MECHANISMS OF RENAL ADAPTATION TO ANOXIA

肾脏适应缺氧的机制

• 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
• 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

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.

该项目的广泛目标是对分子过程的深入理解，通过该过程，未成熟的肾单位受到保护，免受氧气剥夺和缺血性损伤。通过缺血或缺氧诱导的两类基因已被证明在未成熟肾脏，热休克反应（HSR）和缺氧反应途径（HRP）中构成过度表达。这些无处不在的生物系统中的每一个都可能在未成熟的肾单位表现出对损伤的天生宽容中起着基本作用。具体目标1将通过确定对热激转录因子（HSF）的本构激活（HSF）的下调或HSP72和/或HSP25表达的减少来评估HSR，这会损害对未成熟肾脏的损伤的耐受性。具体目标2将通过描述抑制缺氧诱导因子（HIF-1）活性的作用以及在未成熟肾脏中缺氧或缺血性损伤后的抑制作用（HIF-1）活性以及血氧酶和红细胞生成素的合成。这两个具体目的都将检验一个共同的假设：如果不成熟肾单位的耐受性取决于转录因子的活性和/或蛋白质的丰度，与成熟的肾脏相比，这些蛋白质的组成性过表达，那么转录因子活性的衰减和/或抑制特定蛋白质的抑制作用会增加不受欢迎的肾脏的脆弱性或不足的较不受欢迎的否则。拟议的研究利用新技术进行基因沉默 a）允许全球受损由共同转录因子控制的基因家族（寡核苷酸诱饵）和b）特定的抑制所选蛋白合成的特定抑制作用（短干扰RNA，siRNA）。为了确保实验设计的可行性，在LLCPK细胞中已经开发和验证了HSF和HIF-1的转录因子诱饵，以及未成熟近端小管的悬浮液和培养物。此外，已经鉴定出Hsp72 siRNA的两个靶序列，并显示出ATP耗竭后LLCPK细胞中HSP72的合成，而不是HSP25的合成。发育中的肾脏，其中细胞保护蛋白在没有先前压力的情况下被上调，代表了一种理想且有些独特的情况，在其中描绘了分子机制和病理生物学过程，这对于成熟肾脏中的细胞损伤至关重要。此外，对未成熟肾脏的深入了解将很重要，以便可以抵消治疗干预措施 可以避免保护性效果，以照顾急性肾衰竭的婴儿。