MECHANISMS OF RENAL ADAPTATION TO ANOXIA

该项目的广泛目标是对未成熟肾细胞免受缺氧和缺血损伤的分子过程的深入了解。两类由缺血或缺氧诱导的基因在未成熟肾脏中组成性地过表达，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元表现出的先天损伤耐受中发挥基本作用。特异性目的1将通过确定热休克转录因子（HSF）组成激活的下调或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。特异性目的2将通过描述缺氧诱导因子（HIF-1）活性和血氧酶和促红细胞生成素合成在未成熟肾脏缺氧或缺血损伤后的抑制作用来研究HRP。这两个特定的目标都将验证一个共同的假设：如果未成熟肾细胞的耐受性取决于转录因子的活性和/或与成熟肾脏相比组成性过表达的蛋白质的丰度，那么抑制转录因子活性和/或抑制特定蛋白质的合成将增加未成熟肾细胞对缺氧或缺血性损伤的脆弱性。拟议的研究利用了基因沉默的新技术

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.