Genetic dissection of ciliary ARL13B in kidney cystogenesis

这项建议的目的是完善对初级纤毛内信号机制(S)与多囊肾病(PKD)相关的理解。肾囊肿会干扰肾功能，并导致长期的重大健康并发症。PKD及其相关的健康并发症在美国每年造成70亿美元的经济负担，因此确定治疗的分子靶点是一项迫切的未得到满足的临床需求。纤毛是所有肾脏细胞上的细长突起，在细胞信号和囊变过程中发挥关键作用，但其细节和潜在机制尚不清楚。常染色体显性遗传性多囊肾病(ADPKD)主要由纤毛相关多囊蛋白基因PKD1和PKD2突变引起。正常情况下，纤毛中的多囊藻毒素作用于抑制促进包囊的途径。鉴定这一途径的分子组成，称为纤毛依赖囊泡激活剂(CDCA)，代表了对抗ADPKD治疗策略的明确分子靶点。研究CDCA途径，以及更广泛地理解纤毛信号的主要挑战是，该领域的工具过于迟钝。小鼠模型通常会删除基因，导致纤毛和细胞蛋白质池完全丧失，使得任何对纤毛特定功能的解释都是不可能的。我们用纤毛型GTPase Arl13b绕过了这一挑战，它是CDCA的一个拟议的调解人。我们通过改造一只表达纤毛排除的ARL13BV358A变体的小鼠来特异性地从纤毛中移除Ar13b，该变体保留了所有已知的生化活性。Arl13bV358A/V358A小鼠是存活和可生育的，但显示出囊性肾脏，这表明纤毛Arl13b的缺失是肾囊肿形成的特殊关键。我们在这项建议中的目标是检验我们的中心假设，即纤毛Arl13b在调节肾脏囊变中发挥关键作用。这项拟议的工作对于打造初级纤毛内关键信号机制的身份和时机的基础知识非常重要，这可能导致对PKD的治疗干预。

The objective of this proposal is to refine the understanding of the signaling mechanism(s) within the primary cilium in relation to polycystic kidney disease (PKD). Renal cysts interfere with kidney function and lead to major, long-term health complications. PKD and its related health complications result in a $7 billion per year economic burden in the United States so identifying molecular targets for treatment is a pressing unmet clinical need. Cilia, the slender protrusions on all renal cells, play key roles in cell signaling and cystogenesis but the details and underlying mechanism are ill-defined. Autosomal dominant polycystic kidney disease (ADPKD) is primarily due to mutations in the cilia-associated polycystin genes, PKD1 and PKD2. Normally, the polycystins function in cilia to suppress a pathway that promotes cysts. Identification of the molecular components of this pathway, termed the cilia-dependent cyst activator (CDCA), represents a clear molecular target for therapeutic strategies to counter ADPKD. The major challenge in working out the CDCA pathway, and in understanding ciliary signaling more generally, is that the tools in the field are too blunt. Mouse models typically delete genes, resulting in complete loss of both the ciliary and cellular pools of protein, making any interpretation of cilia-specific function impossible. We circumvented this challenge with the ciliary GTPase ARL13B, a proposed mediator of the CDCA. We removed ARL13B specifically from cilia by engineering a mouse expressing a cilia-excluded ARL13BV358A variant that retains all known biochemical activities. Arl13bV358A/V358A mice are viable and fertile yet display cystic kidneys indicating that it is the absence of ciliary ARL13B that is specifically critical to kidney cyst formation. Our goal in this proposal is to test our central hypothesis that ciliary ARL13B plays key roles in regulating kidney cystogenesis. The proposed work is important in forging fundamental knowledge of the identity and the timing of critical signaling mechanisms from within the primary cilium that could lead to therapeutic intervention for PKD.