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

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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), Na +in3 -phosphate cotransport (ΔIsc of 2 to 3 μA/cm2 at 1 mM), and Na +in3 -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.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, 44106-4970, Cleveland, Ohio

    Philip G. Woost, Margaret Finesilver & Ulrich Hopfer

  2. Harvard Medical School, Brigham and Women's Hospital, 4 Blackfan Circle, Room 520, 02115, Boston, Massachusetts

    Robert J. Kolb

  3. Department of Human Physiology and Physiological Chemistry, School of Basic and Applied Medical Sciences, University of Durban-Westville, Private Bag X54001, 4001, Durban, South Africa (I. M.)

    Irene Mackraj

  4. Institute of Zoology, University of Berne, Baltzerstrasse 6, 3012, Berne, Switzerland

    Hans Imboden

  5. Department of Medicine-Nephrology, Duke University and Durham Veterans Affair Medical Centers, 27705, Durham, North Carolina

    Thomas M. Coffman

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  1. Philip G. Woost
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  2. Robert J. Kolb
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  3. Margaret Finesilver
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  4. Irene Mackraj
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  7. Ulrich Hopfer
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Correspondence to Philip G. Woost.

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Woost, P.G., Kolb, R.J., Finesilver, M. et al. Strategy for the development of a matched set of transport-competent, angiotensin receptor-deficient proximal tubule cell lines. In Vitro Cell.Dev.Biol.-Animal 42, 189–200 (2006). https://doi.org/10.1290/0511076.1

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