HYPEROSMOTIC REGULATION OF GENES AND PROTEINS IN INNER MEDULLARY COLLECTING DUCT

内髓集合管基因和蛋白质的高渗调节

• 批准号: 6851012
• 负责人: Tomas Berl
• 金额: $ 36.78万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6851012
• 关键词: JUN kinase; arginine vasopressin; biological signal transduction; body fluid osmolarity; enzyme activity; functional /structural genomics; genetic regulation; hypertonia; kidney cell; laboratory mouse; microarray technology; mitogen activated protein kinase; osmosis; osmotic pressure; proteomics; renal tubule; tissue /cell culture; western blottings

In the antidiuretic state cells that inhabit the inner medulla of terrestrial animals must adapt to a hypertonic environment and to rapidly respond to changes in tonicity. This response involves the activation of a sequential network of protein kinases and ultimately the upregulation of proteins that promote the cellular uptake of the inert osmolytes. It is now well recognized that the adaptive response involves a more complex array of both genes and proteins. The primary aim of this proposal is to examine by both genomic and proteomic approaches, the repertoire of genes and proteins that are specifically upregulated by hypertonicity in inner medullary collecting duct (IMCD3) cells. These studies are designed to establish the temporal sequence for appearance and disappearance of genes over a 24 hour period and to contrast these observations to those seen in cells chronically adapted to hypertonic conditions (600-900 mOsm/kg). The differences in gene-protein expression between IMCD3 and more osmosensitive cortical collecting duct cells (M1) will be examined. Subsequent experiments, employing pharmacologic inhibition will examine the role of the modules of the MAP kinase family (ERKs, JNKs, and P38 MAP kinases) as well as PI3 kinase in the transcription of osmoregulated genes and expression of proteins. We will further focus our attention on the role of the c-JUN N-terminal kinase (JNK). Dominant negative mutants of this kinase are sensitized to hypertonicity and have impaired synthesis of some osmoprotective proteins. However, since dominant negative mutants still exhibit significant JNK activity, we will instead employ cells derived from immorto mice that are entirely deficient in JNK1 or JNK2. In addition to characterizing such cells and analyzing their genomic - proteomic response to hypertonicity, we will examine their osmosensitivity. The physiologic significance of the above observations will be assessed by analyzing the changes in gene-protein expression in rodents in various states of hydration. Furthermore, the importance of selected proteins that are found to be robustly upregulated in the above settings will be studied employing a silencing RNA technique that can render cells incapable of synthesizing selective proteins. The significance of the JNK activation will be tested in JNK1 and JNK2 knockout mice. These animals will be subjected to changing water balance states and to histologic examination of their inner medulla. The described experiments should define genes and proteins involved in osmoregulation and particularly dissect the role of JNK kinase and its isoforms in the adaptation of the mammalian inner medulla to hypertonicity.

在抗利尿状态下，陆生动物内髓细胞必须适应高渗环境并对张力变化做出快速反应。这种反应涉及蛋白激酶连续网络的激活，并最终上调促进细胞吸收惰性渗透剂的蛋白质。现在人们普遍认识到，适应性反应涉及更复杂的基因和蛋白质阵列。该提案的主要目的是通过基因组和蛋白质组学方法检查内髓集合管（IMCD3）细胞中高渗性特异性上调的基因和蛋白质库。这些研究旨在建立 24 小时内基因出现和消失的时间顺序，并对比这些 对长期适应高渗条件（600-900 mOsm/kg）的细胞中观察到的结果进行观察。将检查 IMCD3 和渗透敏感度更高的皮质集合管细胞 (M1) 之间基因蛋白表达的差异。随后的实验将采用药理抑制来检查 MAP 激酶家族模块（ERK、JNK 和 P38 MAP 激酶）以及 PI3 激酶在渗透调节基因转录和蛋白质表达中的作用。我们将进一步关注 c-JUN N 末端激酶 (JNK) 的作用。该激酶的显性失活突变体对高渗敏感，并损害了一些渗透保护蛋白的合成。然而，由于占主导地位 阴性突变体仍然表现出显着的 JNK 活性，我们将使用来自完全缺乏 JNK1 或 JNK2 的永生小鼠的细胞。除了表征这些细胞并分析它们对高渗性的基因组-蛋白质组反应之外，我们还将检查它们的渗透敏感性。上述观察结果的生理意义将通过分析啮齿动物在不同水合状态下基因-蛋白质表达的变化来评估。此外，我们将利用沉默RNA技术来研究在上述环境中发现的被强烈上调的选定蛋白质的重要​​性，该技术可以使细胞无法合成选择性蛋白质。 JNK 激活的意义将在 JNK1 和 JNK2 敲除小鼠中进行测试。这些动物将经历不断变化的水平衡状态并对其内部髓质进行组织学检查。所描述的实验应该 定义了参与渗透调节的基因和蛋白质，特别剖析了 JNK 激酶及其亚型在哺乳动物内髓质适应高渗性中的作用。