Abstract：
High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH.
摘要：
高浓度的尿钙通过激活在集合管细胞的腔膜中表达的钙敏感受体（CaSR）来抵消加压素的作用，这损害了水通道蛋白2（AQP2）的贩运。根据这些发现,我们提供的证据表明，相对于野生型小鼠，模拟常染色体显性低钙血症的CaSR敲入（KI）小鼠，在参与AQP2降解的磷酸化位点Ser261上显示与AQP2磷酸化水平显着升高相关的AQP2总含量显着降低。有趣的是,KI小鼠也有明显较高水平的磷酸化p38MAPK，CaSR的下游效应物，已知在Ser261磷酸化AQP2。此外，ATF1在ser63磷酸化，p38MAPK下游的转录因子，显著高于KI。此外,KI小鼠具有显著更高水平的靶向AQP2的miRNA137,这与AQP2的转录后下调一致。用CaSR拮抗剂钙溶JTT-305体内治疗KI小鼠，在KI小鼠中，AQP2表达增加，靶向AQP2的miRNA137水平降低。一起,这些结果为CaSR在通过增加AQP2-pS261以及AQP2丰度而损害短期加压素反应中的关键作用提供了直接证据。通过p38MAPK-ATF1-miR137途径。关键点：钙敏感受体（CaSR）激活突变是常染色体显性低钙血症（ADH）的主要原因，其特征是肾脏钙排泄不当导致低钙血症和高钙尿症。ADH患者甲状旁腺激素的治疗现状，虽然改善了低钙血症，不要改善高钙尿症或肾钙化。用钙溶JTT-305/MK-5442的体内治疗改善了CaSR敲入小鼠的大多数ADH表型，包括高钙尿症或肾钙化，并逆转了加压素敏感性水通道蛋白2（AQP2）表达的下调，为CaSR在损害加压素反应中的关键作用提供直接证据。钙解剂在降低肾脏钙化风险方面的有益作用可能发生在甲状旁腺激素非依赖性作用中，通过加压素依赖性抑制粗大的上行肢体和收集管中的cAMP合成。改善大多数钙代谢异常，包括高钙尿症，肾钙化,AQP2介导的渗透水重吸收使calcilytic成为ADH的新型治疗剂。
