Intravital analysis of cilia function during injury in the kidney

肾脏损伤期间纤毛功能的活体分析

• 批准号: 10310430
• 负责人: Bradley K. Yoder
• 金额: $ 38.79万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10310430
• 关键词: Abdomen; Ablation; Acute; Acute Renal Failure with Renal Papillary Necrosis; Address; Adolescent; Adult; Affect; Behavior; Biosensor; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cells; Cilia; Confocal Microscopy; Cyst; Data; Defect; Development; Dextrans; Disease; Electrophysiology (science); Epithelial; Epithelial Cells; Fluorescence; Functional disorder; Future; Gene Expression Profile; Genes; Genetic Transcription; Goals; Homeostasis; Human; Image; Impairment; Implant; In Vitro; Injury; Injury to Kidney; Kidney; Label; Lead; Length; Liquid substance; Maintenance; Measures; Mediating; Mediator of activation protein; Modeling; Molecular Weight; Morphology; Mus; Mutant Strains Mice; Mutation; Nature; Nephrons; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Perinatal; Phase; Phenotype; Physiological; Physiology; Polycystic Kidney Diseases; Procedures; Process; Property; Proteins; Regulation; Renal function; Role; Sensory; Signal Transduction; Structure; Testing; Therapeutic Intervention; base; behavior change; ciliopathy; epithelial injury; experience; imaging study; in vivo; injury and repair; innovation; insight; intravenous injection; intravital fluorescence microscopy; intravital imaging; mouse model; mutant; patch clamp; prevent; renal epithelium; repaired; response; response to injury; sensor; tool; transcriptome sequencing; vesicular release

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.

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 发现纤毛破坏会改变急性肾损伤（AKI）后肾脏的修复能力，最终 导致囊肿形成；但纤毛如何与损伤联系起来仍然是个谜。根据体外研究， 初级纤毛被认为是调节流动诱导的 Ca2+ 信号的机械传感器，需要纤毛和 纤毛定位多囊蛋白（Pkd1 和 Pkd2），与人类多囊肾病相关的两个基因 （公钥簿）。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人们对该模型的担忧。第一的， 成年小鼠的纤毛消融在约 8 个月内不会引起囊肿，并且囊肿局部形成，尽管所有小鼠的纤毛都消失了 肾小管上皮。此外，来自纤毛靶向 Ca2+ 生物传感器的数据表明，细胞内没有 Ca2+ 变化。 当轴丝在灌注小管中偏转时，也没有检测到 Pkd2 依赖性 Ca2+ 电流 在纤毛膜片钳研究中。这项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/Pkd1/Pkd2 在防止囊肿快速形成（例如受伤后）方面具有关键作用。因此， 我们认为，损伤可能会引起未受伤状态下不存在的纤毛反应。我们预测 了解在不同生理条件下和受伤后发生的纤毛反应的变化 对于解剖正常纤毛功能、修复不良机制以及纤毛病囊肿形成非常重要， 例如公钥簿。为了分析纤毛的体内作用，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，以及通过手术植入腹部窗口的成像方法 完整肾单位中的纤毛。我们的活体成像表明纤毛通常朝肾小管方向偏转 正常条件下的流量。然而，当流动受损时，纤毛行为会发生变化并开始振荡 并且可以延长或退化。重要的是，受伤后也会出现肾小管血流受损。因此，一个目标 该项目的目的是确定引起纤毛行为这些变化的体内条件。我们将分析 纤毛形态、长度和数量的变化，确定纤毛 Ca2+ 信号传导是否发生变化 以及与流动和纤毛反应改变相关的小管中的转录活性。第二个目标 是为了测量损伤和修复过程中纤毛反应的变化。这将包括测试是否存在 损伤后纤毛 Ca2+ 信号以及这是否依赖于 Pkd2。最后我们来测试一下重要性 通过在损伤和修复过程之前和期间破坏纤毛形成/功能来调节纤毛反应。这样 我们可以确定纤毛是否重新组装并维持适当的纤毛长度（拉长或 缩短）对于上皮在受伤后恢复到静止和分化状态很重要。 应用我们的活体成像策略来解决这些问题是创新的，并且需要理解 肾纤毛功能以及纤毛如何响应和调节与囊肿相关的损伤和修复机制 发展。