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Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide

  • Ion Channels, Transporters
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Abstract

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV40 T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.

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Acknowledgements

Part of this work was presented in an abstract form at the 1998 and 1999 meetings of the American Society of Nephrology. We thank Dr S. Grinstein for antiNHE1 antibody, Dr O. W. Moe for antiNHE3 antibody, Dr P. Méneton for the NHE2 null mice, Dr H. L. Lang for HOECHST 642, C. Rismondo, S. Demaretz and D. Lamblin for excellent technical assistance, M. F. Belair who performed electron microscopy and P. Kitmacher for photographs.

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Authors and Affiliations

  1. INSERM Unité 356, IFR 58 , Université Paris VI, Institut des Cordeliers, 15–21 rue de l'Ecole de Médecine, 75270, Paris Cedex 6, France

    Soline Bourgeois, Patrick Rossignol, Françoise Grelac, Cécile Chalumeau, Christophe Klein, Kamel Laghmani, Régine Chambrey, Josiane Poggioli, Pascal Houillier, Michel Paillard & Marc Froissart

  2. INSERM Unité 430, IFR 58, Université Paris VI, 15 rue de l'Ecole de Médecine , 75270 , Paris Cedex 06, France

    Patrick Bruneval & Jean-Paul Duong

  3. Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, 20 rue Leblanc, 75908, Paris Cedex 15, France

    Patrick Bruneval, Jean-Paul Duong, Pascal Houillier, Michel Paillard & Marc Froissart

  4. CNRS UPR 1983, Institut Lwoff, 7 rue Guy Mocquet, BP8, 94801, Villejuif, France

    Odile Kellermann

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  1. Soline Bourgeois
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Correspondence to Soline Bourgeois.

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Bourgeois, S., Rossignol, P., Grelac, F. et al. Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide. Pflugers Arch - Eur J Physiol 446, 672–683 (2003). https://doi.org/10.1007/s00424-003-1108-x

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