Shaw Stephen, Marples David
School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, United Kingdom.
Am J Physiol Renal Physiol. 2002 Nov;283(5):F1160-6. doi: 10.1152/ajprenal.00207.2002.
AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.
血管加压素（AVP）通过诱导特定水通道蛋白2（AQP2）水通道从细胞质囊泡转运至主细胞的顶端质膜，增加肾集合管的渗透水通透性。在此，我们报道了一种用于研究此现象的新型髓质内肾小管悬浮液，它克服了现有技术面临的一些缺点；髓质内集合管细胞的原代培养物和表达AQP2的细胞系均显示出异常的转运和/或信号通路。肾小管悬浮液通过对从新鲜分离的大鼠肾脏中解剖出的髓质进行蛋白水解消化制备。药物处理后，通过膜分级分离和蛋白质印迹法或免疫细胞化学法确定AQP2的细胞分布。用1 nM AVP处理悬浮液导致AQP2重新分布到主细胞的顶端质膜，这一过程受到微管破坏或蛋白激酶A（PKA）抑制的抑制。我们得出结论，该方法为研究集合管对AVP反应所涉及的细胞机制提供了一种有价值的新方法。
