A novel genetic model for congenital obstructive nephropathy

描述（由申请人提供）：胎儿发育期间尿路梗阻导致先天性梗阻性肾病，这是儿童慢性肾病和终末期肾病最常见的基础。这种疾病通常在产前被发现为肾积水，这是由于尿液积聚在肾盂而导致的肾脏大幅膨胀。大多数先天性梗阻性肾病是由肾盂输尿管连接处（UPJ）梗阻引起的，估计每1000-1500例新生儿中有1例发生，狭窄局限于肾盂与输尿管连接处的上尿路。尽管影响了如此多的儿童，但我们对产前UPJ梗阻的分子和形态学病因了解甚少，缺乏非手术动物模型。我们和其他人已经证明了8蛋白胞囊运输复合物在调节极化上皮细胞的关键方面的重要性，例如紧密的细胞-细胞接触、初级纤毛、囊肿和小管管腔形成。Sec10是胞囊复合体的核心成分，我们最近培育了一种新的转基因小鼠来分析组织特异性Sec10失活的体内后果。我们最初将这种带绒的Sec10菌株与Ksp-Cre小鼠菌株杂交，在肾脏发育过程中诱导输尿管芽源性细胞特异性地敲除Sec10。令人惊讶的是，几乎所有的Sec10FL/FL；Ksp-Cre小鼠出生后很快死亡，表现为严重的双侧肾积水并输尿管梗阻。根据我们的初步分析，我们假设小鼠发育过程中Sec10基因的条件失活引起的尿上皮功能障碍导致先天性UPJ阻塞和肾积水，共享在人类疾病中观察到的致病特征。我们将通过以下具体目的来验证这一假设：(1)确定Sec10FL/FL中观察到的先天性阻塞性肾病的细胞和分子基础；Ksp-Cre老鼠。我们将使用多种分子和免疫组织学方法来描述Sec10FL/FL患者输尿管阻塞及其发生时间；Ksp-Cre胚胎，并鉴定尿上皮细胞和周围平滑肌细胞的异常。(2) Sec10FL/FL组肾损伤比较；具有人类先天性阻塞性肾病常见病理特征的Ksp-Cre小鼠。我们将分析突变肾脏的肌成纤维细胞积聚、肾小球硬化、肾生成减少、肾小管萎缩、炎症和间质纤维化。我们还将分析新生小鼠的生理参数。这些目标的完成将更好地定义一种独特的先天性阻塞性肾病的非手术模型，这对泌尿道发育和疾病的研究领域具有很高的价值。

DESCRIPTION (provided by applicant): Obstruction of the urinary tract during fetal development causes congenital obstructive nephropathy, the most common basis of chronic kidney disease and end stage renal disease in children. This disease is usually detected prenatally as hydronephrosis, which is a large distention of the kidney due to accumulation of urine in the renal pelvis. Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction (UPJ) obstructions, with an estimated incidence of 1 in 1000-1500 births, where the stenosis is localized to the upper urinary tract at the connection between the renal pelvis and ureter. Despite affecting so many children, we have a poor understanding of the molecular and morphologic etiology of prenatal UPJ obstructions, with a scarcity of non-surgical animal models. We, and others, have shown the importance of the eight-protein exocyst trafficking complex in regulating critical aspects of polarized epithelial cells, such as tight cel-cell contacts, primary cilia, and cyst and tubule lumen formation. Sec10 is a central component of the exocyst complex, and we have recently generated a new transgenic mouse to analyze in vivo consequences of tissue- specific Sec10 inactivation. We initially crossed this floxed-Sec10 strain with the Ksp-Cre mouse strain to induce the knockout of Sec10 specifically in ureteric bud-derived cells during kidney development. Surprisingly, nearly all the Sec10FL/FL;Ksp-Cre mice died quickly after birth, displaying severe bilateral hydronephrosis with obstructed ureters. Based on our preliminary analysis, we hypothesize that uroepithelial dysfunction caused by the conditional inactivation of the Sec10 gene during mouse development leads to congenital UPJ obstruction and hydronephrosis, sharing the pathogenic hallmarks observed in human disease. We will test this hypothesis through the following Specific Aims: (1) Determine the cellular and molecular basis of the congenital obstructive nephropathy observed in Sec10FL/FL;Ksp-Cre mice. We will use a variety of molecular and immunohistological approaches to characterize the ureter blockage and its timeframe of occurrence in Sec10FL/FL;Ksp-Cre embryos, and to identify abnormalities in uroepithelial cells and surrounding smooth muscle cells. (2) Compare the renal injury in Sec10FL/FL;Ksp-Cre mice with pathological features commonly observed in human congenital obstructive nephropathy. We will analyze mutant kidneys for myofibroblast accumulation, glomerular sclerosis, reduced nephrogenesis, tubular atrophy, inflammation, and interstitial fibrosis. We will also analyze physiological parameters in the newborn mice. Completion of these Aims will better define a unique non-surgical model of congenital obstructive nephropathy that can be highly valuable to a critically understudied area of urinary tract development and disease.