Methyl-beta-cyclodextrin induces vasopressin-independent apical accumulation of aquaporin-2 in the isolated, perfused rat kidney.
Methyl-beta-cyclodextrin induces vasopressin-independent apical accumulation of aquaporin-2 in the isolated, perfused rat kidney.
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发表时间：
2006
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通讯作者：
L. M. Russo;M. Mckee;Dennis Brown
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作者：
L. M. Russo;M. Mckee;Dennis Brown
加压素通过刺激集合管主细胞中水通道蛋白-2（AQP 2）的质膜积累来增加尿液浓度，允许大量水沿着渗透梯度从管腔穿过集合管流到管腔。血管加压素2型受体（V2 R）的突变导致遗传性X连锁肾源性尿崩症（NDI），这是一种以排尿过多和脱水为特征的疾病。最近，我们发现，胆固醇消耗药物甲基-β-环糊精（mbetaCD）的内吞抑制诱导AQP 2在过表达表位标记的AQP 2的转染肾上皮细胞中的质膜积累。在这里，我们询问mbetaCD是否可以使用离体灌注肾（IPK）原位诱导AQP 2的膜积聚。通过免疫荧光和电子显微镜，我们发现，AQP 2从一个主要的细胞内定位转移到主细胞的顶膜后1小时灌注Sprague-Dawley大鼠肾脏与5 mM mbetaCD。定量染色显示，AQP 2的强度从647+/-114（对照）增加到1，968 +/-299单位（mbetaCD; P<0.001），与用4 nM dDAVP灌注后观察到的效果相似（1，860 +/-298，P<0.001）。在加压素缺乏的Brattleboro大鼠的肾脏灌注mbetaCD后观察到类似的变化。未检测到mbetaCD处理对AQP 3和AQP 4的基底外侧分布的影响。这些数据表明，AQP 2组成性地在主细胞中的顶端膜和胞内囊泡之间原位地粘附，并且通过抑制AQP 2内吞作用诱导顶端AQP 2积累是绕过X连锁NDI中缺陷的V2 R信号传导途径的可行目标。
Vasopressin increases urine concentration by stimulating plasma membrane accumulation of aquaporin-2 (AQP2) in collecting duct principal cells, allowing bulk water flow across the collecting duct from lumen to interstitium down an osmotic gradient. Mutations in the vasopressin type 2 receptor (V2R) cause hereditary X-linked nephrogenic diabetes insipidus (NDI), a disease characterized by excessive urination and dehydration. Recently, we showed that inhibition of endocytosis by the cholesterol-depleting drug methyl-beta-cyclodextrin (mbetaCD) induces plasma membrane accumulation of AQP2 in transfected renal epithelial cells overexpressing epitope-tagged AQP2. Here, we asked whether mbetaCD could induce membrane accumulation of AQP2 in situ using the isolated, perfused kidney (IPK). By immunofluorescence and electron microscopy, we show that AQP2 was shifted from a predominantly intracellular localization to the apical membrane of principal cells following 1-h perfusion of Sprague-Dawley rat kidneys with 5 mM mbetaCD. Quantification of staining revealed that the intensity of AQP2 was increased from 647+/-114 (control) to 1,968+/-299 units (mbetaCD; P<0.001), an effect similar to that seen after perfusion with 4 nM dDAVP (1,860+/-298, P<0.001). Similar changes were observed following mbetaCD perfusion of kidneys from vasopressin-deficient Brattleboro rats. No effect of mbetaCD treatment on the basolateral distribution of AQP3 and AQP4 was detected. These data indicate that AQP2 constitutively recycles between the apical membrane and intracellular vesicles in principal cells in situ and that inducing apical AQP2 accumulation by inhibiting AQP2 endocytosis is a feasible goal for bypassing the defective V2R signaling pathway in X-linked NDI.