State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Peking University, Beijing, China.
PLoS One. 2012;7(9):e45976. doi: 10.1371/journal.pone.0045976. Epub 2012 Sep 21.
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.
利尿剂被广泛用于治疗与水潴留相关的疾病，其中乙酰唑胺 (AZA) 最初作为碳酸酐酶 (CA) 抑制剂发挥作用。水通道蛋白-1 (AQP1) 位于肾近端小管中，是尿液浓缩所必需的。我们实验室之前报道过 AZA 可能调节 AQP1。本研究旨在验证我们的假设，即调节 AQP1 可能介导 AZA 的利尿作用。
方法/主要发现：在体内研究中，我们利用 Sprague Dawley 大鼠以及 AQP1 敲除 (AQP1(-/-)) 小鼠来检测尿量，并用人肾-2 (HK-2) 细胞系进行体外机制研究。在本研究中，我们发现 AZA 最初降低了 CA 的活性，但活性逐渐恢复。相反，利尿作用始终显著。AQP1 蛋白表达在第 7 天和第 14 天显著下降。利用 AQP1(-/-) 小鼠，我们发现 AZA 的利尿作用在第 14 天被取消，而在野生型小鼠中，尿量持续增加。表面等离子体共振 (SPR) 结果表明 AQP1 与肌球蛋白重链 (MHC) 生理性结合，免疫沉淀和免疫荧光结果证实了这种蛋白相互作用。体外研究结果证明 AZA 通过促进与 MHC 的相互作用促进 AQP1 易位到细胞膜上，这依赖于 ERK/肌球蛋白轻链激酶 (MLCK) 途径的激活。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：我们的结果表明，AZA 通过调节 AQP1 发挥利尿作用，通过一种创新的机制证实了其抑制机制是通过促进 MHC 依赖性易位到细胞膜上，然后通过泛素介导的降解，提示了一种新的机制和利尿剂发现的靶标。
