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.
With 12 million patients worldwide (1 in 500), PKD is one of the most common genetic diseases without any distinct bias in demographics or occupations. In addition, PKD is life threatening with no cure;PKD patients resort to symptomatic treatments and undergo dialysis or kidney transplantation, or die in severe cases. Our proposed research will provide a powerful technology to decipher the sensory machinery of kidney cells, extend conventional experimental techniques, and offer novel, far-reaching insights into a diagnostic and cure strategy for PKD.
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