Pathogenesis of PKD in Mice Lacking Renal Cilia

描述（由申请人提供）：多囊肾病（PKD）是人类肾衰竭的最常见遗传原因，其特征在于肾脏和其他上皮器官中充满液体的囊肿积聚。虽然导致PKD的基因已经被确定，但囊肿形成的机制仍不清楚。最近的研究表明，PKD可能是由初级纤毛异常引起的，初级纤毛是一种不动的毛状细胞器，从大多数肾上皮细胞的顶端表面突出。为了验证这一假设，使用Cre/loxP重组来删除转基因小鼠肾脏中的Kif 3a基因。Kif 3a（驱动蛋白家族3a）编码驱动蛋白-II马达蛋白的80/85-kda亚基，其对于纤毛形成是必需的。肾脏特异性Kif 3a缺失导致出生时具有正常肾脏的存活后代。肾囊肿开始出现在出生后第5天（P），肾衰竭发展到P21。囊肿上皮细胞缺乏初级纤毛，细胞增殖、凋亡、极性和β-连环蛋白定位异常。本项目的总体目标是了解肾纤毛的丧失如何导致囊肿形成。诱导型Cre/loxP重组将用于删除成年小鼠中的Kif 3a，以测试成熟肾小管中初级纤毛的丧失是否产生PKD。将检查从Kif 3a突变小鼠建立的囊肿上皮细胞系的纤毛形成、增殖和分化的异常。将阐明增殖增加的机制，重点是β-连环蛋白信号通路的激活。将检查多囊蛋白-1和多囊蛋白-2的表达和亚细胞定位，并将研究具有Kif 3a和Pkd 1或Pkd 2的组合突变的小鼠的表型，以确定Kif 3a是否与Pkd 1和Pkd 2处于相同的囊肿形成途径。将缺乏初级纤毛的Kif 3a突变细胞暴露于流体剪切应力以阐明肾纤毛在细胞内钙的流动依赖性调节中的作用。两者合计，一个新的小鼠模型的PKD的特点有望推进我们的理解人类囊性疾病的分子发病机制和初级纤毛在调节细胞生长和分化的重要作用。

DESCRIPTION (provided by applicant): Polycystic kidney disease (PKD), the most common genetic cause of renal failure in humans, is characterized by the accumulation of fluid-filled cysts in the kidneys and other epithelial organs. Although the genes that cause PKD have been identified, the mechanism of cyst formation remains unclear. Recent studies suggest that PKD may arise from abnormalities of the primary cilium, an immotile, hair-like organelle that project from the apical surface of most renal epithelial cells. To test this hypothesis, Cre/loxP recombination was used to delete the Kif3a gene in the kidneys of transgenic mice. Kif3a (Kinesin family 3a) encodes the 80/85-kda subunit of the kinesin-II motor protein that is essential for cilia formation. Kidney-specific deletion of Kif3a results in viable offspring with normal-appearing kidneys at birth. Renal cysts begin to appear at postnatal day (P)5, and renal failure develops by P21. The cyst epithelial cells lack primary cilia and have abnormalities in cell proliferation, apoptosis, polarity, and (-catenin localization. The overall goal of this project is to understand how the loss of renal cilia leads to cyst formation. Inducible Cre/loxP recombination will be used to delete Kif3a in adult mice to test whether the loss of primary cilia in mature renal tubules produces PKD. Cyst epithelial cell lines established from Kif3a mutant mice will be examined for abnormalities in cilia formation, proliferation, and differentiation. The mechanism of increased proliferation will be elucidated, focusing on activation of the beta-catenin signaling pathway. The expression and subcellular localization of polycystin-1 and polycystin-2 will be examined, and the phenotype of mice with combined mutations of Kif3a and either Pkd1 or Pkd2 will be studied to determine whether Kif3a is in the same pathway of cyst formation as Pkd1 and Pkd2. Kif3a mutant cells that lack primary cilia will be exposed to fluid shear stress to elucidate the role of renal cilia in the flow-dependent regulation of intracellular calcium. Taken together, the characterization of a new mouse model of PKD promises to advance our understanding of the molecular pathogenesis of human cystic diseases and the essential role of primary cilia in regulating cell growth and differentiation.