Pathogenesis of PKD in Mice Lacking Renal Cilia

描述（由申请人提供）：多囊肾病（PKD）是人类肾衰竭最常见的遗传原因，其特征是肾脏和其他上皮器官中充满液体的囊肿积聚。虽然已经确定了导致PKD的基因，但囊肿形成的机制仍不清楚。最近的研究表明PKD可能是由初级纤毛的异常引起的，初级纤毛是一种不运动的毛状细胞器，从大多数肾上皮细胞的顶端表面突出。为了验证这一假设，我们使用Cre/loxP重组来删除转基因小鼠肾脏中的Kif3a基因。Kif3a （Kinesin家族3a）编码Kinesin - ii马达蛋白的80/85-kda亚基，该亚基对纤毛的形成至关重要。肾脏特异性的Kif3a缺失导致后代在出生时具有正常外观的肾脏。肾囊肿在出生后第5天开始出现，在第21天出现肾功能衰竭。囊肿上皮细胞缺乏原发纤毛，细胞增殖、凋亡、极性和（-catenin）定位异常。这个项目的总体目标是了解肾纤毛的丢失是如何导致囊肿形成的。将采用诱导Cre/loxP重组的方法在成年小鼠中删除Kif3a，以检测成熟肾小管中初级纤毛的丢失是否会产生PKD。从Kif3a突变小鼠中建立的囊肿上皮细胞系将被检查纤毛形成、增殖和分化的异常。增殖增加的机制将被阐明，重点是β -连环蛋白信号通路的激活。我们将检测多囊蛋白-1和多囊蛋白-2的表达和亚细胞定位，并研究Kif3a与Pkd1或Pkd2联合突变小鼠的表型，以确定Kif3a是否与Pkd1和Pkd2处于相同的囊肿形成途径。缺乏初级纤毛的Kif3a突变细胞将暴露于流体剪切应力中，以阐明肾纤毛在细胞内钙的流动依赖性调节中的作用。总之，一种新的小鼠PKD模型的表征有望促进我们对人类囊性疾病的分子发病机制和原纤毛在调节细胞生长和分化中的重要作用的理解。

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.