Novel Mouse Models for the Study of Urea Transporters

描述(由申请人提供)：至少从1934年开始，人们就认识到尿素对产生浓缩尿液的重要性。一些研究表明，在缺乏蛋白质的动物和人类中，最大尿液浓缩能力降低，并通过尿素输注恢复。UT-A1/UT-A3、UT-A2和UT-B基因敲除(KO)小鼠均有尿液浓缩缺陷，且UT-A1/UT-A3小鼠的尿液浓缩缺陷严重。因此，任何关于肾脏浓缩尿液机制的假说都需要包括尿素和尿素转运蛋白的一些影响，特别是UT-A1和/或UT-A3。由于UT-A基因的性质，用传统的方法不可能产生只表达UT-A1的KO小鼠。这个应用程序的第一个目标是使用一种新的方法来创建一个仅限UT-A1的鼠标。第二个目标是创造一个仅限UT-A3的鼠标。我们将使用一种创新的方法，从UT-A1/UT-A3KO小鼠开始，然后在内髓集合管(IMCD)特异性启动子的控制下敲入UT-A1或UT-A3，产生只表达UT-A1或UT-A3的小鼠。我们有初步的数据显示，我们已经再生了UT-A1/UT-A3KO小鼠，准备了UT-A1和UT-A3构建体来制造转基因小鼠，并将这些构建体注射到了其中。这些小鼠将允许我们独立确定每个尿素转运体的效果。这项提议的第二个目标是培育一只在IMCD中缺乏蛋白激酶C1(PKC1)的小鼠。我们有初步的数据表明，PKC1是参与其中的特定的PKC同工酶，因为：1)高张激活大鼠IMCDs中的PKC1；以及2)PKC1KO小鼠存在尿液浓缩缺陷和UT-A1蛋白丰度降低。PKC1KO鼠标是一个全球性的KO。因此，PKC1KO小鼠KO小鼠的尿液浓缩缺陷可能是由于IMCD以外的其他部位的PKC1丢失所致，例如大脑丢失导致口渴或加压素调节异常。为了确定聚集性缺陷是否是由于IMCD特异的PKC1丢失所致，我们建议制作IMCD特异的PKC1显性负性小鼠。我们已经获得了一个PKC1显性负性结构，当它与肌细胞特异性启动子一起使用时，被用来使小鼠的心肌细胞中缺乏PKC1。我们建议使用类似的方法来制作IMCD特异性抑制PKC1的小鼠。

DESCRIPTION (provided by applicant): Urea's importance to the generation of a concentrated urine has been appreciated since at least 1934. Several studies show that maximal urine concentrating ability is decreased in protein-deprived animals and humans, and is restored by urea infusion. UT-A1/UT-A3, UT-A2, and UT-B knock-out (KO) mice were each shown to have urine concentrating defects, and the defect in the UT-A1/UT-A3 mouse is severe. Thus, any hypothesis regarding the mechanism by which the kidney concentrates urine needs to include some effect derived from urea and urea transporters, especially UT-A1 and/or UT-A3. Due to the nature of the UT-A gene, it is not possible to generate a KO mouse that only expresses UT-A1 using traditional approaches. The first goal of this application is to use a novel approach to create a UT-A1 only mouse. The second goal is to create a UT-A3 only mouse. We will use an innovative approach by starting with the UT-A1/UT-A3 KO mouse and then knock-in either UT-A1 or UT-A3 under the control of an inner medullary collecting duct (IMCD) specific promoter to generate mice expressing only UT-A1 or UT-A3. We have preliminary data showing that we have regenerated the UT-A1/UT-A3 KO mouse, prepared the UT-A1 and UT-A3 constructs for making the transgenic mice, and have mice in which these constructs have been injected. These mice will allow us to determine the effect of each urea transporter independently. The second goal of this proposal is to generate a mouse lacking protein kinase C1 (PKC1) in the IMCD. We have preliminary data suggesting that PKC1 is the specific PKC isozyme involved since: 1) hypertonicity activates PKC1 in rat IMCDs; and 2) PKC1 KO mice have a urine concentrating defect and a reduction in UT-A1 protein abundance. The PKC1 KO mouse is a global KO. Thus, it is possible that the urine concentrating defect in the PKC1 KO mice KO mouse could be due to loss of PKC1 from a site other than the IMCD, such as loss from the brain that results in abnormalities in thirst or vasopressin regulation. To determine whether the concentrating defect is due to loss of PKC1 specifically from the IMCD, we propose to make an IMCD-specific PKC1 dominant-negative mouse. We have obtained a PKC1 dominant-negative construct, which when used with a myocyte-specific promoter, was used to make a mouse lacking PKC1 in myocytes. We propose to use a similar approach to make a mouse with IMCD-specific inhibition of PKC1.