AT2 mediated Angiotensin II Signaling

描述（由申请人提供）：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脏发育不良，需要透析和移植才能长期生存。Ang II结合至少两种受体AT1和AT2。AT1介导细胞生长和分裂、血管收缩和盐潴留。AT1激活的降低可能介导了缺氧婴儿的某些胎儿毒性。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。在介导细胞死亡、血管舒张和盐排泄方面，AT2似乎与AT1的作用相反，但其下游信号通路尚未被确定，其在胎儿肾形成中的作用尚未被描述。在对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。正常的肾形成涉及后肾间质和入侵的UB之间严格控制的相互作用。过度的UB分支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AT2激活抑制UB分支，并试图确定与此过程相关的下游基因表达的变化。具体目的1：进一步验证AT2抑制培养胎鼠肾脏UB分支的假设。a.使用共聚焦显微镜和凝集素染色，我们将在特异性激活或拮抗AT2.b的条件下检查UB分支。我们将优化最大限度地激活或抑制AT2信号的培养条件，用于后续的减法克隆研究。特异性目的2：确定哪些基因在AT2激活或抑制后差异表达。将在AT2激活或拮抗后的2个时间点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进行测序以确定候选基因。NCBI BLAST搜索和生物信息学将用于将差异表达基因分类为结构和功能类别。定量RT-PCR将证实所选克隆的变化幅度。差异表达克隆的空间表达将使用原位杂交技术进行评估。通过筛选确定的候选基因影响输尿管芽分枝的能力将在未来的研究中进行测试。

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.