MECHANISMS OF RENAL ADAPTATION TO ANOXIA

这个项目的主要目标是对保护未成熟肾单位免受缺氧和缺血损伤的分子过程有更深入的了解。缺血或缺氧诱导的热休克反应（HSR）和缺氧反应途径（HRP）基因在未成熟肾脏中组成性表达。这些无处不在的生物系统中的每一个都可能在对未成熟肾单位表现出的损伤的先天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）的组成性激活或HSP 72和/或HSP 25表达的减少的下调是否会损害未成熟肾单位对损伤的耐受性来评价HSR。具体目标2将通过描述在未成熟肾脏中缺氧或缺血性损伤后抑制缺氧诱导因子（HIF-1）活性以及合成加氧酶和促红细胞生成素的作用来研究HRP。这两个特定的目的将测试一个共同的假设：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表达的蛋白质的丰度，则转录因子活性的抑制和/或特定蛋白质合成的抑制将增加未成熟肾单位对缺氧或缺血性损伤的脆弱性。这项研究利用了基因沉默的新技术 其允许a）由共同转录因子（寡核苷酸诱饵）控制的基因家族的整体抑制和B）选择的蛋白质（短干扰RNA，siRNA）的合成的特异性抑制。为了确保实验设计的可行性，已经在LLCPK细胞以及未成熟近端小管的悬浮液和培养物中开发并验证了HSF和HIF-1的转录因子诱饵。此外，已经鉴定了HSP 72 siRNA的两个靶序列，并显示其在ATP耗尽后特异性抑制LLCPK细胞中HSP 72而非HSP 25的合成。发育中的肾脏，其中细胞保护蛋白在没有预先应激的情况下被上调，代表了一种理想的和有点独特的情况，其中描绘了成熟肾脏中细胞损伤的基本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将是重要的，以便可能抵消未成熟肾脏的治疗干预。 在护理患有急性肾衰竭的婴儿时可避免保护作用。

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.