Role of Insulin Receptors in the Kidney

描述（由申请人提供）：肥胖和糖尿病导致高循环胰岛素水平和高血压。然而，这些关联背后的机制迫切需要澄清。例如，在肾脏中，胰岛素通过自身的受体，可导致钠潴留，并激活上皮钠通道（ENaC）。尽管如此，胰岛素也可以增加一氧化氮（NO）的产生，这已被证明会降低ENaC的活性。因此，胰岛素的这两种作用可能会对BP产生相反的影响。为了更好地阐明胰岛素受体在肾脏中的作用，我们开发了一种“敲除”（KO）或缺失肾远端小管胰岛素受体（IR）的转基因小鼠（IRKO）。这些小鼠存活并正常生长，但具有明显较高的基础血压。在基础状态和胰岛素治疗后，它们快速排泄NaCl负荷的能力也受到损害，并且尿中硝酸盐和亚硝酸盐（尿液中NO的一种形式）的排泄增加变得迟钝。因此，我们的假设是，肾脏中的IR可能在促进体积排泄和维持正常血压方面具有以前未被认识到的作用。我们进一步认为，这些缺陷直接源于一氧化氮生成受损，随后ENaC过度活跃。下面概述了三个主要目标。目的1是确定肾远端小管NOS活性降低是否是IRKO小鼠钠排泄缺陷和血压升高的机制决定因素。将在喂养和禁食的IRKO和WT小鼠的内髓、外髓和皮质以及微解剖的近端小管、厚升肢和收集管中测量肾一氧化氮合酶（NOS）的活性。我们还将测试胰岛素对内髓集管（IMCD）培养中NOS活性和NO水平的直接影响。最后，我们将测试莫西多明作为一氧化氮供体是否能恢复一氧化氮水平，并使IRKO小鼠的血压和钠排泄正常。目的2是确定上皮钠通道（ENaC）活性的增加是否是IRKO小鼠钠排泄缺陷和血压升高的机制决定因素。为此，我们将测试苯甲胺，ENaC的拮抗剂，是否能消除血压和尿钠的差异。ENaC的调节将在原生组织和原代IMCD细胞中进行研究。最后，在Aim 3中，我们确定与ENaC激活和NO生成相关的不同IR信号是否在IRKO小鼠IMCD中发生改变。将评估参与IR信号从受体传递到ENaC和一氧化氮合酶激活的关键蛋白的磷酸化。这些蛋白包括但不限于胰岛素受体底物（IRS）、磷酸肌醇-3激酶（PI-3K）、血清和糖皮质激素调节激酶（SGK1）。总的来说，这些研究将高度阐明胰岛素在远端小管中的作用，以及它在血液控制和钠调节中的作用。此外，它们可能为了解与代谢综合征相关的高血压的病理基础提供见解。

DESCRIPTION (provided by applicant): Obesity and diabetes lead to high circulating levels of insulin and high blood pressure (BP). However, mechanisms underlying these associations are in dire need of clarification. For example, in the kidney, insulin, through its own receptor, can lead to sodium retention, and activate the epithelial sodium channel (ENaC). Nonetheless, insulin can also increase the production of nitric oxide (NO), which has been shown to reduce the activity of ENaC. Thus, these two actions of insulin would be expected to have opposing effects on BP. To better elucidate the role of the insulin receptor in the kidney, we have developed an transgenic mouse with "knockout" (KO) or deletion of the insulin receptor (IR) in the distal tubule of the kidney (IRKO). These mice survive and grow normally, but have significantly higher basal BP. They are also impaired in their ability to rapidly excrete a NaCl load, as well as, have blunted rise in urinary nitrates plus nitrites (a urinary form of NO) excretion in the basal state and after insulin treatment. Thus our hypothesis is that IR in the kidney may have a previously unappreciated role in facilitating volume excretion and in the maintenance of normal BP. We further suggest these deficiencies arise directly as the result of impaired NO production with subsequent over-activity of ENaC. Three main aims are outlined below. Aim 1 is to determine whether reduced renal NOS activity in the distal tubule is a mechanistic determinant of the sodium-excretory defect and elevated BP in the IRKO mice. Renal nitric oxide synthase (NOS) activity will be measured in inner medulla, outer medulla, and cortex, as well as, microdissected proximal tubule, thick ascending limb, and collecting duct from fed and fasted IRKO and WT mice. We will also test the direct effects of insulin on NOS activity and NO levels in inner medullary collecting duct (IMCD) cultures. Finally, we will test whether molsidomine, an NO donor, restores NO levels, as well as normalizes BP and sodium excretion in IRKO mice. Aim 2 is to determine whether increased activity of the epithelial sodium channel (ENaC) is a mechanistic determinant of the sodium-excretory defect and elevated BP in the IRKO mice. For this aim, we will test whether benzamil, an antagonist of ENaC, abolishes differences in BP and natriuresis. ENaC regulation will be examined in native tissue and in primary IMCD cells. Finally, in Aim 3 we determine whether distinct IR signaling relating to ENaC activation and NO generation is altered in the IRKO mouse IMCD. Phosphorylation of critical proteins involved in IR signal transmission from the receptor to the activation of ENaC and nitric oxide synthase will be evaluated. These proteins include, but are not limited to, the insulin receptor substrate (IRS), phosphoinositide-3-kinase (PI-3K), and the serum and glucocorticoid-regulated kinase (SGK1). Overall, these studies will highly elucidate the role of insulin in the the distal tubule with regard to its role in blood control and sodium regulation. Moreover, they may provide insight into the pathology underlying hypertension associated with the metabolic syndrome.