Harvest and primary culture of the murine aldosterone-sensitive distal nephron.
Harvest and primary culture of the murine aldosterone-sensitive distal nephron.
复制标题
鼠醛固酮敏感远端肾单位的收获和原代培养。
DOI：
10.1152/ajprenal.00668.2014
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Bhalla,Vivek
中科院分区：
文献类型：
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作者：
Labarca,Mariana;Nizar,JonathanM;Walczak,ElisabethM;Dong,Wuxing;Pao,AlanC;Bhalla,Vivek
醛固酮敏感性远端肾单位（ASDN）从远曲小管到髓质集合管在结构和功能上具有轴向异质性。离子和水的运输主要分别在ASDN的皮质和髓质之间进行。这一部分的跨细胞运输在健康和疾病中受到高度调节，并在不同的细胞类型中整合。我们目前缺乏一个廉价的，高产的，易于处理的技术来收获和培养细胞的基因表达和生理特性的研究小鼠皮质ASDN。为了解决这一问题，我们收获了肾小管绑定toDolichos biflorus凝集素包被磁珠从肾皮质和这些细胞制剂的特点。我们确定这些细胞富含远曲小管、连接小管和皮质集合管的标记物，包括主细胞和闰细胞。在原代培养中，这些细胞形成具有高电阻（1，000 - 1，500 Ω * cm 2）的极化单层，并保持关键通道的表达和活性。这些细胞表现出阿米洛利敏感的短路电流，可以与醛固酮增强，并保持可测量的钾和阴离子分泌。我们的方法可以很容易地通过研究ASDN的生物学和野生型和转基因小鼠模型之间的表型差异进行调查。
The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound toDolichos biflorus agglutininlectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm2) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.