) v. V2 receptor

antagonist (SR) treated antagonist (SR) treated Brattleboro rats (fig 1B)Brattleboro rats (fig 1B)

Densitometric analysis of immunoblots of Densitometric analysis of immunoblots of the IM & OM + C in control (CTR) & V2 the IM & OM + C in control (CTR) & V2

receptor antagonist (SR) treated receptor antagonist (SR) treated Brattleboro rats (table 1)Brattleboro rats (table 1)

Immunoblots of membrane fractions of Immunoblots of membrane fractions of IM in kidneys from control (CTR) & V2 IM in kidneys from control (CTR) & V2

receptor antagonist (SR) treated receptor antagonist (SR) treated Brattleboro rats using anti-AQP2 Brattleboro rats using anti-AQP2

antibodies (fig 2A)antibodies (fig 2A)

Immunoblots of membrane fractions of Immunoblots of membrane fractions of OM + C in kidneys from control (CTR) & OM + C in kidneys from control (CTR) &

V2 receptor antagonist (SR) treated V2 receptor antagonist (SR) treated Brattleboro rats using anti-AQP2 Brattleboro rats using anti-AQP2

antibodies (fig 2B)antibodies (fig 2B)

Immunoblots of membrane fractions of Immunoblots of membrane fractions of OM + C in kidneys from control (CTR) & OM + C in kidneys from control (CTR) &

V2 receptor antagonist (SR) treated V2 receptor antagonist (SR) treated Brattleboro rats using anti-NKCC2 Brattleboro rats using anti-NKCC2

antibodies (fig 2C)antibodies (fig 2C)


• During OT infusion, urine flow rate During OT infusion, urine flow rate decreased, Uosm increased, & decreased, Uosm increased, & solute-free water reabsorption (Tsolute-free water reabsorption (Tcc

HH2200) )

increasedincreased

• These antidiuretic effects were These antidiuretic effects were reversed by the V2 receptor reversed by the V2 receptor antagonist, but not the OT receptor antagonist, but not the OT receptor antagonistantagonist

Urine flow rate (µl/min/kg BW) in Brattleboro rats: Urine flow rate (µl/min/kg BW) in Brattleboro rats: controls (CTL), oxytocin infused (OT), OT plus V2 controls (CTL), oxytocin infused (OT), OT plus V2 receptor antagonist administration (OT + SR), OT receptor antagonist administration (OT + SR), OT plus OT receptor antagonist administration (OT + plus OT receptor antagonist administration (OT +

GW) (fig 3A)GW) (fig 3A)

Urine osmolality (mosm/kg H2O) in Brattleboro rats: Urine osmolality (mosm/kg H2O) in Brattleboro rats: controls (CTL), oxytocin infused (OT), OT plus V2 controls (CTL), oxytocin infused (OT), OT plus V2 receptor antagonist administration (OT + SR), OT receptor antagonist administration (OT + SR), OT plus OT receptor antagonist administration (OT + plus OT receptor antagonist administration (OT +

GW) (fig 3B)GW) (fig 3B)

Characteristics of the 4 Characteristics of the 4 different groups of Brattleboro different groups of Brattleboro

rats (table 2)rats (table 2)


• OT infusion for 5d OT infusion for 5d

- increased AQP2 protein levels in the IM- increased AQP2 protein levels in the IM & OM + C & OM + C- increased p-AQP2 in the IM & OM + C- increased p-AQP2 in the IM & OM + C- increased AQP3 expression in the IM & - increased AQP3 expression in the IM & OM + COM + C

• These effects were reversed with the V2 These effects were reversed with the V2 receptor antagonist, but not the OT receptor antagonist, but not the OT receptor antagonistreceptor antagonist

Immunoblot & densitometric Immunoblot & densitometric analysis of AQP2 expression in analysis of AQP2 expression in

the IM (fig 4A)the IM (fig 4A)

Immunoblot & densitometric Immunoblot & densitometric analysis of AQP2 expression in the analysis of AQP2 expression in the

OM + C (fig 4B)OM + C (fig 4B)

Immunoblot & densitometric Immunoblot & densitometric analysis of AQP2 expression in analysis of AQP2 expression in

the IM (fig 4C)the IM (fig 4C)


OM + C (fig 4D)OM + C (fig 4D)

Immunoblot & densitometric Immunoblot & densitometric analysis of p-AQP2 expression in analysis of p-AQP2 expression in

the IM (fig 5A)the IM (fig 5A)

Immunoblot & densitometric Immunoblot & densitometric analysis of p-AQP2 expression in analysis of p-AQP2 expression in

the IM (fig 5B)the IM (fig 5B)


IM (fig 5C)IM (fig 5C)


IM (fig 5D)IM (fig 5D)

Summary of densitometric analysis of Summary of densitometric analysis of immunoblots in the IM & OM + C of kidneys of immunoblots in the IM & OM + C of kidneys of

control, OT infused, & OT infused plus V2 control, OT infused, & OT infused plus V2 receptor antagonist administered Brattleboro receptor antagonist administered Brattleboro

rats (table 3)rats (table 3)

Summary of densitometric analysis of Summary of densitometric analysis of immunoblots in the IM & OM + C of kidneys immunoblots in the IM & OM + C of kidneys of control, OT infused, & OT infused plus OT of control, OT infused, & OT infused plus OT

receptor antagonist administered receptor antagonist administered Brattleboro rats (table 4)Brattleboro rats (table 4)


• OT OT - increased apical plasma membrane - increased apical plasma membrane

labeling density of AQP2 & p-AQP2 in the labeling density of AQP2 & p-AQP2 in the principal cells of the IMCD principal cells of the IMCD- increased basolateral staining intensity of - increased basolateral staining intensity of AQP3AQP3 in the principal cells of the IMCD in the principal cells of the IMCD

• The V2 receptor antagonist reversed the The V2 receptor antagonist reversed the aboveabove

• The OT receptor antagonist did not reverse The OT receptor antagonist did not reverse the abovethe above

Immunoperoxidase microscopy of Immunoperoxidase microscopy of AQP2 & p-AQP2 in the IM in the 4 AQP2 & p-AQP2 in the IM in the 4

groups of Brattleboro ratsgroups of Brattleboro rats

Immunoperoxidase microscopy of Immunoperoxidase microscopy of AQP3 in the IM in the 4 groups of AQP3 in the IM in the 4 groups of


ConclusionConclusion

ConclusionConclusion

• The molecular mechanisms of the antidiuretic The molecular mechanisms of the antidiuretic effect of oxytocin involve:effect of oxytocin involve:- increased AQP2 expression- increased AQP2 expression- increased p-AQP2 expression & trafficking- increased p-AQP2 expression & trafficking- increased AQP3 expression- increased AQP3 expression

• These changes are mediated via the V2 receptor These changes are mediated via the V2 receptor on the basolateral membrane of the collecting on the basolateral membrane of the collecting ductduct

• V2 receptor antagonists can potentially be used V2 receptor antagonists can potentially be used to treat symptomatic hyponatremia in pregnant to treat symptomatic hyponatremia in pregnant women receiving oxytocin for labor induction, women receiving oxytocin for labor induction, without affecting uterine contractionswithout affecting uterine contractions

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vasopressin in normal humans. Am J Physiol 259: R53-R60, 1990.vasopressin in normal humans. Am J Physiol 259: R53-R60, 1990.• Briner, VA et al. Comparative effects of arginine vasopressin and Briner, VA et al. Comparative effects of arginine vasopressin and

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• Chou, CL et al. Oxytocin as an antidiuretic hormone: II, role of V2 Chou, CL et al. Oxytocin as an antidiuretic hormone: II, role of V2 vasopressin receptor. Am J Physiol 269: F78-F85, 1995.vasopressin receptor. Am J Physiol 269: F78-F85, 1995.

• Conrad, KP et al. Influence of oxytocin on renal hemodynamics and Conrad, KP et al. Influence of oxytocin on renal hemodynamics and electrolyte and water excretion. Am J Physiol 251: F290-F296, 1986.electrolyte and water excretion. Am J Physiol 251: F290-F296, 1986.

• Kim, GH et al. Vasopressin increases Na-K-2Cl cotransporter Kim, GH et al. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop. Am J Physiol expression in thick ascending limb of Henle’s loop. Am J Physiol 276: F96-F103, 1999.276: F96-F103, 1999.

• Nielsen, S et al. Aquaporins in the kidney: from molecules to Nielsen, S et al. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205-244, 2002.medicine. Physiol Rev 82: 205-244, 2002.

• Pouzet, B et al. Selective blockade of vasopressin V2 receptors Pouzet, B et al. Selective blockade of vasopressin V2 receptors reveals significant V2-mediated water reabsorption in Brattleboro reveals significant V2-mediated water reabsorption in Brattleboro rats with diabetes insipidus. Nephrol Dial Transplant 16: 725-734, rats with diabetes insipidus. Nephrol Dial Transplant 16: 725-734, 2001.2001.

• Serradeil-Le, GC et al. Characterization of SR 121463A, a highly Serradeil-Le, GC et al. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729-2738, 1996.antagonist. J Clin Invest 98: 2729-2738, 1996.

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Molecular Mechanisms of Antidiuretic Effect of Oxytocin Li, C et al. J Am Soc Nephrol 19: 225-232, 2008. Alicia Notkin March 31, 2008