A novel genetic model for congenital obstructive nephropathy

描述(申请人提供)：胎儿发育过程中的尿路梗阻导致先天性梗阻性肾病，这是儿童慢性肾脏疾病和终末期肾脏疾病的最常见基础。这种疾病通常在产前被发现为肾积水，这是由于尿液在肾盆内积聚而导致的肾脏大范围扩张。大多数先天性梗阻性肾病是由肾输尿管连接部(UPJ)梗阻引起的，估计每1000-1500名新生儿中就有1例发生狭窄，狭窄部位位于肾盆和输尿管连接处的上尿路。尽管影响了这么多儿童，但我们对产前UPJ梗阻的分子和形态病因了解很少，缺乏非手术动物模型。我们和其他人已经证明了8蛋白外囊运输复合体在调节极化上皮细胞的关键方面的重要性，如紧密的细胞接触、初级纤毛以及囊腔和管腔的形成。Sec10是外囊复合体的中心成分，我们最近培育了一只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FLOXED-Sec10品系与KSP-CRE小鼠品系杂交，以诱导肾脏发育过程中输尿管芽细胞中特异性的Sec10基因敲除。令人惊讶的是，几乎所有的Sec10FL/FL；KSP-CRE小鼠出生后都很快死亡，表现出严重的双侧肾积水和输尿管阻塞。根据我们的初步分析，我们假设在小鼠发育过程中，Sec10基因的条件失活导致的尿路上皮功能障碍会导致先天性UPJ梗阻和肾积水，这与人类疾病中观察到的致病特征相同。我们将通过以下具体目标来验证这一假设：(1)确定在Sec10FL/FL；KSP-CRE小鼠中观察到的先天性梗阻性肾病的细胞和分子基础。我们将使用各种分子和免疫组织学方法来描述Sec10FL/FL；KSP-CRE胚胎中输尿管阻塞及其发生的时间框架，并确定尿路上皮细胞和周围平滑肌细胞的异常。(2)比较Sec10FL/FL；KSP-CRE小鼠的肾脏损害与人类先天性梗阻性肾病常见的病理特征。我们将分析突变肾脏的肌成纤维细胞聚集、肾小球硬化、肾脏生成减少、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。完成这些目标将更好地定义一种独特的先天性梗阻性肾病的非手术模型，该模型对尿路发育和疾病研究严重不足的领域具有极高的价值。

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.