INFLAMM. /APOPTOSIS--EXPERIMENTAL HEMOLYTIC UREMITIC SYN

描述(由申请人提供)：溶血性尿毒症综合征(HUS)是导致儿童肾功能衰竭的最常见原因。HUS是一种严重的炎症性疾病，由产生志贺毒素的大肠杆菌引起，其特点是肾脏内表达促炎介质。志贺毒素是核毒素大家族中的一员，其对细胞的毒性源于28S核糖体RNA(28S RRNA)的sarcin/ricin环中的单个腺嘌呤的脱嘌呤。28S rRNA的脱氢不仅导致蛋白质翻译的抑制，而且还导致JNK和p38MAPK等应激激活蛋白激酶的强烈和广泛的激活。这些激酶是炎症反应的中心介质，负责诱导促炎细胞因子和趋化因子的转录。对志贺毒素的主要靶组织以及驱动促炎和细胞毒性反应的机制的了解不足，阻碍了HUS介入治疗的发展。对大鼠和小鼠等实验动物注射志贺毒素，未能概括HUS涉及肾小球病理的肾脏特征。我们已经开发了一种由注射蓖麻毒素诱导的HUS小鼠模型。蓖麻毒素是一种核毒素，其核糖体靶标与志贺毒素相同，但它与所有细胞上存在的受体结合。我们将在小鼠和原代培养细胞(巨噬细胞和肾小球来源的细胞)中使用蓖麻毒素，以阐明最初的作用靶点、细胞毒性后果以及导致HUS的细胞和分子机制。一、我们将测定蓖麻毒素和脂多糖对实验性HUS、野生型小鼠和肿瘤坏死因子和白介素I受体缺陷小鼠肾小球基因表达的激活作用：二、我们将测定肾小球培养细胞中被激活的基因和涉及的信号通路。我们将通过阐明顶端caspase 8的参与和确定caspase抑制剂是否可以改善给予蓖麻毒素的细胞毒性后果来表征实验性HUS的凋亡机制。

DESCRIPTION (provided by applicant): Hemolytic-uremic syndrome (HUS) is the most common cause of renal failure in children. HUS is a severe inflammatory disease, caused by Shiga toxin-producing E. coli, and is marked by the expression of proinflammatory mediators within the kidneys. Shiga toxin is a member of a large family of ribotoxins whose toxicity to cells stems from the depurination of a single adenine within the "sarcin/ricin" loop of 28S ribosomal RNA (28S rRNA). The depurination of 28S rRNA results not only in the inhibition of protein translation, but also the intense and extended activation of the stress-activated protein kinases such as JNK and p38 MAPK. These kinases are central mediators of inflammatory responses that are responsible for inducing the transcription of proinflammatory cytokines and chemokines. An insufficient understanding of the primary target tissues of Shiga toxins and the mechanisms that drive the proinflammatory and cytotoxic responses has impeded the development of interventional remedies for HUS. The administration of Shiga toxins to experimental animals such as rats and mice has failed to recapitulate the renal hallmarks of HUS that involve glomerular pathologies. We have developed a mouse model of HUS that is induced by administration of ricin, a ribotoxin that has an identical ribosomal target as Shiga toxins, but that binds to receptors present on all cells. We will employ ricin in mice and primary cultured cells (macrophages and cells derived from glomeruli) to elucidate the initial targets of action, the cytotoxic consequences, and the cellular and molecular mechanisms that lead to HUS. I. We will determine the activation of gene expression induced by ricin and LPS in glomeruli in experimental HUS, in wild-type mice and in mice that are deficient in TNF and IL-I receptors: II. We will determine the genes that are activated, and the signaling pathways involved, in cultured cells of the renal glomerulus. III. We will characterize the apoptotic mechanisms in experimental HUS by elucidating the involvement of apical caspase 8 and by determining whether caspase inhibitors can ameliorate the cytotoxic consequences of ricin administration.