A novel genetic model for congenital obstructive nephropathy

描述（由申请方提供）：胎儿发育期间的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的最常见基础。这种疾病通常在产前被发现为肾积水，这是一种由于尿液积聚在肾盂中而导致的肾脏巨大扩张。大多数先天性梗阻性肾病病例是肾盂输尿管连接部（UPJ）梗阻的结果，估计发病率为1/1000-1500，其中狭窄局限于肾盂和输尿管之间连接处的上尿路。尽管影响这么多的儿童，我们有一个贫穷的了解产前UPJ梗阻的分子和形态学病因，缺乏非手术动物模型。我们和其他人已经证明了八蛋白外囊运输复合物在调节极化上皮细胞的关键方面的重要性，如紧密的细胞-细胞接触，初级纤毛，以及囊肿和小管腔的形成。Sec 10是外囊复合物的核心成分，我们最近产生了一种新的转基因小鼠来分析组织特异性Sec 10失活的体内后果。我们最初将这种被抑制的Sec 10品系与Ksp-Cre小鼠品系杂交，以在肾脏发育过程中诱导特异性地在输尿管芽衍生细胞中敲除Sec 10。令人惊讶的是，几乎所有的Sec 10 FL/FL;Ksp-Cre小鼠出生后迅速死亡，表现出严重的双侧肾积水和输尿管阻塞。基于我们的初步分析，我们假设，在小鼠发育过程中，Sec 10基因的条件性失活引起的尿路上皮功能障碍导致先天性UPJ梗阻和肾盂积水，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的来检验这一假设：（1）确定在Sec 10 FL/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表征输尿管阻塞及其在Sec 10 FL/FL;Ksp-Cre胚胎中发生的时间范围，并确定尿路上皮细胞和周围平滑肌细胞的异常。(2)将Sec 10 FL/FL;Ksp-Cre小鼠的肾损伤与人类先天性梗阻性肾病中常见的病理特征进行比较。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个独特的先天性梗阻性肾病的非手术模型，这对尿路发育和疾病的关键研究领域非常有价值。

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.