Role of guanine nucleotides in ischemic renal injury

描述（由申请人提供）：缺血性肾病仍然是该国死亡率和发病率的主要原因。它是许多血管疾病患者肾功能衰竭的重要病因。虽然缺血性组织损伤的病理生理学是复杂的和多因素的，但已经鉴定了几种损伤介质。其中，ATP耗竭在许多情况下通常被视为急性缺血的标志。事实上，已经在细胞培养系统中使用化学缺氧诱导的ATP消耗的体外模型来了解了许多。然而，在缺血/再灌注或化学缺氧/恢复过程中细胞GTP池的动力学尚未得到系统的研究。因此，我们对鸟嘌呤核苷酸缺失在缺血中的作用的理解仍然存在很大的差距。鸟嘌呤核苷酸的这种作用是由细胞GTP/GDP比率在控制上皮细胞中的运输、极性、细胞骨架和细胞死亡的各种信号转导GTP酶的功能中的重要性所暗示的。特别是，小GTP酶Rho，Rac和Cdc 42正在成为在缺血/再灌注中观察到的损伤修复周期的核心参与者。在这个提议中要测试的中心假设是，在缺血/再灌注期间GTP耗尽是确定所观察到的细胞死亡形式的重要且独立的变量。细胞凋亡越来越被认为是缺血过程中细胞丢失的一种重要方式。我们的假设假定GTP耗竭在调节这种形式的细胞死亡中起主要作用，可能是通过对Rho家族GTP酶的影响。我们建议开发选择性GTP和ATP耗竭的模型，并将其与培养系统中的细胞存活和凋亡性死亡相关联。我们还将研究Rho GTPases在调节细胞凋亡中的作用，使用转染了组成型活性和显性阴性形式的GTPases Rho，Rac和Cdc 42的细胞。最后，我们将研究GTP在肾缺血中的作用，在体内使用控制和HPRT-无效的小鼠，不能挽救鸟苷GTP。将确定增强的GTP水平对形态学、细胞凋亡和肾功能的影响。这些研究将确定GTP作为关键。缺血性损伤的调节剂，并可能导致这种毁灭性疾病的新的治疗选择。

DESCRIPTION (provided by applicant): lschemic renal disease remains a major cause of mortality and morbidity in this country. It is an important etiologic factor in many cases of renal failure in patients with vascular disease. While the pathophysiology of ischemic tissue damage is complex and multifactorial, several mediators of injury have been identified. Among these, ATP depletion is generally viewed as the hallmark of acute ischemia in many settings. Indeed, mush has been learned using in vitro models of chemical anoxia- induced ATP depletion in cell culture systems. However, the dynamics of cellular GTP pools during ischemia/reperIusion or chemical anoxia/recovery have not been investigated systematically. Thus, there remains a big gap in our understanding of the role of guanine nucleotide depletion in ischemia. Such a role for guanine nucleotides is suggested by the importance of the cellular GTP/GDP ratio in the functioning of a variety of signaling GTPases that control trafficking, polarity, the cytoskeleton and cell death in epithelia. In particular, the small GTPases Rho, Rac and Cdc42 are emerging as central participants in the injury-repair cycles observed in ischemia/reperfusion. The central hypothesis to be tested in this proposal is that GTP depletion during ischemia/reperfusion is an important and independent variable in determining the form of cell death observed. Apoptosis is increasingly recognized as a significant mode of cell loss during ischemia. Our hypothesis postulates a major role for GTP depletion in modulating this form of cell death, possibly via an effect on Rho family GTPases. We propose to develop models of selective GTP and ATP depletion and correlate them with cell survival and apoptotic death in culture systems. We will also investigate the role of Rho GTPases in modulating apoptosis using cells transfected with constitutively active and dominant negative forms of the GTPases Rho, Rac and Cdc42. Finally, we will examine the role of GTP in renal ischemia in vivo using control and HPRT-null mice that cannot salvage guanosine to GTP. The effects of enhanced GTP levels on morphology, apoptosis and renal function will be determined. These studies will establish GTP as a key . modulator of ischemic injury and might lead to new therapeutic options for this devastating disease.