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机械感觉的分子机制。发育过程通常是由体液流动驱动的，它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）。在肾脏中，初级纤毛，一个微小的细胞天线，代表了一个特殊的平台来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位于纤毛膜上的钙离子（Ca2+）通道，并诱导细胞质中Ca2+触发的信号事件。血流感觉在肾脏组织完整性和功能中起着至关重要的作用。然而,

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.