Novel Mouse Models for the Study of Urea Transporters

描述（由申请人提供）：至少自1934年以来，尿素对产生浓缩尿液的重要性已被认识到。几项研究表明，在蛋白质缺乏的动物和人类中，最大尿浓缩能力降低，并通过尿素输注恢复。UT-A1/UT-A3、UT-A2和UT-B基因敲除（KO）小鼠均显示具有尿浓缩缺陷，并且UT-A1/UT-A3小鼠中的缺陷是严重的。因此，关于肾脏浓缩尿液的机制的任何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仅表达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UT-A1唯一的鼠标。第二个目标是创建UT-A3专用鼠标。我们将使用一种创新的方法，从UT-A1/UT-A3 KO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，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，制备了用于制备转基因小鼠的UT-A1和UT-A3构建体，并具有注射了这些构建体的小鼠。这些小鼠将使我们能够独立地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（PKC 1）的小鼠。我们有初步的数据表明，PKC 1是特定的PKC同工酶参与，因为：1）高渗激活大鼠IMCD中的PKC 1; 2）PKC 1 KO小鼠有尿浓缩缺陷和UT-A1蛋白丰度减少。PKC 1 KO小鼠是一种全局KO小鼠。因此，PKC 1 KO小鼠中的尿浓缩缺陷可能是由于来自IMCD以外的部位的PKC 1丢失，例如来自大脑的丢失导致口渴或加压素调节异常。为了确定集中缺陷是否是由于IMCD特异性PKC 1的丢失，我们建议制作IMCD特异性PKC 1显性阴性小鼠。我们已经获得了一个PKC 1显性负性的结构，当与肌细胞特异性启动子一起使用时，用于使小鼠在肌细胞中缺乏PKC 1。我们建议使用类似的方法来制造具有IMCD特异性抑制PKC 1的小鼠。 公共卫生相关性：尿素在尿浓缩机制中起着关键作用，因此，在调节水平衡中起着关键作用。我们对尿素转运调节的研究将促进我们对尿浓缩机制的理解。这可能会产生新的治疗肾源性尿崩症，并将成为越来越重要的蛋白激酶C抑制剂治疗糖尿病的发展。

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. PUBLIC HEALTH RELEVANCE: Urea plays a critical role in the urinary concentrating mechanism, and therefore, in the regulation of water balance. Our studies into the regulation of urea transport will advance our understanding of the urinary concentrating mechanism. This could yield new therapies for nephrogenic diabetes insipidus and will become increasingly important with the development of protein kinase C inhibitors for the treatment of diabetes mellitus.