Transcriptional regulation of proteinuria

肾衰竭是美国和世界范围内发病率和死亡率的主要原因。之一 影响肾衰竭进展的主要因素是蛋白尿的量。我们的长期 目的是明确蛋白尿和肾小球疾病的确切机制，以便开发新的 未来的治疗。这项提案的总体目标是了解转录因子的作用 锌指和同源异型盒3（ZHX 3）在蛋白尿发病机制中的作用在具体目标1中，我们将研究 ZHX3在迁移到细胞核之前的翻译后切割和磷酸化。然后我们将 研究阻断ZHX3的核输入或输出是否会导致基因表达谱的改变， ZHX3靶基因。在特异性目标2中，两个ZHX3蛋白片段对基因表达的个体效应均被观察到。 通过真实的时间PCR研究培养细胞中的表达谱。接下来，与其他ZHX家族的互动 成员和其他已知的和推定的相互作用蛋白质进行评估。影响的具体因素 ZHX 3与组织蛋白酶L和足糖萼蛋白启动子的结合将通过以下的组合来研究： 染色质免疫沉淀和定点诱变。其次，个人和综合影响 各种ZHX家族成员及其已知的相互作用伴侣对ZHX启动子活性的影响 在特定目标3中，足细胞特异性条件性ZHX 3敲除小鼠将 研究ZHX3在正常足细胞功能中的作用。小鼠ZHX3基因的选定部分 将被克隆到靶向载体中，该载体将被电穿孔到小鼠胚胎干细胞中。后 筛选同源重组，FLP重组酶将用于去除新霉素抗性 将靶向干细胞与八期胚胎融合，产生嵌合小鼠。经过进一步 繁殖和基因分型后，将floxed ZHX3小鼠与podocin Cre小鼠交配，以产生 几个繁殖和基因分型周期，ZHX 3-/-和+/-小鼠。ZHX 3的尿蛋白排泄 评估缺陷小鼠，并进行详细的形态学表征。 研究啮齿类动物肾脏疾病的发展和尿液中蛋白质的泄漏将有助于我们 更好地了解人类肾脏疾病的过程。这将导致发展适当的 未来的治疗策略。

Kidney failure is a major cause of morbidity and mortality in the United States and worldwide. One of the major factors that influences the progression of kidney failure is the amount of proteinuria. Our long term goal is to define the precise mechanisms of proteinuria and glomerular disease, so as to develop new treatments in the future. The overall goal of this proposal is to understand the role of transcriptional factor Zinc Fingers and Homeoboxes 3 (ZHX3) in the pathogenesis of proteinuria. In Specific Aim 1, we will study the post-translational cleavage and phosphorylation of ZHX3 prior to migration into the nucleus. We will then study whether blocking nuclear import or export of ZHX3 causes alterations in the gene expression profile of ZHX3 target genes. In Specific Aim 2, the individual effects of both ZHX3 protein fragments on the gene expression profile in cultured cells will be studied by real time PCR. Next, interaction with other ZHX family members and other known and putative interacting protein will be assessed. Specific factors that influence the binding of ZHX3 to the Cathepsin L and podocalyxin promoters will be studied by a combination of chromatin immunoprecipitation and site directed mutagenesis. Next, the individual and combined influences of the the various ZHX family members and their known interacting partners on the promoter activity of selected genes will be assesed.ln Specific Aim 3, a podocyte-specific conditional ZHX3 knockout mouse will be generated to study the role of ZHX3 in normal podocyte function. Selected parts of the mouse ZHX3 gene will be cloned into the targeting vector, which will be electroporated into mouse embryonic stem cells. After screening for homologous recombination, FLP recombinase will be used to remove the neomycin resistance cassette, the targeted stem cells fused with eight stage embryos, and chimeric mice generated. After further breeding and genotyping, the floxed ZHX3 mouse will mated with a podocin Cre mouse, to generate, after several cycles of breeding and genotyping, ZHX3 -/- and +/- mice. Urinary protein excretion of the ZHX3 deficient mice will be assessed, and detailed morphological characterization undertaken. Studying the development of kidney disease and the leakage of protein in the urine in rodents will help us better understand the process of kidney disease in humans. This will lead to the devlopment of appropriate treatment strategies in the future.