Intravital analysis of cilia function during injury in the kidney

摘要/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，我们和其他人 发现纤毛断裂最终改变了急性肾损伤(AKI)后肾脏的修复能力 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流动诱导的钙信号的机械传感器，需要纤毛和 纤毛定位的多囊蛋白(PKD1和PKD2)，两个与人类多囊肾病相关的基因 (PKD)。虽然体外数据支持机械传感器的作用，但最近的发现引发了对这一模型的担忧。第一, 成年小鼠的纤毛切除在约8个月内不会引起囊肿，尽管所有小鼠的纤毛都失去了，但囊肿局灶性形成。 小管上皮细胞。此外，纤毛靶向钙生物传感器的数据表明，细胞内的钙没有变化 当轴丝在灌流的小管中偏转时纤毛，也没有检测到依赖于PKD2的钙电流 在纤毛膜片钳研究中。这项研究的一个潜在限制是，它不是在以下条件下进行的 已知cilia/PKD1/PKD2在防止快速囊性形成(如损伤后)方面具有关键作用。因此， 我们认为，损伤可能会诱导非损伤状态下不存在的纤毛反应。我们预测 了解不同生理条件下和损伤后纤毛反应的变化 对研究纤毛疾病的正常纤毛功能、纤毛修复不良机制和包囊形成有重要意义 例如PKD。为了分析纤毛在体内的作用，我们使用了带有荧光标记的纤毛的小鼠系， 活体荧光共聚焦显微镜和外科植入的腹部窗途径成像 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的方向。 在正常情况下流动。然而，当流动受到损害时，纤毛行为发生变化，开始摆动。 并且可以延长或退缩。重要的是，损伤后也会发生小管血流受损。因此，一个目标是 这个项目的目的是确定体内条件导致纤毛行为的这些变化。我们将分析 纤毛形态、长度和数量的变化决定了纤毛钙信号是否有变化 以及与血流和纤毛反应改变有关的小管中的转录活动。第二个目标 是测量在损伤和修复过程中纤毛反应的变化。这将包括测试是否有 纤毛损伤后的钙信号以及这是否依赖于PKD2。最后，我们将测试 纤毛在损伤和修复过程之前和期间通过扰乱纤毛的形成/功能来作出反应。以这种方式 我们可以确定纤毛的重新组装和保持适当的纤毛长度(拉长或 缩短)对于损伤后上皮恢复静止和分化状态很重要。 应用我们的体内成像策略来解决这些问题是创新的，需要理解 肾纤毛功能及其对囊性损伤和修复机制的反应和调节 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.