Inducible Dysplastic Nephropathy in B2-Deficient Mice

描述(由申请人提供)：基因与环境的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，这种模型是明确的基因-环境相互作用的产物。更好地了解基因-环境相互作用导致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。我们先前的研究表明，孕期盐应激可导致缓激肽B2受体缺失(BdkrB2-/-)胚胎发生肾脏发育不全，其原因是P53介导的肾祖细胞的凋亡和终末分化程序的抑制。在肾脏器官发生过程中，肿瘤抑制蛋白P53和BdkrB2之间存在生理性的相互作用。因此，虽然BdkrB2是P53介导的转录激活的靶标，但BdkrB2需要阻止检查点激酶1(Chk1)对Ser23上P53的磷酸化。P-p53Ser23是BdkrB2-/-突变小鼠肾发育不全的中枢调节因子。 在这项竞争性更新应用中要测试的总体假设是，Chk1-P53途径的激活是细胞自主的，它介导了BdkrB2突变体对肾脏发育不全的易感性，并且胚胎盐应激在表观遗传中作用于抑制遗传敏感宿主的末端分化程序。特定目的1验证BdkrB2受体在输尿管芽系中介导肾保护的假设。为此，我们培育了条件性BdkrB2lox/lox小鼠，它们将用于通过Cre介导的切除来产生输尿管芽或后肾间充质中缺乏受体的后代。我们预计BdkrB2从集合管中的丢失足以通过激活Chk1-P53途径来概括传统的BdkrB2缺失的表型。特殊目的2验证了这样的假设，即BdkrB2缺失的胚胎暴露在盐胁迫下与Pax-2的进行性表观遗传沉默有关，Pax-2是正常上皮肾单位分化所必需的转录因子。与报告鼠的遗传杂交将揭示Bdkrb2突变体中Pax2基因转录的时空动态。Pax2启动子上组蛋白编码的特征将确定BdkrB2-/-肾脏中的进行性转录抑制伴随着组蛋白脱乙酰酶-1招募、启动子组蛋白H3去乙酰化和赖氨酸4(H3K4)去甲基化和H3K9/27甲基化，随后是CpG岛的高甲基化。我们推测，组蛋白密码中的这些修饰抑制了转录因子与Pax2启动子的结合。最后，我们将确定这些对基因表达的表观遗传效应是否可以通过有条件地灭活HDAC1来挽救。特异性目标3验证了p-p53Ser23在发育不良的BdkrB2-/-肾中积聚，抑制肝细胞核因子-1(HNF1？)基因转录的假设。这些研究将证明，HNF1的抑制有助于上皮-间充质转化，从而导致异型增生。我们认为，用非磷酸化版本的P53(p53S/A23)替换内源性P53基因可以挽救盐胁迫下BdkrB2-/-胚胎中HNF1的表达和末端分化。 公共卫生相关性：在4岁以下儿童慢性肾功能衰竭中，肾脏和尿路先天畸形占40%。除了DNA序列的突变外，肾脏发育异常也可能是由于“表观遗传密码”的改变造成的。短暂的环境暴露导致的表观遗传密码的稳定变化可能会永久性地扰乱胎儿的基因表达程序。因此，了解明确的基因突变(例如BdkrB2-/-)如何与明确的胚胎应激源(妊娠盐)相互作用以改变肾脏发育的调节，可能会为治疗或预防肾脏和尿路畸形开辟新的途径。

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.