Pathogenesis of PKD in Mice Lacking Renal Cilia

描述(申请人提供)：多囊肾病(PKD)是人类肾功能衰竭最常见的遗传原因，其特征是肾脏和其他上皮器官中积聚充满液体的囊肿。虽然引起PKD的基因已经被确定，但囊形成的机制仍不清楚。最近的研究表明，PKD可能是由于初级纤毛的异常引起的，初级纤毛是一种从大多数肾上皮细胞的顶面突起的不活动的毛状细胞器。为了验证这一假设，Cre/loxP重组被用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，它是纤毛形成所必需的。肾脏特异性Kif3a基因的缺失会导致后代存活，出生时肾脏外观正常。肾囊肿生后第5天开始出现，21天时出现肾功能衰竭。囊上皮细胞缺乏原生纤毛，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丢失是如何导致囊性形成的。在成年小鼠中，将使用可诱导的Cre/loxP重组来删除Kif3a，以测试成熟肾小管中初级纤毛的丢失是否会产生PKD。从Kif3a突变小鼠建立的囊上皮细胞系将被检查纤毛形成、增殖和分化的异常。促进增殖的机制将被阐明，重点是激活β-连环蛋白信号通路。将检测多囊蛋白-1和多囊蛋白-2的表达和亚细胞定位，并将研究Kif3a与PKD1或PKD2联合突变的小鼠的表型，以确定Kif3a是否与PKD1和PKD2处于相同的囊性形成途径。缺乏原生纤毛的Kif3a突变细胞将暴露在流体剪应力下，以阐明肾纤毛在细胞内钙流动调节中的作用。综上所述，一种新的PKD小鼠模型的特征有望促进我们对人类囊性疾病的分子发病机制以及初级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.