NaCI Balance and Targeted Insulin Receptor Knockout Mice

描述（由申请人提供）：胰岛素是肾脏中的一种抗利钠激素。胰岛素增加钠在近端小管、粗升肢和远端小管的重吸收。然而，其作用的分子机制尚未得到很好的描述。此外，由于胰岛素抵抗引起的高胰岛素血症与高血压高度相关，而高血压往往先于慢性肾脏疾病。然而，由于这些病理状态的复杂性，胰岛素通过其自身受体在肾脏中的直接作用很难解析出来。因此，这些研究旨在开发两种新的动物模型，使我们能够更好地解决胰岛素在肾脏中的直接作用及其在NaCI平衡，血压和肾脏NaCI转运蛋白调节中的作用。为了实现这些目标，我们的计划是通过育种，使用CRE/IoxP方法，开发两种转基因小鼠系，其中胰岛素受体（IR）分别从收集管主细胞或厚升肢细胞中被敲除。这些小鼠（以及从其肾脏制备的细胞培养物）将被评估胰岛素敏感的NaCI转运、血压变化和靶转运蛋白的调节。在本研究中，我们验证了一个整体假设，即在小鼠的集束管主细胞或厚升肢细胞中由于选择性敲除基因表达而缺乏胰岛素受体的小鼠，在基础或刺激条件下，这些部位的NaCI重吸收会减少。在具体目标1中，我们计划开发集管主细胞胰岛素受体敲除小鼠（CD-IR KO），并评估基础条件下NaCI的运输特性。在具体目标2中，我们计划开发厚升肢细胞胰岛素受体敲除小鼠（TAL-IR KO），并同样评估基础NaCI运输。最后，在具体的目的3中，我们计划在体内和体外评估高胰岛素血症条件下上述小鼠的血压、NaCI平衡、NaCI转运以及肾脏NaCI转运蛋白的调节。我们计划通过两种方式在小鼠中实现高胰岛素血症：1)我们将通过颈静脉注入胰岛素；2)我们将喂养一种糖尿病饮食，这种饮食应该会诱导胰岛素抵抗，从而提高内源性胰岛素水平。总的来说，我们相信这些模型将为胰岛素诱导的高血压这一已知几十年的生理现象提供新的分子见解，并具有明确的临床后果。

DESCRIPTION (provided by applicant): Insulin is an anti-natriuretic hormone in the kidney. Insulin increases sodium reabsorption in the proximal tubule, thick ascending limb, and distal tubule. However, the molecular mechanisms underlying its actions are not well described. In addition, hyperinsulinemia, due to insulin resistance, is highly correlated with hypertension, which often precedes chronic renal disease. Nevertheless, because of the complexity of these pathological states, the direct effects of insulin, in the kidney through its own receptor, are difficult to parse out. Thus, these studies are aimed at developing two new animal models that will allow us to better address the direct role of insulin in the kidney and its role in NaCI balance, blood pressure, and the regulation of renal NaCI transport proteins. In order to achieve these aims our plan is to develop, through breeding, using the CRE/IoxP approach, two lines of transgenic mice in which the insulin receptor (IR) is knocked out of either the collecting duct principal cells or the thick ascending limb cells, respectively. These mice (and cell cultures prepared from their kidneys) will be evaluated for insulin-sensitive NaCI transport, blood pressure changes, and the regulation of target transport proteins. In this proposal, we test the overall hypothesis that mice that lack insulin receptors in either their collecting duct principal or thick ascending limb cells due to selective knock out of gene expression in these cells will have decreased NaCI reabsorption at these sites under basal or stimulated conditions. In specific aim 1, we plan to develop the collecting duct principal cell insulin receptor knock-out mouse (CD-IR KO) and evaluate NaCI transporting characteristics under basal conditions. In specific aim 2, we plan to develop the thick ascending limb cell insulin receptor knock-out mouse (TAL-IR KO) and, likewise, evaluate basal NaCI transport. Finally, in specific aim 3, we plan to evaluate blood pressure, NaCI balance, NaCI transport, and the regulation of renal NaCI transport proteins in the above mice under hyperinsulinemic conditions in vivo and ex vivo. We plan to achieve hyperinsulinemia in the mouse in two ways: 1) we will infuse insulin via the jugular vein; and 2) we will feed a diabetogenic diet that should induce insulin resistance and thus raise endogenous insulin levels. Overall, we believe these models will provide fresh molecular insight into a physiological phenomenon that has been known for decades, insulin induced hypertension, with clear clinical ramifications.