title: High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.
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 (86)Rb(+) 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.
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Amendola 165/A, Bari 70126, Italy. monica.carmosino@uniba.it
authors.forename: Monica
authors.initials: M
authors.lastname: Carmosino
authors.forename: Federica
authors.initials: F
authors.lastname: Rizzo
authors.forename: Silvia
authors.initials: S
authors.lastname: Torretta
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Procino
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D004847:Epithelial Cells / Q000378:metabolism*; D019085:Fluorescent Antibody Technique, Direct; D057166:High-Throughput Screening Assays; D018374:LLC-PK1 Cells; D008856:Microscopy, Fluorescence; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013552:Swine; D014162:Transfection
chemical_list: D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1