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

Outline Background Background Purpose Purpose Methods Methods Results Results Conclusion Conclusion

Background

Location of aquaporins in the kidney (Nielsen 2002)

Vasopressin & aquaporins Vasopressin  increased AQP2 expression long- term Vasopressin  increased AQP2 expression long- term Vasopressin is involved in trafficking of AQP2 to the apical membrane of the principal cells short- term Vasopressin is involved in trafficking of AQP2 to the apical membrane of the principal cells short- term Vasopressin  increased AQP3 expression on the basolateral membrane of the principal cells Vasopressin  increased AQP3 expression on the basolateral membrane of the principal cells Vasopressin  upregulation of the NKCC2 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 Have a single base deletion in the vasopressin gene Lack endogenous vasopressin (behave like central DI) Lack endogenous vasopressin (behave like central DI) Are profoundly polyuric Are profoundly polyuric Have lower AQP2 & AQP3 expression 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 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  Vasopressin administration in these animals  - significant increases in AQP2 & AQP3 expression - reverses polyuria - 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) 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) 9 amino acid peptide hormone secreted by the posterior pituitary (like vasopressin) 7 out of 9 AAs are identical between oxytocin & 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 Both peptides form a cyclic structure with disulfide bonds between cysteines at similar positions

Amino acid sequence in oxytocin & vasopressin

Background: oxytocin Used in pregnancy to induce labor (previously administered in 5% dextrose solution, though now recommended in LR or NS) 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 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 vitro: increases osmotic water permeability in microdissected renal inner medullary collecting ducts (reversible by a V2 receptor antagonist) In vivo: causes an antidiuresis in vasopressin- deficient Brattleboro rats (reversible by a V2 receptor antagonist) In vivo: causes an antidiuresis in vasopressin- deficient Brattleboro rats (reversible by a V2 receptor antagonist)

Purpose

Purpose of this paper To understand the molecular mechanisms behind the antidiuretic effect of oxytocin 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 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 Animals given 1 week to acclimate to high altitude of Denver 12 hour light-dark cycle 12 hour light-dark cycle Standard diet Standard diet Free access to water Free access to water

Methods: protocol 1 8 Brattleboro rats: 4 in 1 st group (control group) & 4 in 2 nd group (experimental group – given SR121463B, a V2-selective nonpeptide vasopressin receptor antagonist, at a dose of 1mg/kg/d sc 2x/d x 3d) 8 Brattleboro rats: 4 in 1 st group (control group) & 4 in 2 nd 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) 4 Sprague-Dawley rats matched as normal controls (to measure plasma OT levels)

Methods: protocol 1 Urine was collected & osmolality & creatinine were measured Urine was collected & osmolality & creatinine were measured Trunk blood was collected for serum osmolality, sodium, & creatinine Trunk blood was collected for serum osmolality, sodium, & creatinine

Methods: protocol 2 4 groups, each with 6 Brattleboro rats 4 groups, each with 6 Brattleboro rats 1 st group: sham-operated controls – had an osmotic minipump delivering saline (instead of OT) to the peritoneum 1 st 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) 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 24 hours later the rats were decapitated Urine was collected & osmolality & creatinine were measured Urine was collected & osmolality & creatinine were measured Trunk blood was collected for serum osmolality, sodium, & creatinine Trunk blood was collected for serum osmolality, sodium, & creatinine Kidneys were removed, dissected into OM + C & IM regions, homogenized, & centrifuged Kidneys were removed, dissected into OM + C & IM regions, homogenized, & centrifuged Protein concentration was determined & immunoblotting for AQP channels & Na & urea transporters was performed Protein concentration was determined & immunoblotting for AQP channels & Na & urea transporters was performed Immunolabeling was performed on paraffin-embedded sections Immunolabeling was performed on paraffin-embedded sections

Results

Results: protocol 1 Brattleboro rats had marked polyuria & decreased Uosm Brattleboro rats had marked polyuria & decreased Uosm Plasma OT concentrations were increased in Brattleboro rats compared to Sprague-Dawley rats (statistically significant) 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  increased urine flow rate & decreased Uosm V2 receptor antagonist administration  decreased AQP2 in the IM & OM + C & the NKCC2 in the OM + C 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 (T c H 2 0 ) increased During OT infusion, urine flow rate decreased, Uosm increased, & solute-free water reabsorption (T c H 2 0 ) increased These antidiuretic effects were reversed by the V2 receptor antagonist, but not the OT receptor antagonist 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  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 - 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 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  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 - 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 V2 receptor antagonist reversed the above The OT receptor antagonist did not reverse 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 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 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 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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