AT2 mediated Angiotensin II Signaling

AT2 介导的血管紧张素 II 信号转导

• 批准号: 6751512
• 负责人: TERI J MAUCH
• 金额: $ 14.95万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6751512
• 关键词: angiotensin II; angiotensin receptor; confocal scanning microscopy; developmental genetics; embryo /fetus tissue /cell culture; expression cloning; genetic screening; growth media; in situ hybridization; laboratory rat; losartan; mesenchyme; molecular biology information system; nephrogenesis; organ culture; receptor expression; subtraction hybridization; ureter

DESCRIPTION (provided by applicant): Fetuses deprived of the vasoactive peptide and potent growth factor Angiotensin II (Ang II) are born with renal dysplasia, and they require dialysis and transplantation for long term survival. Ang II binds at least two receptors, AT1, and AT2. AT1 mediates cell growth and division, vasoconstriction, and salt retention. Decreased AT1 activation likely mediates some of the fetotoxicity in Ang U -deprived babies. AT2, however, is the predominant Ang II receptor in the fetal kidney, and its expression declines at birth. In mediating cell death, vasodilation, and salt excretion, AT2 seems to oppose AT1 action, but its downstream signaling pathways have yet to be identified, and its role in fetal nephrogenesis has not been delineated. In preliminary studies using cultured rat metanephroi, isolated from confounding variables, we found that a) Ang II stimulated ureteric bud (UB) branching, b) AT1 blockade decreased UB branching, whereas c) AT2 antagonism increased UB branching. Normal nephrogenesis involves tightly controlled reciprocal interactions between the metanephric mesenchyme and the invading UB. Excessive UB branching results in abnormal and ectopic induction of mesenchyme, whereas insufficient UB branching causes renal hypoplasia. We hypothesize that AT2 activation inhibits UB branching, and we seek to identify changes in downstream gene expression associated with this process.Specific Aim 1: To further test the hypothesis that AT2 inhibits UB branching in cultured fetal rat kidneys. a. Using confocal microscopy and lectin staining we will examine UB branching under conditions that specifically activate or antagonize AT2.b. We will optimize culture conditions that maximally activate or suppress AT2 signaling for subsequent subtraction cloning studies.Specific Aim 2: To identify which genes are differentially expressed following AT2 activation or suppression.a. Subtraction cloning will be performed between kidneys at 2 time points following AT2 activation or antagonismb. Hybridization analysis will be used to confirm differentially expressed clones.c. Differentially expressed cDNAs will be sequenced to identify candidate genes. NCBI BLAST searches and bioinformatics will be used to sort differentially expressed genes into structural and functional categories.d. Quantitative RT-PCR will confirm the magnitude of change of selected clones.e. The spatial expression of differentially expressed clones will be assessed using in situ hybridization.The ability of candidate genes identified by this screen to influence ureteric bud branching will be tested in future studies.

描述(申请人提供)：缺乏血管活性多肽和强大的生长因子血管紧张素II(Ang II)的胎儿出生时患有肾发育不良，他们需要透析和移植才能长期存活。血管紧张素Ⅱ与至少两个受体AT1和AT2结合。AT1调节细胞的生长和分裂、血管收缩和盐分滞留。AT1活性降低可能是ANG-U缺乏儿的一些胎儿毒性的调节因素。然而，AT2是胎儿肾脏中主要的Ang II受体，其表达在出生时下降。在调节细胞死亡、血管扩张和盐分排泄方面，AT2似乎与AT1的作用相反，但其下游信号通路尚未确定，其在胎儿肾脏形成中的作用也尚未阐明。在从混合变量分离的培养大鼠后肾的初步研究中，我们发现a)Ang II刺激输尿管芽(UB)分支，b)AT1阻断减少UB分支，而c)AT2拮抗增加UB分支。正常的肾脏发生包括后肾间充质和侵袭性UB之间严格控制的相互作用。UB分支过多会导致间充质的异常和异位诱导，而UB分支不足则会导致肾发育不良。我们假设AT2的激活抑制了UB分支，我们试图确定与这一过程相关的下游基因表达的变化。具体目标1：进一步验证AT2抑制培养的胎鼠肾脏UB分支的假设。使用共聚焦显微镜和凝集素染色，我们将研究在特定激活或拮抗AT2.b的条件下UB的分支。我们将优化培养条件，为后续的消减克隆研究最大限度地激活或抑制AT2信号。具体目标2：确定哪些基因在AT2激活或抑制后有差异表达。在AT2激活或拮抗后的2个时间点，在肾脏之间进行消减克隆。杂交分析将用于确认差异表达克隆。差异表达的cDNA将被测序以确定候选基因。NCBI BLAST搜索和生物信息学将被用于将差异表达的基因分类为结构和功能类别。定量RT-PCR将确认所选克隆的变化幅度。差异表达克隆的空间表达将通过原位杂交进行评估。通过该筛选确定的候选基因影响输尿管芽分支的能力将在未来的研究中得到测试。