Regulation of Renal Inner Medullary Function

描述(申请人提供)：肾源性尿崩症(NDI)是一种疾病，其特征是由于肾脏无法对加压素做出反应而产生大量稀释的尿液。NDI可以是先天的，也可以是后天的。先天性NDI由90%的2型加压素受体(V2R)突变和另外10%的AQP2突变引起(已知突变)。获得性NDI最常见的原因是锂治疗，这是一种干扰cAMP信号的药物。在V2R突变或服用锂的患者中，AQP2或UT-A1蛋白都没有突变，这表明如果有可能磷酸化这些蛋白并增加它们的顶膜堆积，而不依赖血管加压素或cAMP，那么人们可能能够治疗或至少减轻NDI的严重程度。因此，我们寻找一种可以独立于加压素而同时磷酸化AQP2和UT-A1的激酶。一磷酸腺苷激酶(AMPK)是一种能量敏感的激酶，可被渗透应激和低氧所刺激。AMPK从未在内髓中被研究过，因为内髓通常是高渗和低氧的。我们有初步的数据表明，AMPK在体外和在自然大鼠的内髓集合管中都能磷酸化AQP2和UT-A1。我们也有初步数据显示，二甲双胍增加了缺乏V2R的小鼠的尿液渗透压，V2R是先天性NDI的小鼠模型。在这一应用中，我们将检验AMPK的假设，即AMPK独立于加压素，通过增加AQP2和UT-A1的磷酸化、顶端质膜堆积和功能来增加尿液浓缩能力。特定目标1将测试AQP2和UT-A1是否为AMPK磷酸化的底物。特异性目标2将测试AMPK对AQP2和UT-A1的磷酸化是否增加了这两种蛋白的顶膜积累。具体目标3将在NDI动物模型中测试AMPK的激活是否会增加尿素和水的通透性，以及尿液浓缩能力。如果我们的假设是 正确，那么AMPK激活剂将是治疗由于V2R突变引起的先天性NDI以及由于干扰cAMP而导致的获得性NDI的新的治疗选择。

DESCRIPTION (provided by applicant): Nephrogenic diabetes insipidus (NDI) is a disease characterized by the production of very large quantities of dilute urine from an inability of the kidney to respond to vasopressin. NDI can be either congenital or acquired. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in 90% of families (in which the mutation is known) and in AQP2 in the other 10%. The most common cause of acquired NDI is lithium treatment, a medication that interferes with cAMP signaling. In patients with V2R mutations or taking lithium, there are no mutations in either the AQP2 or UT-A1 proteins, suggesting that if it is possible to phosphorylate these proteins and increase their apical membrane accumulation independent of vasopressin or cAMP, then one may be able to treat, or at least lessen the severity of, the NDI. Therefore, we looked for a kinase that could phosphorylate both AQP2 and UT-A1 independent of vasopressin. Adenosine monophosphate kinase (AMPK) is an energy-sensing kinase that can be stimulated by osmotic stress and hypoxia. AMPK has never been studied in inner medulla, which is normally hypertonic and hypoxic. We have preliminary data showing that AMPK phosphorylates AQP2 and UT-A1, both in vitro and in native rat inner medullary collecting ducts. We also have preliminary data showing that metformin increases urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. In this application, we will test the hypothesis that AMPK, independent of vasopressin, increases urine concentrating ability through increases in AQP2 and UT-A1 phosphorylation, apical plasma membrane accumulation, and function. Specific Aim 1 will test whether AQP2 and UT-A1 are substrates for phosphorylation by AMPK. Specific Aim 2 will test whether phosphorylation of AQP2 and UT-A1 by AMPK increases the apical membrane accumulation of these two proteins. Specific Aim 3 will test whether activation of AMPK increases urea and water permeabilities, and urine concentrating ability in animal models of NDI. If our hypothesis is correct, then AMPK activators would be a novel therapeutic option for congenital NDI due to V2R mutations, and for acquired forms of NDI due to interference with cAMP.