Guanine nucleotides in ischemic renal injury

描述(由申请人提供)：在这个国家，缺血性肾脏疾病仍然是死亡和发病的主要原因。它是许多血管疾病患者发生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机制是复杂的和多因素的，但已经确定了几种损伤的介质。其中，在许多情况下，ATP耗竭通常被视为急性缺血的标志。事实上，利用细胞培养系统中化学缺氧导致的ATP耗竭的体外模型已经了解到MUSH。然而，细胞内GTP池在缺血/再灌流或化学缺氧/恢复过程中的动态变化还没有得到系统的研究。因此，我们对鸟嘌呤核苷酸耗竭在缺血中的作用的理解仍然有很大的差距。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的运输、极性、细胞骨架和细胞死亡的各种信号GTP酶的功能中的重要性而提出的。特别是，小的GTP酶Rho、Rac和CDC42正在成为缺血/再灌注损伤修复循环的中心参与者。在这项建议中要检验的中心假设是，缺血/再灌流期间GTP的耗竭是决定所观察到的细胞死亡形式的一个重要的独立变量。细胞凋亡日益被认为是缺血时细胞丢失的一种重要方式。我们的假说假定GTP耗竭在调节这种形式的细胞死亡中起主要作用，可能是通过影响Rho家族GTP酶。我们建议建立选择性GTP和ATP耗竭的模型，并将它们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 GTP酶在调节细胞凋亡中的作用，方法是将Rho、Rac和CDc42的活性GTP酶和显性阴性GTP酶分别导入细胞。最后，我们将使用对照组和不能将鸟苷恢复为GTP的HPRT缺失小鼠来研究GTP在活体肾缺血中的作用。GTP水平升高对细胞形态、细胞凋亡和肾功能的影响将被确定。这些研究将确立GTP是一个关键因素。它是缺血性损伤的调节剂，并可能为这一毁灭性疾病带来新的治疗选择。

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.