AT2 mediated Angiotensin II Signaling

AT2 介导的血管紧张素 II 信号转导

• 批准号: 6558095
• 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 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6558095
• 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（由申请方提供）：缺乏血管活性肽和强效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，需要透析和移植才能长期存活。 血管紧张素II结合至少两种受体，AT 1和AT 2。AT 1介导细胞生长和分裂、血管收缩和盐潴留。AT 1激活的降低可能介导缺乏Ang U的婴儿的某些胎儿毒性。然而，AT 2是胎儿肾脏中主要的Ang II受体，其表达在出生时下降。在介导细胞死亡，血管舒张和盐排泄，AT 2似乎反对AT 1的行动，但其下游信号通路尚未确定，其在胎儿肾发生的作用尚未划定。在使用培养的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）AT 1阻断减少UB分支，而c）AT 2拮抗增加UB分支。正常的肾发生涉及后肾间充质和侵袭性UB之间严格控制的相互作用。 UB分支过多导致间质异常和异位诱导，而UB分支不足导致肾发育不全。我们假设，AT 2激活抑制UB分支，我们试图确定下游基因表达的变化与此processing.Specific Aim 1：为了进一步测试的假设，AT 2抑制UB分支在培养的胎鼠肾脏。a.使用共聚焦显微镜和凝集素染色，我们将检查UB分支的条件下，特异性激活或拮抗AT2.b。我们将优化培养条件，最大限度地激活或抑制AT 2信号转导，用于后续的消减克隆研究。具体目标2：鉴定哪些基因在AT 2激活或抑制后差异表达。在AT 2激活或拮抗后的2个时间点b，在肾脏之间进行减法克隆。杂交分析将用于确认差异表达的克隆。将对差异表达的cDNA进行测序以鉴定候选基因。将使用NCBI BLAST搜索和生物信息学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采用原位杂交进行评估，通过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进行测试。