Oxytocin - Molecular Mechanisms of Molecular Mechanisms of...

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Unformatted text preview: Molecular Mechanisms of Molecular Mechanisms of Antidiuretic Effect of Oxytocin Li, C et al. J Am Soc Nephrol 19: 225­232, 2008. Alicia Notkin March 31, 2008 Outline Outline • • • • • Background Purpose Methods Results Conclusion Background Background Location of aquaporins in the Location of aquaporins in the kidney (Nielsen 2002) Vasopressin & aquaporins Vasopressin & aquaporins • Vasopressin increased AQP2 expression • • • long­term 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 upregulation of the NKCC2 Signal Transduction Via the V2 Signal Transduction Via the V2 Receptor (Brenner & Rector 2004) Brattleboro rats 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 1/3­1/2 of normal AQP expression suggests a probable vasopressin­independent effect Brattleboro rats Brattleboro rats • Vasopressin administration in these animals ­ significant increases in AQP2 & AQP3 expression ­ reverses polyuria Brattleboro rats 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 vasopressin­deficient) Background: oxytocin 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 & Amino acid sequence in oxytocin & vasopressin http://www.neurosci.pharm.utoledo.edu/MBC3320/vasopressin.htm Background: oxytocin 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 vasopressin­ deficient Brattleboro rats (reversible by a V2 receptor antagonist) Purpose Purpose Purpose of this paper 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 Methods Methods • Animals given 1 week to acclimate to high altitude of Denver • 12 hour light­dark cycle • Standard diet • Free access to water Methods: protocol 1 Methods: protocol 1 • 8 Brattleboro rats: 4 in 1st group (control group) & 4 in 2nd group (experimental group – given SR121463B, a V2­selective nonpeptide vasopressin receptor antagonist, at a dose of 1mg/kg/d sc 2x/d x 3d) • 4 Sprague­Dawley rats matched as normal controls (to measure plasma OT levels) Methods: protocol 1 Methods: protocol 1 • Urine was collected & osmolality & creatinine were measured • Trunk blood was collected for serum osmolality, sodium, & creatinine Methods: protocol 2 Methods: protocol 2 • 4 groups, each with 6 Brattleboro rats 1st group: sham­operated 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 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 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 paraffin­embedded sections Results Results Results: protocol 1 Results: protocol 1 • Brattleboro rats had marked polyuria & • • • decreased Uosm Plasma OT concentrations were increased in Brattleboro rats compared to Sprague­Dawley 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 Urine flow rate ( control (CTR) v. V2 receptor antagonist (SR) treated Brattleboro rats (fig 1A) Urine osmolality (mOsm/kg H20) in 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 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 Immunoblots of membrane fractions of IM in kidneys from control (CTR) & V2 receptor antagonist (SR) treated Brattleboro rats using anti­AQP2 antibodies (fig 2A) Immunoblots of membrane fractions of OM + C Immunoblots of membrane fractions of OM + C in kidneys from control (CTR) & V2 receptor antagonist (SR) treated Brattleboro rats using anti­AQP2 antibodies (fig 2B) Immunoblots of membrane fractions of OM + C Immunoblots of membrane fractions of OM + C in kidneys from control (CTR) & V2 receptor antagonist (SR) treated Brattleboro rats using anti­NKCC2 antibodies (fig 2C) Results: protocol 2 Results: protocol 2 • During OT infusion, urine flow rate decreased, Uosm increased, & solute­free 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 Urine flow rate ( (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 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 Characteristics of the 4 different groups of Brattleboro rats (table 2) Results: protocol 2 Results: protocol 2 • OT infusion for 5d ­ increased AQP2 protein levels in the IM & OM + C ­ increased p­AQP2 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 Immunoblot & densitometric analysis of AQP2 expression in the IM (fig 4A) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of AQP2 expression in the OM + C (fig 4B) Immunoblot & densitometric analysis Immunoblot & densitometric analysis of AQP2 expression in the IM (fig 4C) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of AQP2 expression in the OM + C (fig 4D) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of p­AQP2 expression in the IM (fig 5A) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of p­AQP2 expression in the IM (fig 5B) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of AQP3 expression in the IM (fig 5C) Immunoblot & densitometric analysis of Immunoblot & densitometric analysis of AQP3 expression in the IM (fig 5D) Summary of densitometric analysis of immunoblots in 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 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 Results: protocol 2 • OT ­ increased apical plasma membrane labeling density of AQP2 & p­AQP2 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 Immunoperoxidase microscopy of AQP2 & p­AQP2 in the IM in the 4 groups of Brattleboro rats Immunoperoxidase microscopy of Immunoperoxidase microscopy of AQP3 in the IM in the 4 groups of Brattleboro rats Conclusion Conclusion Conclusion Conclusion • The molecular mechanisms of the antidiuretic effect of • • oxytocin involve: ­ increased AQP2 expression ­ increased p­AQP2 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 References References • Andersen, LJ et al. Antidiuretic effect of subnormal levels of arginine • • • • • • • vasopressin in normal humans. Am J Physiol 259: R53­R60, 1990. Briner, VA et al. Comparative effects of arginine vasopressin and oxytocin in cell culture systerms. Am J Physiol 263: F222­F227, 1992. Chou, CL et al. Oxytocin as an antidiuretic hormone: II, role of V2 vasopressin receptor. Am J Physiol 269: F78­F85, 1995. Conrad, KP et al. Influence of oxytocin on renal hemodynamics and electrolyte and water excretion. Am J Physiol 251: F290­F296, 1986. Kim, GH et al. Vasopressin increases Na­K­2Cl cotransporter expression in thick ascending limb of Henle’s loop. Am J Physiol 276: F96­F103, 1999. Nielsen, S et al. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205­244, 2002. Pouzet, B et al. Selective blockade of vasopressin V2 receptors reveals significant V2­mediated water reabsorption in Brattleboro rats with diabetes insipidus. Nephrol Dial Transplant 16: 725­734, 2001. Serradeil­Le, GC et al. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729­2738, 1996. ...
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This note was uploaded on 12/24/2011 for the course STEP 1 taught by Professor Dr.aslam during the Fall '11 term at Montgomery College.

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