Novel Mouse Models for the Study of Urea Transporters

描述（由申请人提供）：尿素对浓尿生成的重要性至少从1934年开始就被认识到。一些研究表明，蛋白质剥夺的动物和人类的最大尿浓缩能力下降，并通过尿素输注恢复。UT-A1/UT-A3、UT-A2和UT-B敲除（KO）小鼠均显示有尿浓缩缺陷，且UT-A1/UT-A3小鼠的缺陷较为严重。因此，任何关于肾脏浓缩尿液机制的假设都需要包括来自尿素和尿素转运蛋白的一些作用，特别是UT-A1和/或UT-A3。由于UT-A基因的性质，使用传统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颖的方法来创建只使用UT-A1的鼠标。第二个目标是创建只支持UT-A3的鼠标。我们将采用一种创新的方法，从UT-A1/UT-A3 KO小鼠开始，然后在内髓收集管（IMCD）特异性启动子的控制下敲入UT-A1或UT-A3，以产生仅表达UT-A1或UT-A3的小鼠。我们有初步的数据表明，我们已经再生了UT-A1/UT-A3 KO小鼠，准备了用于制造转基因小鼠的UT-A1和UT-A3构建体，并在小鼠体内注射了这些构建体。这些小鼠将使我们能够独立确定每种尿素转运蛋白的作用。该提议的第二个目标是产生在IMCD中缺乏蛋白激酶C1 （PKC1）的小鼠。我们有初步的数据表明PKC1是特定的PKC同工酶，因为：1)高渗性激活大鼠imcd中的PKC1；2) PKC1 KO小鼠存在尿浓缩缺陷和UT-A1蛋白丰度降低。PKC1 KO鼠标是一个全局KO。因此，PKC1 KO小鼠的尿浓缩缺陷可能是由于除了IMCD以外的其他部位的PKC1缺失所致，例如大脑中的PKC1缺失导致口渴或抗利尿激素调节异常。为了确定集中缺陷是否是由于IMCD特异性的PKC1缺失引起的，我们建议制造一只IMCD特异性PKC1显性阴性小鼠。我们已经获得了PKC1显性阴性构建体，当与肌细胞特异性启动子一起使用时，可以使小鼠在肌细胞中缺乏PKC1。我们建议使用类似的方法制造具有imcd特异性PKC1抑制的小鼠。

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.