Urea transport in MDCK cells that are stably transfected with UT-A1
Urea transport in MDCK cells that are stably transfected with UT-A1
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DOI：
10.1152/ajpcell.00499.2003
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发表时间：
2004-06-01
影响因子：
5.5
通讯作者：
Gunn, RB
中科院分区：
文献类型：
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作者：
Fröhlich, O;Klein, JD;Gunn, RB
由于缺乏合适的细胞培养系统，对尿素转运蛋白的细胞生物学研究进展受阻。本研究的目的是建立一个极化上皮细胞系，稳定表达最大的大鼠肾尿素转运蛋白UT-A亚型，UT-A1。将UT-A1基因克隆到pcDNA 5/FRT中，并转染具有整合的Flp重组靶位点的Madin-Darby犬肾（MDCK）细胞。将来自单个克隆的细胞在胶原蛋白包被的膜上生长至汇合，直到阻力> 1，500 Omega.cm（2）。在37 ℃下，在含5 mM尿素的HCO 3-/CO2缓冲液（pH 7.4）中测量跨上皮[C-14]尿素通量。未刺激的UT-A1转染的MDCK细胞和未转染或假转染的MDCK细胞之间的基线通量没有差异。然而，只有在UT-A1转染细胞的UT-A1蛋白表达（通过Western印迹分析测量）和尿素转运刺激毛喉素或精氨酸加压素。毛喉素和精氨酸加压素也增加了UT-A1的磷酸化。硫代烟酰胺、二甲基脲和根皮素抑制毛喉素刺激的UT-A1转染MDCK细胞中的[C-14]尿素通量。这些特征与大鼠终末内髓集合管相似。这种新的极化上皮细胞系稳定表达UT-A1，并再现了在大鼠终末内髓集合管中观察到的几种生理反应。
Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was > 1,500 Omega.cm(2). Transepithelial [C-14] urea fluxes were measured at 37degreesC in a HCO3-/CO2 buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed ( as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [C-14]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.