MECHANISMS OF RENAL ADAPTATION TO ANOXIA

肾脏适应缺氧的机制

基本信息

批准号：
7479724
负责人：
Michael Kashgarian
金额：
$ 25.58万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7479724
关键词：
Acute Kidney Failure Address Affinity Anoxia Apoptotic Biological Biological Phenomena Brain Brain Hypoxia-Ischemia Cell Death Induction Cells Cellular Structures Chronic Class Connexins Depth Down-Regulation Ensure Epithelial Cells Experimental Designs Gene Family Gene Proteins Gene Silencing Genes Heart Heat shock proteins Heat-Shock Response Homeostasis Hypoxia Hypoxia Inducible Factor Hypoxia Pathway Imaging Techniques Infant Care Injury Intercalated disc Ischemia Kidney Metabolic Metabolism Microarray Analysis Molecular Natural regeneration Nephrons Neuroglia Neurons Numbers Oligonucleotides Outcome Measure Oxygen Pathway interactions Play Preparation Process Proteins Proteomics RNA Interference Rattus Research Research Design Resistance Resistance development Role Stress Suspension Culture System Techniques Testing Therapeutic Intervention base cell injury deprivation design heat shock transcription factor injured kidney cell protective effect protein expression renal epithelium response transcription factor

项目摘要

The broad objective of this project is an advanced understanding of molecular processes by which the immature nephron is protected from both oxygen deprivation and ischemic insults. Two classes of genes which are induced by ischemia or hypoxia have been shown to be over expressed constitutively in the immature kidney, the heat shock response (HSR) and the hypoxia responsive pathway (HRP). Each of these ubiquitous biological systems may play a fundamental role in the innate tolerance to injury manifested by the immature nephron. Specific Aim 1 will evaluate the HSR by determining if down-regulation of either constitutive activation of Heat Shock Transcription Factor (HSF) or diminution of HSP72 and/or HSP25 expression will impair tolerance to injury in immature nephrons. Specific Aim 2 will investigate HRP by delineating effects of inhibition of Hypoxia Inducible Factor (HIF-1) activity and synthesis of hemoxygenase and erythropoitin following hypoxic or ischemic insults in immature kidneys. Both specific aims will test a common hypothesis: if tolerance of the immature nephron is dependent on the activity of transcription factors and/or abundance of proteins which are constitutively over-expressed compared to the mature kidney, then dampening of transcription factor activity and/or inhibition of synthesis of specific proteins will increase the vulnerability of the immature nephron to a hypoxic or ischemic injury. The proposed studies utilize new techniques for gene silencing which allow a) global dampening of families of genes controlled by a common transcription factor (oligonucleotide decoys) and b) specific inhibition of synthesis of selected proteins (short interference RNA, siRNA). To ensure the feasibility of the experimental design, transcription factor decoys for HSF and HIF-1 have been developed and validated in LLCPK cells, as well as suspensions and cultures of immature proximal tubules. In addition, two target sequences for HSP72 siRNA have been identified and shown to specifically inhibit synthesis of HSP72 but not HSP25 in LLCPK cells after ATP depletion. The developing kidney, in which cytoprotective proteins are up-regulated without prior stress, represents an ideal and somewhat unique circumstance in which to delineate molecular mechanisms and pathobiologic processes which are fundamental to cellular injury in the mature kidney. Moreover, an in-depth understanding in the immature kidney will be important so that therapeutic interventions which might offset the protective effects can be avoided in care of infants with acute renal failure.

该项目的总体目标是深入了解保护未成熟肾单位免受缺氧和缺血性损伤的分子过程。两类由缺血或缺氧诱导的基因已被证明在未成熟的肾脏中组成型过度表达，即热休克反应（HSR）和缺氧反应途径（HRP）。这些普遍存在的生物系统中的每一个都可能在未成熟肾单位表现出的先天损伤耐受性中发挥重要作用。具体目标 1 将通过确定热休克转录因子 (HSF) 组成型激活的下调或 HSP72 和/或 HSP25 表达的减少是否会损害未成熟肾单位对损伤的耐受性来评估 HSR。具体目标 2 将通过描述未成熟肾脏缺氧或缺血损伤后抑制缺氧诱导因子 (HIF-1) 活性以及血氧合酶和红细胞生成素合成的影响来研究 HRP。这两个具体目标都将检验一个共同的假设：如果未成熟肾单位的耐受性取决于转录因子的活性和/或与成熟肾相比组成性过度表达的蛋白质的丰度，那么转录因子活性的抑制和/或抑制特定蛋白质的合成将增加未成熟肾单位对缺氧或缺血性损伤的脆弱性。拟议的研究利用基因沉默新技术它允许 a) 对由共同转录因子（寡核苷酸诱饵）控制的基因家族进行全局抑制，以及 b) 对选定蛋白质的合成进行特异性抑制（短干扰 RNA，siRNA）。为了确保实验设计的可行性，HSF 和 HIF-1 的转录因子诱饵已在 LLCPK 细胞以及未成熟近端小管的悬浮液和培养物中进行了开发和验证。此外，HSP72 siRNA 的两个靶序列已被鉴定，并显示在 ATP 耗尽后特异性抑制 LLCPK 细胞中 HSP72 的合成，但不抑制 HSP25。发育中的肾脏，其中细胞保护蛋白在没有先前应激的情况下上调，代表了一种理想且有些独特的环境，在这种环境中描述了成熟肾脏中细胞损伤的基础分子机制和病理生物学过程。此外，对未成熟肾脏的深入了解也很重要，这样治疗干预措施可能会抵消在护理患有急性肾功能衰竭的婴儿时可以避免保护作用。