Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct


Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of α-1, β-1, and β-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased α-1, β-1, and β-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of β-1, but not that of α-1 or β-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.


  1. 1

    Masilamani S, Knepper MA, Burg MB : Urine concentration and dilution, in Brenner BM ( ed): The Kidney, 6th ed. Philadelphia, WB Saunders, 2000, pp 595–635.

    Google Scholar 

  2. 2

    Knepper M, Burg M : Organizationof nephron function. Am J Physiol 1983; 244: F579–F589.

    CAS  PubMed  Google Scholar 

  3. 3

    Bertorello AM, Katz AI : Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am J Physiol 1994; 265: F743–F755.

    Google Scholar 

  4. 4

    Feraille E, Doucet A : Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 2001; 81: 345–418.

    CAS  Article  Google Scholar 

  5. 5

    Blanco G, Mercer RW : Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 1998; 275: F633–F650.

    CAS  Article  Google Scholar 

  6. 6

    Sweadner KJ : Isozymes of the Na+-K +-ATPase. Biochim Biophys Acta 1989; 988: 185–220.

    CAS  Article  Google Scholar 

  7. 7

    Herrera VLM, Emanuel JR, Ruiz-Opazo N, Levenson N, Nadal-Ginard B : Three differentially expressed Na,K-ATPase a subunit isoforms: structural and functional implications. J Cell Biol 1987; 105: 1855–1865.

    CAS  Article  Google Scholar 

  8. 8

    Martin-Vassallo P, Dackowski W, Emanuel JR, Levenson R : Identification of a putative isoform of the Na,K-ATPase β subunit. Primary structure and tissue-specific expression. J Biol Chem 1989; 264: 4613–4618.

    Google Scholar 

  9. 9

    Mercer RW, Schneider JW, Savita A, Emanuel J, Benz EJ, Levenson R : Rat brain Na,K-ATPase β-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells. Mol Cell Biol 1986; 6: 3884–3890.

    CAS  Article  Google Scholar 

  10. 10

    Orlowski J, Lingrel JB : Tissue-specific and developmental regulation of rat Na+-K+-ATPase catalytic α-isoform and β-subunit mRNAs. J Biol Chem 1988; 263: 10436–10442.

    CAS  PubMed  Google Scholar 

  11. 11

    Shull GE, Greeb J, Lingrel JB : Molecular cloning of three distinct forms of the Na+-K+-ATPase α-subunit from rat brain. Biochemistry 1986; 25: 8125–8132.

    CAS  Article  Google Scholar 

  12. 12

    Young RM, Shull GE, Lingrel JB : Multiple mRNAs from rat kidney and brain encode a single Na+,K +-ATPase β subuint protein. J Biol Chem 1987; 262: 4905–4910.

    CAS  PubMed  Google Scholar 

  13. 13

    Mercer RW, Biemesderfer D, Bliss DP Jr, Collins JH, Forbush B 3rd : Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase. J Cell Biol 1993; 121: 579–586.

    CAS  Article  Google Scholar 

  14. 14

    Ahn KY, Madsen KM, Tisher CC, Kone BC : Differential expression and cellular distribution of mRNAs encoding α- and β-isoforms of Na+-K +-ATPase in rat kidney. Am J Physiol 1994; 265: F792–F801.

    Google Scholar 

  15. 15

    Farman N, Coutry N, Logvinenko N, Blot-Chabaud M, Bourbouze R, Bonvalet JP : Adrenalectomy reduces α1 and not β1 Na+-K+-ATPase mRNA expression in rat distal nephron. Am J Physiol 1992; 263: C810–C817.

    CAS  Article  Google Scholar 

  16. 16

    Tumlin JA, Hoban CA, Medford RM, Sands JM : Expression of Na-K-ATPase α- and β-subunit mRNA and protein isoforms in the rat nephron. Am J Physiol 1994; 266: F240–F245.

    CAS  PubMed  Google Scholar 

  17. 17

    Takayama M, Nonoguchi H, Yang T, et al: Acute and chronic effects of hyperosmolality on mRNA and protein expression and the activity of Na-K-ATPase in the IMCD. Exp Nephrol 1999; 7: 295–305.

    CAS  Article  Google Scholar 

  18. 18

    Pu HX, Cluzeaud F, Goldshleger R, Karlish SJD, Farman N, Blostein R : Functioinal role and immunocytochemical localization of the γa and γb forms of the Na,K-ATPase γ subunit. J Biol Chem 2001; 276: 20370–20378.

    CAS  Article  Google Scholar 

  19. 19

    Arystarkhova E, Wetzel RK, Sweadner KJ : Distribution and oligomeric association of splice forms of Na+-K +-ATPase regulatory γ-subunit in rat kidney. Am J Physiol Renal Physiol 2002; 282: F393–F407.

    CAS  Article  Google Scholar 

  20. 20

    Greger R : Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev 1985; 65: 760–797.

    CAS  Article  Google Scholar 

  21. 21

    Woods RL, Johnston CI : Contribution of vasopressin to the maintenance of blood pressure during dehydration. Am J Physiol 1983; 245: F615–F621.

    CAS  PubMed  Google Scholar 

  22. 22

    Imbert M, Chabardes D, Montegut M, Clique A, Morel F : Vasopressin dependent adenylate cyclase in single segments of rabbit kidney tubule. Pflügers Arch 1975; 357: 173–186.

    CAS  Article  Google Scholar 

  23. 23

    Ecelbarger CA, Kim G-H, Wade J, Knepper MA : Regulation of abundance of renal sodium transporters and channels by vasopressin. Exp Neurol 2001; 171: 227–234.

    CAS  Article  Google Scholar 

  24. 24

    Imanishi K, Nonoguchi H, Nakayama Y, Machida K, Ikebe M, Tomita K : Type 1A angiotensin II receptor is regulated differently in proximal and distal nephron segments. Hypertens Res 2003; 26: 405–411.

    CAS  Article  Google Scholar 

  25. 25

    Nonoguchi H, Tomita K, Marumo F : Effects of atrial natriuretic peptide and vasopressin on chloride transport in long- and short-looped medullary thick ascending limbs. J Clin Invest 1992; 90: 349–357.

    CAS  Article  Google Scholar 

  26. 26

    O'Neil RG, Dubinsky WP : Micromethodology for measuring ATPase activity in renal tubules: mineralocorticiod influence. Am J Physiol 1984; 247: C314–C320.

    CAS  Article  Google Scholar 

  27. 27

    Tomita K, Owada A, Iino Y, Yoshiyama N, Shiigai T : Effect of vasopressin on Na+-K+-ATPase activity in rat cortical collecting duct. Am J Physiol 1987; 253: F874–F879.

    CAS  PubMed  Google Scholar 

  28. 28

    Fort P, Marty L, Picchaczyk M, et al: Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigene family. Nucl Acids Res 1985; 13: 1431–1442.

    CAS  Article  Google Scholar 

  29. 29

    Owada A, Nonoguchi H, Terada Y, Marumo F, Tomita K : Microlocalization and effects of adrenomedullin in nephron segments and in mesangial cells of the rat. Am J Physiol Renal Physiol 1997; 272: F691–F697.

    CAS  Article  Google Scholar 

  30. 30

    Nonoguchi H, Knepper MA, Manganiello VC : Effects of atrial natriuretic factor on cyclic guanosine monophosphate and cyclic adenosine monophosphate accumulation in microdissected nephron segments from rats. J Clin Invest 1987; 79: 500–507.

    CAS  Article  Google Scholar 

  31. 31

    Tian G, Dang C, Lu Z : The change and significance of the Na+-K+-ATPase alpha-subunit in ouabain-hypertensive rats. Hypertens Res 2001; 24: 729–734.

    CAS  Article  Google Scholar 

  32. 32

    Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F : Different localization and regulation of two types of vasopressin receptor messenger RNA in microdissected rat nephron segments using reverse transcription polymerase chain reaction. J Clin Invest 1993; 92: 2339–2345.

    CAS  Article  Google Scholar 

  33. 33

    Tashima Y, Kohda Y, Nonoguchi H, et al: Intranephron localization and regulation of the V1a vasopressin receptor during chronic metabolic acidosis and dehydration in rats. Pflügers Arch 2001; 442: 652–661.

    CAS  Article  Google Scholar 

  34. 34

    Kikeri D, Sun A, Zeidel MK, Hebert SC : Cell membrane impermeable to NH3 . Nature 1989; 339: 478–480.

    CAS  Article  Google Scholar 

  35. 35

    Bankir L, de Rouffignac C : Urinary concentrating ability: insights from comparative anatomy. Am J Physiol 1985; 249: R643–R666.

    CAS  PubMed  Google Scholar 

  36. 36

    Hinojos CA, Doris PA : Altered subcellular distribution of Na+,K+-ATPase in proximal tubules in young spontaneously hypertensive rats. Hypertension 2004; 44: 95–100.

    CAS  Article  Google Scholar 

  37. 37

    Tsuchida H, Imai G, Shima Y, Satoh T, Owada S : Mechanism of sodium load-induced hypertension in non-insulin dependent diabetes mellitus model rats: defective dopaminergic system to inhibit Na-K-ATPase activity in renal epithelial cells. Hypertens Res 2001; 24: 127–135.

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Hiroshi Nonoguchi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sakuma, Y., Nonoguchi, H., Takayama, M. et al. Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct. Hypertens Res 28, 671–679 (2005). https://doi.org/10.1291/hypres.28.671

Download citation


  • hyperosmolality
  • Na-K-ATPase
  • cAMP
  • medullary thick ascending limb
  • outer medullary collecting duct

Further reading