Background: The kidney-specific isoform of the Na-K-2Cl cotransporter NKCC2 is involved in the Na+ reabsorption in the 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. Indeed both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for hypertension treatment. Accordingly a high-throughput screening for NKCC inhibitors is extremely useful in the development and characterization of new anti-hypertensive drugs. So far the high-throughput screening of NKCC transporters activity has been done by 86Rb+ influx assays. Methods: We developed a Tallium (Tl+) based fluorescent influx assay that can accurately and rapidly measure NKCC transport activity in adherent epithelial cells in the high-throughput Flex station device (FLEXA). 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). Results: In the absence of Cl- in the assay buffer, Tl+ addition did not induce any increase in fluorescence. However a robust Tl+ influx was observed after Cl-addition in c-NKCC2 transfected cells but not in mock-transfected or in parental LLC-PK1 cells suggesting that the Tl+ influx is actually mediated by the c-NKCC2 cotransporter. The c-NKCC2-driven FLEXA signal displays a rapid linear increased phase within the first 20 s after Cl- addition followed by a slower increase and a plateau phase. The initial rate of Tl+-dependent Cl- influx observed in c-NKCC2 transfected cells is about 3-fold over the background signal in mock-transfected LLC-PK1 cells. The preincubation with furosemide prevented the Cl-dependent Tl+ influx confirming the specificity of the NKCC-mediated Tl+ influx. Conclusions: We demonstrated that this assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, as a continuous assay, it offers improved sensitivity over endpoint NKCC functional assay. 
High-Throughput NKCC Functional Assay in Adherent Epithelial Cells
PROCINO, Giuseppe;SVELTO, Maria
2012-01-01
Abstract
Background: The kidney-specific isoform of the Na-K-2Cl cotransporter NKCC2 is involved in the Na+ reabsorption in the 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. Indeed both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for hypertension treatment. Accordingly a high-throughput screening for NKCC inhibitors is extremely useful in the development and characterization of new anti-hypertensive drugs. So far the high-throughput screening of NKCC transporters activity has been done by 86Rb+ influx assays. Methods: We developed a Tallium (Tl+) based fluorescent influx assay that can accurately and rapidly measure NKCC transport activity in adherent epithelial cells in the high-throughput Flex station device (FLEXA). 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). Results: In the absence of Cl- in the assay buffer, Tl+ addition did not induce any increase in fluorescence. However a robust Tl+ influx was observed after Cl-addition in c-NKCC2 transfected cells but not in mock-transfected or in parental LLC-PK1 cells suggesting that the Tl+ influx is actually mediated by the c-NKCC2 cotransporter. The c-NKCC2-driven FLEXA signal displays a rapid linear increased phase within the first 20 s after Cl- addition followed by a slower increase and a plateau phase. The initial rate of Tl+-dependent Cl- influx observed in c-NKCC2 transfected cells is about 3-fold over the background signal in mock-transfected LLC-PK1 cells. The preincubation with furosemide prevented the Cl-dependent Tl+ influx confirming the specificity of the NKCC-mediated Tl+ influx. Conclusions: We demonstrated that this assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, as a continuous assay, it offers improved sensitivity over endpoint NKCC functional assay. I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
