High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.
High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.
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DOI：
10.1186/1471-2121-14-16
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发表时间：
2013-03-18
期刊：
影响因子：
--
通讯作者：
Svelto M
中科院分区：
文献类型：
--
作者：
Carmosino M;Rizzo F;Torretta S;Procino G;Svelto M
肾脏特异性NKCC协同转运蛋白亚型NKCC 2参与Thich Ascending Limb（TAL）细胞中的Na+重吸收和体液体积的调节。相比之下，亚型NKCC 1代表了内皮细胞中Cl-进入的主要途径，在细胞体积调节和血管张力中起着至关重要的作用。重要的是，这两种NKCC亚型都参与血压的调节，并代表了治疗高血压的重要潜在药物靶点。利用现有的基于铊（Tl+）的试剂盒，我们建立了基于Tl+流入的荧光测定，其可以使用高通量Flex站装置准确且快速地测量粘附上皮细胞中的NKCC转运蛋白活性。我们评估了该试验在肾上皮LLC-PK 1细胞中的可行性，该细胞稳定转染了先前表征的嵌合NKCC 2构建体（c-NKCC 2）。我们证明了该测定具有高度可重复性，提供了NKCC介导的离子通量曲线的高时间分辨率，重要的是，作为一种连续测定，它提供了比以前的终点NKCC功能测定更高的灵敏度。到目前为止，NKCC转运蛋白活性的筛选已经通过86 Rb+内流测定进行。事实上，基于荧光的高通量筛选方法用于测试NKCC抑制剂将是非常有用的开发和表征新的抗高血压药物。
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. 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. 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.
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影响因子：
3.3
作者：
Carmosino, Monica;Gimenez, Ignacio;Forbush, Biff
通讯作者：
Forbush, Biff
DOI：
10.1152/ajprenal.00107.2002
发表时间：
2002-11-01
影响因子：
4.2
作者：
Gagnon, É;Forbush, B;Isenring, P
通讯作者：
Isenring, P
影响因子：
5.8
作者：
Karczewski, Jerzy;Kiss, Laszlo;Spencer, Robert H.
通讯作者：
Spencer, Robert H.
影响因子：
1.8
作者：
Schmalhofer, William A.;Swensen, Andrew M.;Garcia, Maria L.
通讯作者：
Garcia, Maria L.
影响因子：
4.8
作者：
Benziane, Boubacar;Demaretz, Sylvie;Laghmani, Kamel
通讯作者：
Laghmani, Kamel