Intravital analysis of cilia function during injury in the kidney

总结/摘要 初级纤毛存在于大多数肾上皮细胞上，但其功能尚不清楚。最近，我们和其他 发现纤毛破坏改变了肾脏在急性肾损伤（阿基）后的修复能力， 导致囊肿形成;但纤毛如何与损伤联系仍然是个谜。基于体外研究， 初级纤毛被认为是调节需要纤毛的流动诱导的Ca 2+信号的机械传感器， 纤毛定位的多囊蛋白（Pkd 1和Pkd 2），与人类多囊肾病相关的两个基因 （PKD）.虽然体外数据支持机械传感器的作用，但最近的研究结果引起了对该模型的关注。第一、 成年小鼠的纤毛消融在约8个月内不会引起囊肿，并且囊肿形成局灶性，尽管纤毛全部丧失， 小管上皮此外，来自纤毛靶向Ca 2+生物传感器的数据表明，在细胞中没有Ca 2+变化。 纤毛时，轴丝在灌注小管偏转，也没有检测到Pkd 2依赖的Ca 2+电流 在纤毛膜片钳研究中。本研究的一个潜在局限性是，它不是在以下条件下进行的： 已知纤毛/Pkd 1/Pkd 2在防止快速囊肿形成（例如在损伤后）中具有关键作用。因此，在本发明中， 我们认为，损伤可能会引起纤毛反应，而非损伤状态中不存在这种反应。我们预测 了解在不同生理条件下和损伤后纤毛反应的变化 将是重要的解剖正常纤毛功能，修复不良的机制，和囊肿形成的纤毛病变， 例如PKD。为了分析纤毛在体内的作用，我们使用带有荧光标记纤毛的小鼠品系， 活体荧光共聚焦显微镜和手术植入腹腔窗方法成像 纤毛在完整的肾单位。我们的活体成像显示纤毛通常偏向小管方向 正常情况下的流量。然而，当血流受损时，纤毛行为改变，它们开始振荡 并且可以伸长或后退。重要的是，受损的小管流动也发生在损伤后。因此，一个目标 这个项目的目的是确定在体内条件下，诱导纤毛行为的这些变化。我们将分析 纤毛形态、长度和数量的变化，确定纤毛Ca 2+信号传导是否发生变化 以及与流动和改变的纤毛反应相关的小管中的转录活性。第二个目标 是测量损伤和修复过程中纤毛反应的变化。这将包括测试， 损伤后的纤毛Ca 2+信号以及这是否依赖于Pkd 2。最后，我们将测试 通过在损伤和修复过程之前和期间破坏纤毛形成/功能来引起纤毛反应。以这种方式 我们可以确定纤毛的重组和适当纤毛长度的维持（延长或 缩短）对于上皮在损伤后恢复到静止和分化状态是重要的。 应用我们的活体成像策略来解决这些问题是创新的，需要了解 肾纤毛功能以及纤毛如何响应和调节囊肿相关损伤和修复机制 发展

SUMMARY/ABSTRACT Primary cilia are present on most renal epithelial cells but their function is unknown. Recently, we and others found that cilia disruption alters the ability of the kidney to repair following acute kidney injury (AKI), eventually leading to cyst formation; but how cilia are connected with injury remains enigmatic. Based on in vitro studies, primary cilia were thought to be mechanosensors regulating a flow-induced Ca2+ signal requiring the cilia and cilia localized polycystin proteins (Pkd1 and Pkd2), two genes associated with human polycystic kidney disease (PKD). While in vitro data support a mechanosensor role, recent findings raise concerns with this model. First, cilia ablation in adult mice does not cause cysts for ~8 months and the cysts form focally, despite cilia loss on all tubule epithelium. Additionally, data from a cilia targeted Ca2+ biosensor indicate there is no Ca2+ change in the cilium when the axoneme is deflected in perfused tubules, nor is there a Pkd2 dependent Ca2+ current detected in cilia patch clamp studies. A potential limitation of this study is that it was not performed under conditions where cilia/Pkd1/Pkd2 are known to have critical roles in preventing rapid cyst formation (e.g. following injury). Thus, we propose that injury may induce a cilia response not present in non-injured states. We predict that understanding changes in cilia responses that occur under differing physiological conditions and following injury will be important for dissecting normal cilia function, mechanisms of mal-repair, and cyst formation in ciliopathies, such as PKD. To analyze in vivo roles of the cilium, we are using mouse lines with fluorescently tagged cilia, intravital fluorescence confocal microscopy, and a surgically implanted abdominal window approach to image cilia in intact nephrons. Our intravital imaging indicate that cilia are typically deflected in the direction of tubule flow under normal conditions. However, when flow is impaired, cilia behavior change and they begin to oscillate and can elongate or regress. Importantly, impaired tubule flow also occurs following injury. Thus, one objective of this project is to ascertain in vivo conditions that induce these changes in cilia behavior. We will analyze changes in cilia morphology, length, and number, determine whether there are changes in cilia Ca2+ signaling and transcriptional activity in the tubules associated with flow and altered cilia responses. The second objective is to measure changes in cilia responses during the injury and repair process. This will include testing if there is a cilia Ca2+ signal following injury and whether this is dependent on Pkd2. Finally, we will test the importance of cilia responses by disrupting cilia formation/function prior to and during the injury and repair process. In this way we can determine whether reassembly of the cilium and maintenance of proper cilia length (elongated or shortened) are important for the epithelium to return to a quiescent and differentiated state following injury. Applying our intravital imaging strategy to address these questions is innovative and is needed to understand renal cilia function and how the cilia respond to and regulate injury and repair mechanisms associated with cyst development.