Strategy for the development of a matched set of transport-competent, angiotensin receptor-deficient proximal tubule cell lines |
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Authors: | Philip G Woost Robert J Kolb Margaret Finesilver Irene Mackraj Hans Imboden Thomas M Coffman Ulrich Hopfer |
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Institution: | (1) Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, 44106-4970 Cleveland, Ohio;(2) Harvard Medical School, Brigham and Women's Hospital, 4 Blackfan Circle, Room 520, 02115 Boston, Massachusetts;(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.);(4) Institute of Zoology, University of Berne, Baltzerstrasse 6, 3012 Berne, Switzerland;(5) Department of Medicine-Nephrology, Duke University and Durham Veterans Affair Medical Centers, 27705 Durham, North Carolina |
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Abstract: | In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT(1) and AT(2). 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(R), which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F(2) 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, (AT(1A) -/-], Tag +/-]), (AT(1B) -/-], Tag +/-]), and (AT(1A) -/-], AT(1B) -/-], 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 microA/cm2), basal monolayer conductance (G, 2 to 10 mS/cm2), Na3(+)-phosphate cotransport (DeltaIsc of 2 to 3 microA/cm(2) at 1 mM), and Na(3)(+)-succinate cotransport (DeltaIsc of 1 to 9 microA/cm(2) 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 beta-arrestin 2 translocation and showed an Ang II-mediated response in wild type but not (AT(1A) -/ -], AT(1B) -/-]) cells. Cell lines were amplified, yielding a virtually unlimited supply of highly differentiated, transport-competent, angiotensin receptor-deficient PCT cell lines. |
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Keywords: | cilium electrolyte transport epithelial cell line Immortomouse? proximal tubule SV40 large T-antigen |
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