Email updates

Keep up to date with the latest news and content from BMC Cell Biology and BioMed Central.

Open Access Methodology article

High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells

Monica Carmosino12*, Federica Rizzo1, Silvia Torretta1, Giuseppe Procino1 and Maria Svelto1

Author affiliations

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Amendola 165/A, Bari, 70126, Italy

2 Department of Sciences, University of Basilicata, Via dell’Ateneo Lucano, Potenza, 85100, Italy

For all author emails, please log on.

Citation and License

BMC Cell Biology 2013, 14:16  doi:10.1186/1471-2121-14-16

Published: 18 March 2013

Abstract

Background

The kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na+ reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension.

Results

Taking advantage of an existing Thallium (Tl+)-based kit, we set up a Tl+ influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays.

Conclusions

So far the screening of NKCC transporters activity has been done by 86Rb+ influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

Keywords:
NKCC activity; Fluorescent assay; High-throughput; Drug screening