Strategy for the development of a matched set of transport-competent, angiotensin receptor-deficient proximal tubule cell lines

  • Philip G. Woost
  • Robert J. Kolb
  • Margaret Finesilver
  • Irene Mackraj
  • Hans Imboden
  • Thomas M. Coffman
  • Ulrich Hopfer
Articles Cell and Tissue Models

Summary

In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT1 and AT2. Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor-deficient mice were bred with an Immortomouse®, which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F2 mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor-deficient cell lines, (AT1A [−/−], Tag [+/−]), (AT1B[−/−], Tag [+/−]),and (AT1B[−/−], Tag [+/+]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; −3 to 3 μA/cm2), basal monolayer conductance (G, 2 to 10 mS/cm2), Nain3+-phosphate cotransport (ΔIsc of 2 to 3 μA/cm2 at 1 mM), and Nain3+-succinate contransport (ΔIsc of 1 to 9 μA/cm2 at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II-stimulated \-arrestin 2 translocation and showed an Ang II-mediated response in wild type but not (AT1A [−/ −], AT1B [−/−]) cells. Cell line were amplified, yielding a virtually unlimited supply of highly differentiated, transportcompetent, angiotensin receptor-deficient PCT cell lines.

Key words

cilium electrolyte transport epithelial cell line Immortomouse® proximal tubule SV40 large T-antigen 

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References

  1. Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular biology of the cell: cell junctions, cell adhesion, and the extracellular matrix, 4th ed. New York: Garland Science (Taylor & Francis Group); 2002:1065–1125.Google Scholar
  2. Allen, C. B.; Schneider, K.; White, C. W. Limitations to oxygen diffusion and equilibration in in vitro cell exposure sustems in hyperoxia and hypoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L1021-L1027; 2001.PubMedGoogle Scholar
  3. Barak, L. S.; Ferguson, S. S. G.; Zhang, J.; Caron, M. G. A \-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J. Biol. Chem. 272:27497–27500; 1997.PubMedCrossRefGoogle Scholar
  4. Baum, M.; Quigley, R. Maturation of rat proximal tubule chloride permeability. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289:R1659-R1664; 2005.PubMedGoogle Scholar
  5. de Casparo, M.; Catt, K. J.; Inagami, T.; Wright, J. W.; Unger, T. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol. Rev. 52:415–472; 2000.Google Scholar
  6. Dickman, K. G.; Mandel, L. J. Glycolytic and oxidative metabolism in primary renal proximal tubule cultures. Am. J. Physiol. Cell Physiol. 257:C333-C340; 1989.Google Scholar
  7. Dinh, D. T.; Frauman, A. G.; Johnston, C. I.; Fabian, M. E. Angiotensin receptors: distribution, signalling and function. Clin. Sci. (Lond.) 100:481–492; 2001.Google Scholar
  8. du Cheyron, D.; Chalumeau, C.; Defontaine, N.; Kleein, C.; Kellerman, O.; Paillard, M.; Poggioli, J. Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: role of PI 3-kinase. Kidney Int. 64:939–949; 2003.PubMedCrossRefGoogle Scholar
  9. Erkan, E.; Devarajan, P.; Schwartz, G. J. Apoptotic response to albumin overload: proximal vs. distal/collecting tubule cells. Am. J. Nephrol. 25:121–131; 2005.PubMedCrossRefGoogle Scholar
  10. Ferguson, S. S. G.; Caron, M. G. Green fluorescent protein-tagged \-arrestin translocation as a measure of G protein-coupled receptor activation. In: Smrcka, A. V., ed. methods in molecular biology: G protein signaling methods and protocols. Totowa, NJ, Humana Press; 2004; 121–126.Google Scholar
  11. Frei, N.; Weissenberger, J.; Beck-Sickinger, A. G.; Höfliger, M.; Weis, J.; Imboden, H., Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monolonal antibodies in the rat adrenal gland. Regul. Pept. 101:149–155; 2001.PubMedCrossRefGoogle Scholar
  12. Gross, E.; Hawkins, K.; Abuladze, N.; Puskhin, A.; Cotton, C. U.; Hopfer, U.; Kurtz, I. The stoichiometry of the electronic sodium bicarbonate cotransporter NBCI is cell-type dependent. J. Physiol. 531:597–603; 2001.PubMedCrossRefGoogle Scholar
  13. Hopfer, U.; Jacobberger, J. W.; Gruenert, D. C.; Eckert, R. L.; Jat, P. S.; Whitsett, J. A. Immortalization of epithelial cells. Am. J. Physiol. 270 (Cell Physiol. 39):C1-C11; 1996.PubMedGoogle Scholar
  14. Ichiki, T.; Labosky, P. A.; Shiota, C., et al. Effects on blood pressure and exploratory behavior of mice lacking angiotensin II type-2 receptor. Nature 377:748–750, 1995.PubMedCrossRefGoogle Scholar
  15. Inagami, T. Molecular biology and signaling of angiotensin receptors: an overview. J. Am. Soc. Nephrol. Suppl. 11:S2-S7; 1999.Google Scholar
  16. Ito, M.; Oliverio, M. I.; Mannon, P. J.; Best, C. F.; Maeda, N.; Smithies, O.; Coffman, T. M. Regalation of blood pressure by the type 1A angiotensin II receptor gene. Proc. Natl. Acad. Sci. USA 92:3521–3525; 1995.PubMedCrossRefGoogle Scholar
  17. Jat, P. S.; Noble, M. D.; Ataliotis, P.; Tanaka, Y.; Yannoutsos, N.; Larsen, L.; Kioussis, D. Direct derivation of conditionally immortal cell lines from an H-2K b-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA 88:5096–5100; 1991.PubMedCrossRefGoogle Scholar
  18. Kolb, R. J.; Woost, P. G.; Hopfer, U. Membrane trafficking of angiotensin receptor type-1 and mechanochemical signal transduction in proximal tubule cells. Hypertension 44:352–359; 2004.PubMedCrossRefGoogle Scholar
  19. Kriz, W.; Knissling, B. Structural organization of the mammalian kidney. In: Seldin, D. W.; Giebisch, G., ed. The kidney: physiology and pathophysiology, Vol. 1, 2nd ed. New York: Raven Press; 1992: 707–777.Google Scholar
  20. Lefkowitz, R. J.; Shenoy, S. K., Transduction of receptor signals by \-arrestins. Science 308:512–517; 2005.PubMedCrossRefGoogle Scholar
  21. Lin, F.; Cordes, K.; Li, L.; Hood, L.; Couser, W. G.; Shankland, S. J.; Igarashi, P. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-repefusion injury in mice. J. Am. Soc. Nephrol. 14:1188–1199; 2003.PubMedCrossRefGoogle Scholar
  22. Lutz, M. D.; Cardinal, J.; Burg, M. B. Electrical resistance of renal proximal tubule perfused in vitro. Am. J. Physiol. 225:729–734; 1973.PubMedGoogle Scholar
  23. Marshansky, V.; Bourgoin, S.; Londono, I.; Bendayan, M.; Vinay, P.. Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules. Electrophoresis 18:538–547; 1997.PubMedCrossRefGoogle Scholar
  24. Morais, C.; Westhuyzen, J.; Pat, B.; Gobe, G.; Healy, H. High ambient glucose is effect neutral on cell death and proliferation in human proximal tubule epithelial cells. Am. J. Physiol. Renal Physiol. 289:F401-F409; 2005.PubMedCrossRefGoogle Scholar
  25. Muller-Berger, S.; Nesterov, VV; Fromter, E. Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na−HCO3 cotransport stoichiometry and of response to acetazolamide. Pffugers Arch. 434:383–391; 1997.CrossRefGoogle Scholar
  26. Murer, H.; Hernando, N.; Forster, I.; Biber, J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol. Rev. 80:1373–1409; 2000.PubMedGoogle Scholar
  27. National Institutes of Health. Public health service policy on humane care and use of laboratory animals. National Institutes of Health: Office of Laboratory Animal Welfarel. Bethesda, MD; 2002.Google Scholar
  28. Navar, L. G.; Imig, J. D.; Zou, L.; Wang, C. T. Intrarenal production of angiotensin II Semin. Nephrol. 17:412–422; 1997.PubMedGoogle Scholar
  29. Nowak, G.; Schnellmann, R. G. Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells. Am. J. Physiol. 268(4) Pt 1):C1053-C1061; 1995.PubMedGoogle Scholar
  30. Oliverio, M. I.; Coffman, T. M. Angiotensin II receptor physiology using gene targeting. News Physio. Sci. 15:171–175; 2000.Google Scholar
  31. Oliverio, M. I.; Kim, H.-S.; Ito, M., et al. Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc. Natl. Acad. Sci. USA 95:15496–15501; 1998.PubMedCrossRefGoogle Scholar
  32. Orosz, D. E.; Woost, P.G.; Kolb, R. J., et al. Growth, immortalization, and differention potential of normal adult human proximal tubule cells. In Vitro Cell. Dev. Biol. 40A:22–34; 2004.CrossRefGoogle Scholar
  33. Pajor, A. M. Molecular properties of sodium/dicarboxylate cotransporters, J. Membrane Biol. 175:1–8; 2000.CrossRefGoogle Scholar
  34. Panchapakesan, U.; Sumual, S.; Pollock, C. A.; Chen, X. PPARgamma agonists exert antifibrotic effects in renal tubular cells exposed to high glucose. Am. J. Physiol. Renal Physiol. 289:F1153-F1158; 2005.PubMedCrossRefGoogle Scholar
  35. Praetorius, H. A.; Frokiaer, J.; Neilsen, S.; Spring, K. R. Bending the primary cilium opens Ca2+-sensitive intermediate-conductance K+ channels in MDCK cells. J. Membr. Biol. 191:193–200; 2003.PubMedCrossRefGoogle Scholar
  36. Praetorius, H. A.; Spring, K. R. Bending of the MDCK primary cilium increases intracellular calcium. J Membr.. Biol. 184:71–79; 2001.PubMedCrossRefGoogle Scholar
  37. Quan, A.; Baum, M. Regulation of proximal tubule transport by endogenously produced angiotensin II. Nephron 84:103–110; 2000.PubMedCrossRefGoogle Scholar
  38. Romero, M. F.; Douglas, J. G.; Eckert, R. L.; Hopfer, U.; Jacobberger, J. W. Development and characterization of rabbit proximal tubular epithelial cell lines. Kidney Int. 42:1130–1144; 1992.PubMedGoogle Scholar
  39. Scherberich, J. E.; Gauhl, C.; Mondorf, W. Biochemical, immunological and ultrastructural studies on brush-border membranes of human kidney. Curr. Probl. Clin. Biochem. 8:85–95; 1977.PubMedGoogle Scholar
  40. Shenoy, S. K.; Lefkowitz, R. J. Receptor-specific ubiquination of \-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes. J. Biol. Chem. 280:15315–15324; 2005.PubMedCrossRefGoogle Scholar
  41. Todd, J. H.; Sens, M. A.; Hazen-Martin, D. J.; Bylander, J. E.; Smyth, B. J.; Sens, D. A. Variation in the electrical properties of cultured human proximal tubule cells. In Vitro Cell. Dev. Biol. 29A:371–378; 1993.Google Scholar
  42. Touyz, R. M.; Berry, C. Recent advances in angiotensin II signaling. Braz. J. Med. Biol. Res. 35:1001–1015; 2002.PubMedCrossRefGoogle Scholar
  43. Wolff, M.; Fandrey, J.; Jelkmann, W. Microelectrode measurements of pericellular P02 in erythropoietin-producing huma hepatoma cell cultures. Am. J. Physiol. Cell Physiol. 265:C1266-C1270; 1993.Google Scholar
  44. Woost, P. G.; Orosz, D. E.; Jin, W.; Frisa, P. S.; Jacobberger, J. W.; Douglas, J. G.; Hopfer, U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int. 50:125–134; 1996.PubMedGoogle Scholar
  45. Xu, J.; Li, X. X.; Albrecht, F. E.; Hopfer, U.; Carey, R. M.; Jose, P. A. Dopamine receptor, G and Na+-H+ exchanger interactions in the kidney in hypertension. Hypertension 36:395–399; 2000.PubMedGoogle Scholar
  46. Yeager, T. R.; Reddel, R. R. Constructing immortalized human cell lines. Curr. Opin. Biotech. 10:465–469; 1999.PubMedCrossRefGoogle Scholar
  47. Yu, P.; Asico, L. D.; Eisner, G. M.; Hopfer, U.; Felder, R. A.; Jose, P. A. Renal protein phosphatase 2A activity and spontaneous hypertension in rats. Hypertension 36:1053–1058; 2000.PubMedGoogle Scholar
  48. Zeng, C.; Asico, L. D.; Wang, X.; Hopfer, U.; Eisner, G. M.; Felder, R. A.; Jose, P. A. Angiotensin II regulation of AT1 and D3 dopamine receptors in renal proximal tubule cells of SHR. Hypertension 41:724–729; 2003a.PubMedCrossRefGoogle Scholar
  49. Zeng, C.; Luo, Y.; Asico, L. D.; Hopfer, U.; Eisner, G.M.; Felder, R. A.; Jose, P. A. Perturbation of D1 dopamine and AT1 receptor interaction in spontaneously hypertensive rats. Hypertension 42:787–792; 2003b.PubMedCrossRefGoogle Scholar
  50. Zeng, C.; Wang, D.; Asico, L. D.; Welch, W. J.; Wilcox, C. S.; Hopfer, U.; Eisner, G. M.; Felder, R. A.; Jose, P. A. Aberrant D1 and D3 dopamine receptor transregulation in hypertension. Hypertension 43:654–660; 2004.PubMedCrossRefGoogle Scholar
  51. Zhu, Z.; Zhang, S. H.; Wagner, C.; Kurtz, A.; Maeda, N.; Coffman, T.; Arendshorst, W. J. Angiotensin AT1B receptor mediates calcium signaling in vascular smooth muscle cells of AT1A receptor-deficient mice. Hypertension 31:1171–1177; 1998.PubMedGoogle Scholar

Copyright information

© Society for In Vitro Biology 2006

Authors and Affiliations

  • Philip G. Woost
    • 1
  • Robert J. Kolb
    • 2
  • Margaret Finesilver
    • 1
  • Irene Mackraj
    • 3
  • Hans Imboden
    • 4
  • Thomas M. Coffman
    • 5
  • Ulrich Hopfer
    • 1
  1. 1.Department of Physiology and BiophysicsCase Western Reserve UniversityCleveland
  2. 2.Harvard Medical SchoolBrigham and Women's HospitalBoston
  3. 3.Department of Human Physiology and Physiological Chemistry, School of Basic and Applied Medical SciencesUniversity of Durban-WestvilleDurbanSouth Africa (I. M.)
  4. 4.Institute of ZoologyUniversity of BerneBerneSwitzerland
  5. 5.Department of Medicine-NephrologyDuke University and Durham Veterans Affair Medical CentersDurham

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