Transfection Alters Ion Transport in MDCK Cells |
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Authors: | DM Kaji J Bates JD Goyzueta K Prasadan H Yu S Kumar |
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Institution: | (1) Renal Section, Veterans Affairs Medical Center, 130 W. Kingsbridge Rd, Bronx, NY 10468, US;(2) Mount Sinai School of Medicine, New York, NY 10029, US;(3) ACGT, Northbrook, IL 60062, US;(4) Warren Medical Research Institute, University of Oklahoma, Oklahoma City, OK, US |
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Abstract: | In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection
of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an
``empty' plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to
transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection
procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished
in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected
with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the
transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied
by a proportional reduction in the abundance of Na-K pump units as assessed by 3H] ouabain binding. 3H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the
reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected
cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection
itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially
faulty and misleading interpretation.
Received: 25 April 1995/Revised: 25 September 1995 |
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Keywords: | : Uromodulin — Cation transport — Na-K-ATPase — Anion-dependent cotransport — MDCK — mTAL |
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