MECHANISM OF DNA DAMAGE IN HYPOXIA/REOXYGENATION INJURY

一般认为，体内缺血/再灌注损伤和 体外缺氧/复氧损伤特征在于 细胞内部稳态的崩溃。然而，DNA损伤作为一种 细胞活力丧失之前的早期事件以前没有 在这个模型中描述。本提案的核心假设是 DNA损伤是肾脏缺氧/复氧损伤的早期事件 近端小管或体内肾脏缺血/再灌注损伤，和 氧化应激和核酸内切酶激活是主要的 DNA损伤的决定因素。我们的初步研究 支持这一假设。DNA损伤（通过碱性解旋测量） 试验）发生，缺氧少至5分钟，然后 再氧合至新鲜分离的大鼠近端肾小管（PT）。 DNA损伤的两种充分描述的机制是氧化应激和 核酸内切酶激活。在我们的初步研究中， 氧代谢物（ROM）以及核酸内切酶抑制剂是保护性的 抗缺氧/复氧诱导的DNA损伤。这两 DNA损伤、氧化应激和核酸内切酶激活的机制， 并不一定是相互排斥的。事实上，在我们最近的研究中， 已经表明核酸内切酶激活是DNA的重要机制， LLC-PK1细胞（肾小管上皮细胞）的损伤和细胞死亡 线）暴露于过氧化氢。 本提案的目标将通过以下方式实现： 新鲜分离的大鼠PT，用于体外研究和体内研究 利用经受缺血/再灌注的大鼠肾脏的方法如下： I.目的：探讨体外缺氧/复氧损伤中DNA损伤的特点， 体内缺血/再灌注损伤。 二.研究核酸内切酶激活在DNA损伤和细胞凋亡中的作用， 体外缺氧/复氧损伤及缺血/再灌注损伤 体内损伤。 三.研究钙离子在缺氧/复氧诱导的DNA损伤中的作用 损伤和细胞损伤。 四.研究活性氧代谢产物在DNA损伤中的作用， 体外缺氧/复氧损伤中的细胞损伤， 体内缺血/再灌注损伤。 V.研究铁在DNA损伤和细胞损伤中的作用， 体外缺氧/复氧损伤和缺血/再灌注损伤 vivo.

It is generally accepted that ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro are characterized by a rapid collapse of internal homeostasis of the cell. However, DNA damage as an early event prior to loss of cell viability has not been previously described in this model. The central hypothesis of the present proposal is that DNA damage is an early event in hypoxia/reoxygenation injury to renal proximal tubules or in ischemia/reperfusion injury to kidney in vivo, and that oxidative stress and endonuclease activation are the major determinants of the DNA damage. Our preliminary studies lend strong support to this hypothesis. DNA damage (measured by the alkaline unwinding assay) occurs with as little as 5 min. of hypoxia followed by reoxygenation, to freshly isolated rat proximal renal tubules (PT). The two well described mechanisms of DNA damage are oxidant stress and endonuclease activation. In our preliminary studies scavengers of reactive oxygen metabolites (ROM) as well as endonuclease inhibitors are protective against the DNA damage induced by hypoxia/reoxygenation. These two mechanisms of DNA damage, oxidative stress and endonuclease activation, are not necessarily mutually exclusive. Indeed, in our recent study we have shown that endonuclease activation is an important mechanism of DNA damage and cell death in LLC-PK1 cells (a renal tubular epithelial cell line) exposed to hydrogen peroxide. The objectives of the present proposal, which will be carried out using freshly isolated rat PT for in vitro studies, and for in vivo studies utilizing rat kidneys subjected to ischemia/reperfusion are the following: I. To characterize DNA damage in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. II. To examine the role of endonuclease activation in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. III. To examine the role of calcium in hypoxia/reoxygenation-induced DNA damage and cell injury. IV. To examine the role of reactive oxygen metabolites in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. V.To examine the role of iron in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo.