Inducible Dysplastic Nephropathy in B2-Deficient Mice

描述（由申请人提供）：基因-环境相互作用与先天性疾病有关。因此，迫切需要开发人类疾病的动物模型，这是确定的基因-环境相互作用的产物。更好地了解基因-环境相互作用诱导先天性缺陷的机制对于推进器官发育不全的预防和治疗策略是必要的。我们以前证明，缓激肽B2受体无效（BdkrB 2-/-）胚胎暴露于妊娠期盐应激发展肾发育不全的结果，p53介导的肾祖细胞凋亡和终末分化程序的抑制。在肾器官发生过程中，肿瘤抑制蛋白p53和BdkrB 2之间存在生理串扰。因此，虽然BdkrB 2是p53介导的转录激活的靶点，但需要BdkrB 2来防止检查点激酶1（Chk 1）对Ser 23上的p53的磷酸化。p-p53 Ser 23是BdkrB 2-/-突变小鼠肾发育不全的中心介质 在该竞争性更新申请中待检验的总体假设是，Chk 1-p53通路（其介导BdkrB 2突变体对肾发育不全的易感性）的激活是细胞自主的，并且胚胎盐胁迫在表观遗传学上起作用以抑制遗传易感宿主中的终末分化程序。特异性目的1检验了BdkrB 2受体介导输尿管芽谱系中的肾保护的假设。为此，我们已经开发了条件BdkrB 2lox/lox小鼠，这将被用来产生后代缺乏受体的输尿管芽或后肾间充质通过铬介导的切除。我们预计，BdkrB 2从集合管的损失是足以重演传统的BdkrB 2-空表型激活Chk 1- p53途径。具体目标2测试的假设，暴露BdkrB 2-null胚胎盐胁迫与渐进的Pax-2，一个转录因子，是正常的上皮肾单位分化所需的表观遗传沉默。与报告小鼠的遗传杂交将揭示Bdkrb 2突变体中Pax 2基因转录的时空动态。在Pax 2启动子处的组蛋白编码的表征将确定BdkrB 2-/-肾脏中的进行性转录抑制伴随着组蛋白脱乙酰酶-1募集、启动子组蛋白H3脱乙酰化和赖氨酸4（H3 K4）脱甲基化和H3 K9/27甲基化，随后是CpG岛的超甲基化。我们推测，这些修改组蛋白编码抑制转录因子结合的Pax 2启动子。最后，我们将确定这些基因表达的表观遗传效应是否可以通过HDAC 1的条件失活来挽救。特异性目的3检验了以下假设：在发育不良的BdkrB 2-/-肾脏中积累的p-p53 Ser 23抑制肝细胞核因子-1（HNF 1？）基因转录。这些研究将证明HNF 1的抑制有助于上皮-间充质转化，从而导致发育不良。我们认为，用p53的非磷酸化版本（p53 S/A23）替换内源性p53基因可以挽救盐胁迫下BdkrB 2-/-胚胎中HNF 1？的表达和终末分化。 公共卫生相关性：在小于4岁的儿童中，肾脏和泌尿道的先天性畸形占慢性肾衰竭的40%。除了DNA序列的突变，肾脏发育异常可能是由于“表观遗传密码”的改变。由短暂环境暴露引起的表观遗传密码的稳定改变可以永久地破坏胎儿基因表达程序。因此，了解一个定义的基因突变（例如，BdkrB 2-/-）与确定的胚胎应激物（妊娠盐）相互作用以改变肾脏发育的调节，可能为治疗或预防肾脏和尿路畸形开辟新途径。

DESCRIPTION (provided by applicant): Gene-environment interactions are implicated in congenital disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. A better understanding of the mechanisms by which gene-environment interactions induce congenital defects is necessary to advance preventive and therapeutic strategies for organ dysgenesis. We previously demonstrated that bradykinin B2 receptor-null (BdkrB2-/-) embryos exposed to gestational salt stress develop renal dysgenesis as a result of p53-mediated apoptosis of nephron progenitors and repression of the terminal differentiation program. A physiological cross-talk operates between the tumor suppressor protein, p53, and the BdkrB2 during renal organogenesis. Thus, while BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 is required to prevent the phosphorylation of p53 on Ser23 by Checkpoint kinase 1 (Chk1). p-p53Ser23 is the central mediator of renal dysgenesis in BdkrB2-/- mutant mice. The Overall hypothesis to be tested in this Competitive Renewal application is that activation of the Chk1-p53 pathway, which mediates the susceptibility of BdkrB2 mutants to renal dysgenesis, is cell autonomous, and that embryonic salt stress acts epigenetically to repress the terminal differentiation program in a genetically susceptible host. Specific Aim 1 tests the hypothesis that BdkrB2 receptors mediate nephroprotection in the ureteric bud lineage. To this end, we have developed conditional BdkrB2lox/lox mice, which will be used to produce progeny lacking the receptor from the ureteric bud or metanephric mesenchyme by Cre-mediated excision. We anticipate that loss of BdkrB2 from the collecting duct is sufficient to recapitulate the conventional BdkrB2-null phenotype by activating the Chk1- p53 pathway. Specific Aim 2 tests the hypothesis that exposure of BdkrB2-null embryos to salt stress is associated with progressive epigenetic silencing of Pax-2, a transcription factor that is required for normal epithelial nephron differentiation. Genetic crosses to reporter mice will reveal the spatiotemporal dynamics of Pax2 gene transcription in Bdkrb2-mutants. Characterization of the histone code at the Pax2 promoter will determine that progressive transcriptional repression in BdkrB2-/- kidneys is accompanied by histone deacetylase-1 recruitment, promoter histone H3 deacetylation and lysine 4 (H3K4) demethylation and H3K9/27 methylation, followed by hypermethylation of CpG islands. We postulate that these modifications in the histone code inhibit transcription factor binding to the Pax2 promoter. Finally, we will determine if these epigenetic effects on gene expression can be rescued by conditional inactivation of HDAC1. Specific Aim 3 tests the hypothesis that p-p53Ser23, which accumulates in dysplastic BdkrB2-/- kidneys, represses Hepatocyte Nuclear Factor-1 (HNF1¿) gene transcription. These studies will demonstrate that repression of HNF1¿ contributes to epithelial-mesenchymal transition and thus to dysplasia. We propose that replacement of the endogenous p53 gene with a non-phosphorytable version of p53 (p53S/A23) rescues HNF1¿ expression and terminal differentiation in salt-stressed BdkrB2-/- embryos. PUBLIC HEALTH RELEVANCE: Congenital malformations f the kidney and urinary tract account for up to 40% of chronic kidney failure in children less than 4 years of age. In addition to mutations in the DNA sequence, abnormal kidney development may result from alterations in the "epigenetic code". Stable alterations in the epigenetic code resulting from transient environmental exposures can permanently disrupt the fetal gene expression program. Thus, understanding how a defined genetic mutation (e.g., BdkrB2-/-) interacts with a defined embryonic stressor (gestational salt) to alter the regulation of kidney development may open new avenues to the treatment or prevention of kidney and urinary tract malformations.