Flow sensation by kidney cells

描述(由申请人提供)：本提案的目的是阐明肾脏细胞对尿流进行机械感觉的分子机制。发育过程通常是由体液流动驱动的，体液流动传递的信息是机械的(剪切、阻力、压力)或化学的(营养物质、代谢物、生长因子)。在肾脏，初级纤毛是一个微小的细胞天线，代表着一个专门的平台，可以感知和整合尿流中的复杂信息。流体的剪切力激活了位于纤毛膜的钙离子通道，并在胞浆中诱导了钙离子触发的信号事件。流动感对肾脏的组织完整性和功能起着至关重要的作用。然而， 在分子和细胞水平上将细胞外机械信号转化为睫状体内化学信号的机制仍然知之甚少。这主要是由于缺乏实验技术来可视化和操纵初级纤毛内的化学信号。最近，我们开发了一系列分子传感器和致动器，首次分别实现了睫状体钙信号的可视化和睫状体结构成分的快速扰动。根据给药后纤毛弯曲的轮廓，我们推测纤毛的底部会受到较大的应力，如膜的张力和压缩，从而打开机械敏感的钙通道，从而启动该区域的钙信号。为了测试这一点，我们将可视化流诱导的钙信号在 在空间和时间上的高分辨率，这将被开发的分子传感器所利用，从而可以精确地描绘肾细胞初级纤毛内的钙动态。然后，我们将使用传统的和我们新开发的分子致动器来确定赋予流体感觉所需的机械性能的结构组件。CA2信号也调节纤毛的物理性质，提示以脱敏的形式进行反馈调节。因此，我们将研究血流诱导的钙信号如何调节初级纤毛的物理性质。然后，我们将把这项研究扩展到多囊肾病(PKD)，该疾病表现为肾脏细胞无法正确感知尿流。特别是，我们将确定PKD肾脏细胞中受损的机械感觉步骤，以期获得对PKD进展机制的深入了解。对于实验，我们将使用小鼠(MIMCD3)和狗(MDCK)的肾脏收集管道上皮细胞，并进行或不进行PKD1和/或PKD2的基因操作。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the molecular mechanism underlying mechanosensation of urine flow by kidney cells. Developmental processes are often driven by body fluid flow which delivers information that is mechanical (shear, drag, pressure) or chemical (nutrients, metabolites, growth factors). In the kidney, the primary cilium, a tiny cellular antenna, represents a specialized platform to sense and integrate such complex information in urine flow. Shear forces from fluid flow activate calcium ion (Ca2+) channels that reside in the ciliary membrane and induce Ca2+-triggered signaling events in the cytosol. Flow sensation plays a critical role in tissue integrity and functions of kidney. However, the mechanism converting extracellular mechanical cues into intraciliary chemical signaling at the molecular and cellular levels remains poorly understood. This is primarily due to a lack of experimental techniques to visualize and manipulate chemical signaling inside primary cilia. Recently, we have developed a series of molecular sensors and actuators that for the first time enabled visualization of ciliary Ca2+ signaling and rapid perturbation of ciliary structural components, respectively. Based on the bending profile of primary cilia upon flow administration, we hypothesize that the base of cilia experience a large stress such as membrane tension and compression which opens mechanosensitive Ca2+ channels to initiate the Ca2+ signaling in this region. To test this, we will visualize flow-induced Ca2+ signaling at a high resolution in space and time, which will be leveraged by the developed molecular sensors whereby Ca2+ dynamics can be precisely mapped within the primary cilia of kidney cells. We will then determine structural components that confer the mechanical properties required for flow sensation using conventional as well as our newly developed molecular actuators. Ca2+ signaling also regulates the physical properties of the cilium, suggesting a feedback regulation in the form of desensitization. Therefore, we will investigate how flow-induced Ca2+ signaling modulates the physical properties of the primary cilium. We will then extend this study to polycystic kidney disease (PKD), which manifests an inability of kidney cells to properly sense the urine flow. In particular, we will determine the mechanosensation steps impaired in the PKD kidney cells with an aim to obtain insights into the PKD progression mechanism. For experiments, we will use kidney collecting duct epithelial cells from mice (mIMCD3) and dogs (MDCK) with or without genetic manipulation of PKD1 and/or PKD2.