Alicia Notkin March 31, 2008

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

2. Outline • Background • Purpose • Methods • Results • Conclusion

3. Background

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

5. Vasopressin & aquaporins • Vasopressin  increased AQP2 expression long-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  upregulation of the NKCC2

6. Signal Transduction Via the V2 Receptor (Brenner & Rector 2004)

7. 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

8. Brattleboro rats • Vasopressin administration in these animals  - significant increases in AQP2 & AQP3 expression- reverses polyuria

9. 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)

10. 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

11. Amino acid sequence in oxytocin & vasopressin http://www.neurosci.pharm.utoledo.edu/MBC3320/vasopressin.htm

12. 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)

13. Purpose

14. 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

15. Methods

16. Methods • Animals given 1 week to acclimate to high altitude of Denver • 12 hour light-dark cycle • Standard diet • Free access to water

17. 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)

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

19. 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)

20. Methods: protocol 2

21. 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

22. Results

23. 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

24. Urine flow rate (µl/min/kg BW) in control (CTR) v. V2 receptor antagonist (SR) treated Brattleboro rats (fig 1A)

25. Urine osmolality (mOsm/kg H20) in control (CTR) v. V2 receptor antagonist (SR) treated Brattleboro rats (fig 1B)

26. Densitometric analysis of immunoblots of the IM & OM + C in control (CTR) & V2 receptor antagonist (SR) treated Brattleboro rats (table 1)

27. Immunoblots of membrane fractions of IM in kidneys from control (CTR) & V2 receptor antagonist (SR) treated Brattleboro rats using anti-AQP2 antibodies (fig 2A)

28. 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)

29. 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)

30. 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

31. 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)

32. 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)

33. Characteristics of the 4 different groups of Brattleboro rats (table 2)

34. 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

35. Immunoblot & densitometric analysis of AQP2 expression in the IM (fig 4A)

36. Immunoblot & densitometric analysis of AQP2 expression in the OM + C (fig 4B)

37. Immunoblot & densitometric analysis of AQP2 expression in the IM (fig 4C)

38. Immunoblot & densitometric analysis of AQP2 expression in the OM + C (fig 4D)

39. Immunoblot & densitometric analysis of p-AQP2 expression in the IM (fig 5A)

40. Immunoblot & densitometric analysis of p-AQP2 expression in the IM (fig 5B)

41. Immunoblot & densitometric analysis of AQP3 expression in the IM (fig 5C)

42. Immunoblot & densitometric analysis of AQP3 expression in the IM (fig 5D)

43. 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)

44. 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)

45. 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

46. Immunoperoxidase microscopy of AQP2 & p-AQP2 in the IM in the 4 groups of Brattleboro rats

47. Immunoperoxidase microscopy of AQP3 in the IM in the 4 groups of Brattleboro rats

48. Conclusion

49. 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

50. References • Andersen, LJ et al. Antidiuretic effect of subnormal levels of arginine vasopressin in normal humans. Am J Physiol 259: R53-R60, 1990. • Briner, VA et al. Comparative effects of arginine vasopressin and oxytocin in cell culture systerms. Am J Physiol 263: F222-F227, 1992. • Chou, CL et al. Oxytocin as an antidiuretic hormone: II, role of V2 vasopressin receptor. Am J Physiol 269: F78-F85, 1995. • Conrad, KP et al. Influence of oxytocin on renal hemodynamics and electrolyte and water excretion. Am J Physiol 251: F290-F296, 1986. • Kim, GH et al. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop. Am J Physiol 276: F96-F103, 1999. • Nielsen, S et al. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205-244, 2002. • Pouzet, B et al. Selective blockade of vasopressin V2 receptors reveals significant V2-mediated water reabsorption in Brattleboro rats with diabetes insipidus. Nephrol Dial Transplant 16: 725-734, 2001. • Serradeil-Le, GC et al. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729-2738, 1996.