Nephrogenic diabetes insipidus (NDI) is caused by impairment of vasopressin (VP) receptor type 2 signaling. Because potential therapies for NDI that target the canonical VP/cAMP/protein kinase A pathway have so far proven ineffective, alternative strategies for modulating aquaporin 2 (AQP2) trafficking have been sought. Successful identification of compounds by our high-throughput chemical screening assay prompted us to determine whether EGF receptor (EGFR) inhibitors stimulate AQP2 trafficking and reduce urine output. Erlotinib, a selective EGFR inhibitor, enhanced AQP2 apical membrane expression in collecting duct principal cells and reduced urine volume by 45% after 5 days of treatment in mice with lithium-induced NDI. Similar to VP, erlotinib increased exocytosis and decreased endocytosis in LLC-PK1 cells, resulting in a significant increase in AQP2 membrane accumulation. Erlotinib increased phosphorylation of AQP2 at Ser-256 and Ser-269 and decreased phosphorylation at Ser-261 in a dose-dependent manner. However, unlike VP, the effect of erlotinib was independent of cAMP, cGMP, and protein kinase A. Conversely, EGF reduced VP-induced AQP2 Ser-256 phosphorylation, suggesting crosstalk between VP and EGF in AQP2 trafficking and a role of EGF in water homeostasis. These results reveal a novel pathway that contributes to the regulation of AQP2-mediated water reabsorption and suggest new potential therapeutic strategies for NDI treatment.

译文

:肾性尿崩症(NDI)是由2型加压素(VP)受体信号转导受损引起的。由于迄今为止已证明靶向经典VP / cAMP /蛋白激酶A途径的NDI潜在疗法无效,因此寻求了调节水通道蛋白2(AQP2)转运的替代策略。通过我们的高通量化学筛选测定法成功鉴定化合物,促使我们确定EGF受体(EGFR)抑制剂是否刺激AQP2转运并减少尿量。在用锂诱导的NDI治疗的小鼠中,经过5天的治疗,选择性的EGFR抑制剂厄洛替尼增强了AQP2顶膜在收集导管主细胞中的表达,并使尿液量减少了45%。与VP相似,厄洛替尼增加LLC-PK1细胞的胞吐作用并减少其内吞作用,从而导致AQP2膜积聚明显增加。厄洛替尼以剂量依赖性方式增加在Ser-256和Ser-269处AQP2的磷酸化,并在Ser-261处减少磷酸化。但是,与VP不同,厄洛替尼的作用独立于cAMP,cGMP和蛋白激酶A。相反,EGF减少了VP诱导的AQP2 Ser-256磷酸化,表明VP和EGF之间在AQP2转运中起串扰作用,以及EGF在水中的作用稳态。这些结果揭示了有助于调节AQP2介导的水重吸收的新途径,并提出了NDI治疗的新潜在治疗策略。

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