A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing
A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing
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DOI：
10.1007/s00424-013-1321-1
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发表时间：
2014-02-01
影响因子：
4.5
通讯作者：
Devuyst, Olivier
中科院分区：
文献类型：
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作者：
Glaudemans, Bob;Terryn, Sara;Devuyst, Olivier
亨利氏袢的粗升支（TAL）的上皮细胞内衬进行必要的运输过程和分泌尿调素，在正常尿液中最丰富的蛋白质。缺乏分化的细胞培养系统阻碍了TAL功能的研究。在这里，我们报告了一种方法，以产生分化的原代培养的TAL细胞，从小鼠肾脏中获得的微解剖小管开发。TAL小管在渗透性滤膜上培养约12天形成极化的汇合单层。TAL细胞保持分化，并在顶膜表达功能标记物，如尿调蛋白、NKCC 2和ROMK。对原代TAL单层的电生理学测量显示管腔阳性跨上皮电位（+9.4 +/- 0.8 mV/cm（2））和跨上皮电阻与体内记录的相似。在从ROMK敲除小鼠获得的原代培养物中，顶端布美他尼消除了跨上皮电位。原代TAL细胞对尿调素的加工、成熟和顶端分泌与体内观察到的相同。原代TAL细胞对缺氧、高渗和去氨加压素刺激有适当的反应，并且它们可以被转染。这种原代培养系统的建立将允许对从转基因小鼠模型中获得的TAL细胞进行研究，为了解该部分在健康和疾病中的作用提供了关键工具。
The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in similar to 12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 +/- 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.