Aquaporin-1 translocation and degradation mediates the water transportation mechanism of acetazolamide.
Aquaporin-1 translocation and degradation mediates the water transportation mechanism of acetazolamide.
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Aquaporin-1 易位和降解介导乙酰唑胺的水运输机制
DOI：
10.1371/journal.pone.0045976
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Zhang J;An Y;Gao J;Han J;Pan X;Pan Y;Tie L;Li X
背景技术利尿剂广泛用于治疗水潴留相关疾病，其中乙酰唑胺(AZA)最初作为碳酸酐酶(CA)抑制剂。尿液浓缩需要位于肾近曲小管的水通道蛋白-1 (AQP1)。此前我们实验室曾报道过 AZA 可能调节 AQP1。本研究的目的是验证我们的假设，即调节 AQP1 可能介导 AZA 的利尿作用。方法/主要发现 对于体内研究，我们利用 Sprague Dawley 大鼠以及 AQP1 敲除 (AQP1−/−) 小鼠来检查尿量，并使用人肾 2 (HK-2) 细胞系进行体外机制研究。在我们目前的研究中，我们发现AZA最初降低了CAs活性，但活性逐渐恢复。相反，利尿作用始终显着。 AQP1蛋白表达在第7天和第14天显着降低。通过使用AQP1−/−小鼠，我们发现AZA的利尿作用在第14天被取消，而野生型小鼠的尿量持续增加。表面等离子共振 (SPR) 结果表明 AQP1 在生理上与肌球蛋白重链 (MHC) 结合，免疫沉淀和免疫荧光结果证实了这种蛋白质相互作用。体外研究结果证明，AZA 通过促进与 MHC 的相互作用，依赖于 ERK/肌球蛋白轻链激酶 (MLCK) 通路激活，促进 AQP1 易位到细胞膜上。 MHC抑制剂BDM和ERK抑制剂U0126均消除了AZA的上述作用。最终，AZA 诱导 AQP1 泛素化，而蛋白酶体抑制剂 MG132 逆转了 AZA 对 AQP1 的下调作用。结论/意义我们的研究结果确定了AZA通过调节AQP1的创新机制发挥利尿作用，并验证了其抑制机制是通过促进MHC依赖性易位到细胞膜上，然后泛素介导的降解，暗示了利尿剂发现的新机制和靶点。
Background Diuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA. Methodology/Principal Findings For in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1−/−) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1−/− mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1. Conclusions/Significance Our results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering.
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发表时间：
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期刊：
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影响因子：
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