Studies with a novel mouse model of X-linked nephrogenic diabetes insipidus

我们最近建立了第一个可存活的XNDI小鼠模型，在该模型中，V2R基因可以在成年小鼠的肾脏中以有条件的(4-OH-他莫昔芬诱导的)方式缺失。由此产生的V2R KO小鼠显示出XNDI的所有关键症状，包括产生大量稀释尿(多尿)和多饮(Li JH等人。J·克莱恩。投资。119、3115-3126、2009)。 为了确定治疗XNDI的有效药物，我们决定采取全面和系统的方法。水通道的合成和功能允许肾脏集合管中的水再吸收(水通道蛋白2)，需要肾脏集合管细胞中的cAMP水平达到一定水平。然而，在XNDI小鼠中，由于缺乏V2Rs，cAMP水平较低。此外，由于cAMP水平低，肾脏集合管细胞表达的水通道蛋白2的数量显著减少(Li JH等人。J·克莱恩。投资。119、3115-3126、2009)。 因此，我们推测，能够刺激肾脏集合管细胞内cAMP水平的药物可能有相当大的治疗XNDI的潜力。为了大大增加识别合适的XNDI药物的可能性，我们开始与NIH国家基因组学中心(NGC)合作，对大型药物库进行筛选。在与NGC的合作中，我们使用与肾脏集合管细胞(mpkCCD细胞)具有相似特性的培养肾细胞进行筛查。这些筛选可以识别能够提高这些细胞中cAMP水平的化合物。 到目前为止，已经对以下文库进行了筛选，以寻找可以提高mpkCD细胞中cAMP水平的化合物： 1.Lopac图书馆(药理活性化合物图书馆) 这是一个来自Sigma-Aldrich的化合物文库，包含1280个药理上已知的化合物。 2.FDA文库 这是FDA批准的所有化合物以及欧洲和日本批准的化合物(约3800种具有药理活性的化合物)的资料库。 这项筛选工作已经确定了许多能够提高mpkCCD细胞内cAMP水平的化合物，具有很高的效力和效率。在XNDI小鼠身上测试这些化合物之前，我们目前正在确认这些药物在收集新鲜从小鼠肾脏制备的管子方面也具有活性。这是一个关键的步骤，因为目前还不完全清楚在培养中生长的mpkCCD细胞在多大程度上模仿了实际的收集管细胞。 此外，我们亦正设立一套系统，让市民可以利用刚制备好的收集风管，筛选上述图书馆。这些小管将从大鼠的肾脏中制备，因为可以从小鼠身上获得的组织数量非常有限。 一旦在这些体外筛选中确定了活性化合物，这些药物将在XNDI小鼠身上进行测试。

We recently generated the first viable mouse model of XNDI in which the V2R gene can be deleted in a conditional (4-OH-tamoxifen-inducible) fashion in the kidneys of adult mice. The resulting V2R KO mice showed all key symptoms of XNDI, including the production of large amounts of dilute urine (polyuria) and polydipsia (Li JH et al. J. Clin. Invest. 119, 3115-3126, 2009). To identify drugs useful for the treatment of XNDI, we decided to take a comprehensive and systematic approach. The synthesis and function of the water channel that allows water reuptake in kidney collecting ducts (aquaporin 2) requires that the cAMP levels in the renal collecting duct cells reach a certain level. However, in XNDI mice, cAMP levels are low due to the absence of V2Rs. Moreover, due to low cAMP levels, the amount of aquaporin 2 protein expressed by renal collecting duct cells is dramatically reduced (Li JH et al. J. Clin. Invest. 119, 3115-3126, 2009). We therefore reasoned that agents that can stimulate cAMP levels in renal collecting duct cells may have considerable potential for the treatment of XNDI. To greatly increase the likelihood to identify suitable XNDI drugs, we started to collaborate with the NIH National Genomics Center (NGC) to carry out screens of large drug libraries. In collaboration with the NGC, we carried out screens using cultured renal cells that have similar properties as renal collecting duct cells (mpkCCD cells). These screens can identify compounds that are able to raise cAMP levels in these cells. So far, the following libraries have been screened for compounds that can increase cAMP levels in mpkCCD cells: 1. LOPAC library (Library of Pharmacologically Active Compounds) This is a compound library from Sigma-Aldrich with 1280 pharmacologically known compounds. 2. FDA library This is a library of all compounds approved by the FDA as well as compounds approved in Europe and Japan (about 3800 pharmacologically active compounds). This screening effort has identified many compounds that can increase cAMP levels in mpkCCD cells with high potency and efficacy. Before testing these compounds in the XNDI mice, we are currently in the process of confirming that these drugs are also active in collecting duct tubules freshly prepared form mouse kidneys. This is a critical step since it is not fully clear to which extent mpkCCD cells grown in culture mimic actual collecting duct cells. We are also setting up a system that will allow the screening of the above libraries with freshly prepared collecting duct tubules. These tubules will be prepared from RAT kidneys since the amount of tissue that can be obtained from the mouse is very limited. Once active compounds have been identified in these in vitro screens, these drugs will be tested in XNDI mice.