Molecular Pathogenesis of Vesicoureteral Reflux

描述（由申请方提供）：本项目的主要目的是使用条件性Robo 2基因敲除等位基因的反流小鼠模型，确定ROBO 2缺陷在膀胱输尿管反流（VUR）和反流性肾病发病机制中的作用。VUR是儿童中最常见的遗传性疾病之一，发病率约为1：100。其特征是尿液从膀胱回流到输尿管和肾脏，并导致肾皮质瘢痕形成。VUR患者可能在以后的生活中出现反流性肾病，这是一种以蛋白尿、高血压和局灶性肾小球硬化为特征的疾病，约占终末期肾衰竭病例的10%。尽管VUR在儿科人群中的发病率很高，但VUR和反流性肾病的分子基础仍然未知。ROBO 2是SLIT配体的跨膜蛋白，其控制轴突伸长和树枝化。我们已经证明ROBO 2也参与尿路发育，并且在VUR患者的一个子集中发生突变。我们已经产生并研究了一个有条件的Robo 2基因敲除小鼠模型，该模型表现出惊人的尿路异常，与人类VUR非常相似。我们还发现Robo 2在发育中的小鼠肾小球中表达，其表达模式表明其位于足细胞中。此外，Robo 2缺陷型小鼠表现出肾单位数量减少和损伤后蛋白尿以及输尿管分支异常和输尿管延长缺陷。因此，我们的研究为ROBO 2突变参与人类VUR提供了强有力的证据，并为我们提供了一个可行的反流小鼠模型，以进一步研究Robo缺陷在VUR病因学中的作用，并确定肾小球Robo 2的缺失是否赋予反流性肾病的易感性。为了研究VUR和反流性肾病的潜在独特致病机制，我们建议首先描述Robo 2缺陷小鼠的反流和反流性肾病表型，并确定Robo 2缺失是否会导致异常分支形态发生和低肾单位禀赋，这可能会导致反流性肾病的风险。其次，我们建议研究肾小球形成过程中Robo 2的正常定位，以及Robo 2缺失对发育中足细胞和成熟肾脏的结构和功能的影响。这将检验以下假设：足细胞中ROBO 2的原发性异常可能使肾脏在面对VUR或梗阻时易受损伤。最后，鉴于Robo 2缺陷小鼠的输尿管分支和输尿管延长异常缺陷以及Robo/Slit信号在神经发育中的实质性作用，我们将研究Robo 2是否控制输尿管结构和功能以及尿路神经支配。总之，这些实验将严格定义ROBO 2在VUR和反流性肾病发病机制中的作用。他们将在体内和体外对Robo在肾脏和泌尿道正常和异常发育过程中的作用产生相当大的机理见解。这些研究的结果将提供VUR发展前因的疾病机制的新知识，这可能有助于我们预测谁有反流性肾病的风险，并确定新的治疗策略。公共卫生相关性：膀胱输尿管反流（VUR）是一种常见的儿童疾病，可导致反复尿路感染和肾脏瘢痕形成。很大一部分VUR患者会发生进行性肾损伤，导致反流性肾病和终末期肾衰竭。了解VUR和反流性肾病的潜在致病机制将提供新的方法来检测患者的风险，并确定新的治疗策略。

DESCRIPTION (provided by applicant): The primary objective of this project is to define the role of ROBO2 deficiency in the pathogenesis of vesicoureteral reflux (VUR) and reflux nephropathy using a reflux mouse model with a conditional Robo2 knockout allele. VUR is one of the commonest genetic disorders found in children, with an incidence of about 1:100. It is characterized by reflux of urine from the bladder into the ureters and kidneys and leads to scarring of the kidney cortex. Patients with VUR may present later in life with reflux nephropathy, a condition characterized by proteinuria, hypertension and focal glomerulosclerosis, which accounts for about 10% of cases of end-stage renal failure. Despite the high incidence of VUR in the pediatric population, the molecular basis of VUR and reflux nephropathy remains unknown. ROBO2 is a transmembrane protein for SLIT ligand that controls axon elongation and arborization. We have shown that ROBO2 is also involved in urinary tract development and is mutated in a subset of patients with VUR. We have generated and studied a conditional Robo2 knockout mouse model, which exhibits striking urinary tract abnormalities closely resembling those in human VUR. We also found that Robo2 is expressed in developing mouse glomeruli in a pattern that suggests a location in podocytes. In addition, Robo2 deficient mice exhibit low nephron number and post-injury proteinuria as well as abnormal ureteric branching and defective elongation of the ureters. Thus, our studies have provided strong evidence for the involvement of ROBO2 mutations in human VUR and provided us with a viable reflux mouse model to further investigate the role of Robo deficiency in the etiology of VUR and determine if loss of glomerular Robo2 confers susceptibility to reflux nephropathy. To examine potentially unique pathogenic mechanisms of VUR and reflux nephropathy, we propose first to characterize the reflux and reflux nephropathy phenotype in Robo2 deficient mice and to determine if Robo2 deletion leads to abnormal branching morphogenesis and low nephron endowment, which could confer risk of reflux nephropathy. Second, we propose to investigate the normal localization of Robo2 during glomerulogenesis and the structural and functional effects of Robo2 deletion in developing podocytes and in mature kidney. This will test the hypothesis that a primary abnormality of ROBO2 in the podocyte may render the kidney susceptible to injury in the face of VUR or obstruction. Lastly, given the abnormal ureteric branching and ureter elongation defects in Robo2 deficient mice and substantial actions of Robo/Slit signaling in neural development, we will examine if Robo2 controls ureteral structure and function and urinary tract innervation. In sum, these experiments will rigorously define the role of ROBO2 in the pathogenesis of VUR and reflux nephropathy. They will yield considerable mechanistic insights in vivo and ex vivo on the role of Robo in normal and abnormal developmental processes of the kidney and urinary tract. Results from these studies will provide new knowledge of disease mechanisms underlying developmental antecedents of VUR, which may assist us to predict who is at risk of reflux nephropathy and identify novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Vesicoureteral reflux (VUR) is a common condition in childhood that causes substantial morbidity from recurrent urinary infection and scarring of the kidneys. A significant proportion of patients with VUR will develop progressive kidney damage leading to reflux nephropathy and end-stage kidney failure. Understanding the underlying pathogenic mechanism of VUR and reflux nephropathy will provide novel approaches to detect patients at risk and identify novel therapeutic strategies.