Marples D, Christensen B M, Frokiaer J, Knepper M A, Nielsen S
Physiology Department, University of Leeds, Leeds LS2 9NQ, United Kingdom.
Am J Physiol. 1998 Sep;275(3):F400-9. doi: 10.1152/ajprenal.1998.275.3.F400.
To examine the involvement of vasopressin and dehydration in the regulation of aquaporin-2 (AQP2) expression in rat kidney, we investigated the effects of treatment for 60 h with the specific V2-receptor antagonist OPC-31260 (OPC), alone and in conjunction with dehydration for the last 12 h. Changes in AQP2 protein and mRNA expression in kidney inner medulla were determined by Western and Northern blotting, and AQP2 distribution was analyzed by immunocytochemistry and immunoelectron microscopy. Treatment with OPC increased urine output fourfold, with a reciprocal decrease in urine osmolality. AQP2 expression decreased to 52 +/- 11% of control levels (n = 12, P < 0.05), and AQP2 was found predominantly in intracellular vesicles in collecting duct principal cells. This is consistent with efficient blockade of the vasopressin-induced AQP2 delivery to the plasma membrane and with the observed increased diuresis. Consistent with this, AQP2 mRNA levels were also reduced in response to prolonged OPC treatment (30 +/- 10% of control levels, n = 9). Five days of treatment with furosemide, despite producing even greater polyuria than OPC, was not associated with downregulation of AQP2 levels, demonstrating that AQP2 downregulation is not secondary to increased urine flow rate or loss of medullary hypertonicity. During 12-h thirsting in the continued presence of OPC, urine output dropped dramatically, to levels not significantly different from that seen in (nonthirsted) control animals. In parallel with this, AQP2 levels rose to control levels. Control experiments confirmed continued effective receptor blockade. These results indicate that the V2-receptor antagonist causes a modest decrease in AQP2 expression that is not a consequence of increased urine flow rate or washout of medullary hypertonicity. However, this decrease is much less marked than that seen in some forms of acquired nephrogenic diabetes insipidus. In conjunction with the effects of thirsting, this suggests that modulation of AQP2 expression is mediated partly, but not exclusively, via V2 receptors.
为研究血管加压素和脱水在大鼠肾脏水通道蛋白-2（AQP2）表达调节中的作用，我们分别研究了用特异性V2受体拮抗剂OPC-31260（OPC）单独处理60小时以及在最后12小时联合脱水处理的效果。通过蛋白质免疫印迹法和Northern印迹法测定肾内髓质中AQP2蛋白和mRNA表达的变化，并用免疫细胞化学和免疫电子显微镜分析AQP2的分布。用OPC处理使尿量增加了四倍，尿渗透压相应降低。AQP2表达降至对照水平的52±11%（n = 12，P < 0.05），且AQP2主要存在于集合管主细胞的细胞内囊泡中。这与血管加压素诱导的AQP2向质膜转运的有效阻断以及观察到的利尿增加一致。与此相符的是，长期用OPC处理后AQP2 mRNA水平也降低（为对照水平的30±10%，n = 9）。用速尿处理五天，尽管产生的多尿比OPC更严重，但并未导致AQP2水平下调，表明AQP2下调并非继发于尿流率增加或髓质高渗性丧失。在持续给予OPC的情况下，12小时禁水期间尿量急剧下降，降至与（未禁水的）对照动物无显著差异的水平。与此同时，AQP2水平升至对照水平。对照实验证实受体持续有效阻断。这些结果表明，V2受体拮抗剂使AQP2表达适度降低，这并非尿流率增加或髓质高渗性消除的结果。然而，这种降低远不如某些形式的获得性肾性尿崩症明显。结合禁水的影响，这表明AQP2表达的调节部分但非完全通过V2受体介导。
