Role of Insulin Receptors in the Kidney

胰岛素受体在肾脏中的作用

• 批准号: 7887108
• 负责人: Carolyn Mary Ecelbarger
• 金额: $ 37.9万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887108
• 关键词: 1-Phosphatidylinositol 4-Kinase; Acute; Blood; Blood Pressure; Breeding; Cadherins; Carrier Proteins; Cells; Centrifugation; Defect; Diabetes Mellitus; Distal; Distal convoluted renal tubule structure; Dose; Duct (organ) structure; Epithelial; Etiology; Excretory function; Fasting; Financial compensation; Generations; Genotype; Glucose; Hour; Hyperinsulinism; Hypertension; Immunohistochemistry; Incidence; Insulin; Insulin Receptor; Insulin Resistance; Kidney; Knock-out; Lead; Limb structure; Maintenance; Measures; Metabolic syndrome; Metabolism; Molsidomine; Mus; Natriuresis; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Obesity; Organ; Pancreas; Pathology; Pathway interactions; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Plasma; Play; Production; Proteins; Receptor Signaling; Regulation; Renal tubule structure; Role; Saline; Sgk protein; Signal Pathway; Signal Transduction; Site; Sodium; Sodium Channel; System; Testing; Thick; Tissues; Transgenic Mice; Tubular formation; Urine; Vasodilator Agents; Western Blotting; Wild Type Mouse; Zucker Rats; benzamil; blood pressure regulation; cell type; epithelial Na+ channel; feeding; inhibitor/antagonist; insight; mouse model; public health relevance; receptor; receptor expression; recombinase; response; saluretic; transmission process; uptake; urinary

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. PUBLIC HEALTH RELEVANCE: Insulin resistance is associated with high circulating plasma levels of insulin, as well as, increased blood pressure; however, cellular mechanisms relating the two are not well understood. The kidney, a major regulator of blood pressure, expresses insulin receptors along the renal tubule; however, their role there is undefined. These studies are aimed at elucidating insulin's role in the distal tubule, which will hopefully allow for earlier and better targeted treatments in obesity and diabetes.

描述(申请人提供)：肥胖和糖尿病导致循环中胰岛素水平高和高血压(BP)。然而，这些联系背后的机制亟需澄清。例如，在肾脏中，胰岛素通过其自身的受体可导致钠滞留，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，这已被证明可以降低ENaC的活性。因此，预计胰岛素的这两种作用将对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，我们开发了一种在肾脏远端小管中敲除或缺失胰岛素受体(IR)的转基因小鼠(IRKO)。这些小鼠存活并正常生长，但基础血压明显较高。他们快速排泄氯化钠负荷的能力也受到了损害，在基础状态和胰岛素治疗后，他们的尿硝酸盐和亚硝酸盐(NO的一种尿液形式)排泄的增加也变得迟钝。因此，我们的假设是，肾脏中的IR在促进容量排泄和维持正常血压方面可能具有以前未被认识的作用。我们进一步认为，这些缺陷的直接原因是NO的产生受损，随后ENaC过度活性。以下概述了三个主要目标。目的1是确定肾脏远端小管一氧化氮合酶活性降低是否是导致IRKO小鼠钠排泄缺陷和血压升高的机制决定因素。在饲养和禁食的IRKO和WT小鼠的内髓、外髓和皮质，以及显微解剖的近端小管、粗大的升肢和集合管，将测量肾脏一氧化氮合酶(NOS)的活性。我们还将测试胰岛素对内髓集合管(IMCD)培养中NOS活性和NO水平的直接影响。最后，我们将测试NO供体Molsidomine是否能恢复IRKO小鼠的NO水平，并使血压和钠的排泄正常化。目的2是确定上皮性钠通道(ENaC)活性增加是否是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ENaC的拮抗剂苯扎米是否能消除血压和钠尿的差异。ENAC的调节将在天然组织和原代IMCD细胞中进行检测。最后，在目标3中，我们确定了IRKO小鼠IMCD中与ENaC激活和NO生成相关的不同IR信号是否发生了改变。参与从受体到ENaC和一氧化氮合酶激活的IR信号传递的关键蛋白质的磷酸化将被评估。这些蛋白质包括但不限于胰岛素受体底物(IRS)、磷脂酰肌醇-3-激酶(PI-3K)以及血清和糖皮质激素调节的激酶(SGK1)。总体而言，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，它们可能提供对代谢综合征相关高血压的潜在病理的洞察。 公共卫生相关性：胰岛素抵抗与高循环血浆胰岛素水平以及血压升高有关；然而，与这两者相关的细胞机制尚不清楚。肾脏是血压的主要调节器，沿着肾小管表达胰岛素受体；然而，它们在那里的作用尚不明确。这些研究旨在阐明胰岛素在远端小管中的作用，这有望使肥胖和糖尿病的早期和更有针对性的治疗成为可能。