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

我们最近产生了第一个可行的XNDI小鼠模型，其中V2 R基因可以在成年小鼠肾脏中以条件性（4-OH-他莫昔芬诱导）方式缺失。所得的V2 R KO小鼠显示出XNDI的所有关键症状，包括产生大量稀尿（多尿）和烦渴（Li JH等人，J. Clin. Invest. 119，3115-3126，2009）。 为了确定对治疗XNDI有用的药物，我们决定采取全面和系统的方法。允许肾集合管中水再摄取的水通道（水通道蛋白2）的合成和功能需要肾集合管细胞中的cAMP水平达到一定水平。然而，在XNDI小鼠中，由于缺乏V2 R，cAMP水平较低。此外，由于低cAMP水平，肾集合管细胞表达的水通道蛋白2蛋白的量显著降低（Li JH等人，J. Clin. Invest. 119，3115-3126，2009）。 因此，我们推断，可以刺激肾集合管细胞中cAMP水平的药物可能具有相当大的治疗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。与NGC合作，我们使用具有与肾集合管细胞（mpkCCD细胞）相似特性的培养肾细胞进行了筛选。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，已经筛选了以下文库中可以增加mpkCCD细胞中cAMP水平的化合物： 1.药理活性化合物库（Library of Pharmacologically Active Compounds） 这是来自Sigma-Aldrich的化合物库，具有1280种已知化合物。 2. FDA图书馆 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种生物活性化合物）的库。 这项筛选工作已经鉴定出许多可以以高效力和功效增加mpkCCD细胞中cAMP水平的化合物。在XNDI小鼠中测试这些化合物之前，我们目前正在确认这些药物在从小鼠肾脏新鲜制备的集合管小管中也具有活性。这是一个关键步骤，因为尚不完全清楚在培养物中生长的mpkCCD细胞在多大程度上模拟实际的集合管细胞。 我们还建立了一个系统，可以用新鲜制备的集合管筛选上述文库。这些小管将从大鼠肾脏中制备，因为可从小鼠中获得的组织量非常有限。 一旦在这些体外筛选中鉴定出活性化合物，将在XNDI小鼠中测试这些药物。

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.