Flow sensation by kidney cells

描述（由申请人提供）：本提案的目的是阐明肾细胞对尿流的机械感觉的分子机制。发育过程通常由体液流动驱动，体液流动传递机械（剪切力、阻力、压力）或化学（营养物质、代谢物、生长因子）信息。在肾脏中，初级纤毛是一种微小的细胞天线，代表了一个专门的平台，可以感知和整合尿流中的复杂信息。来自流体流动的剪切力激活存在于睫状体膜中的钙离子（Ca 2+）通道并诱导胞质溶胶中的Ca 2+触发的信号传导事件。血流感觉对肾脏组织的完整性和功能起着至关重要的作用。然而，在这方面， 在分子和细胞水平上将细胞外机械信号转化为纤毛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，我们已经开发了一系列的分子传感器和致动器，第一次使可视化的睫状体Ca 2+信号和睫状体结构组件的快速扰动，分别。基于流动给药时初级纤毛的弯曲轮廓，我们假设纤毛基部经历大的应力，例如膜张力和压缩，其打开机械敏感性Ca 2+通道以启动该区域中的Ca 2+信号传导。为了测试这一点，我们将在一个特定的时间点观察流动诱导的Ca 2+信号传导。 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，从而可以在肾细胞的初级纤毛内精确地绘制Ca 2+动力学。然后，我们将确定结构组件，赋予使用传统的以及我们新开发的分子致动器的流动感觉所需的机械性能。Ca 2+信号还调节纤毛的物理性质，表明脱敏形式的反馈调节。因此，我们将研究如何流动诱导的Ca 2+信号调节初级纤毛的物理特性。然后，我们将这项研究扩展到多囊肾病（PKD），这表明肾细胞无法正确感知尿流。特别是，我们将确定PKD肾细胞中受损的机械感觉步骤，旨在深入了解PKD进展机制。对于实验，我们将使用来自小鼠（mIMCD 3）和狗（MDCK）的肾集合管上皮细胞，进行或不进行PKD 1和/或PKD 2的遗传操作。

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.