Understanding the dynamic interplay of altered calcium signaling and actin remodeling in podocytes in FSGS using in vivo imaging

足细胞丢失是局灶性节段性肾小球硬化(FSGS)发展的中心，并导致邻近足细胞肥大以维持滤过屏障。然而，肥大的效果是有限的，一旦足细胞丢失超过20%的门槛，这将导致肾小球硬化。对于足细胞向肥大的调节、维持精细的足细胞结构和防止基底膜脱离，肌动蛋白细胞骨架的动态能力是最重要的。因此，在这项研究中，我们将解决钙信号改变的相互作用和肌动蛋白细胞骨架的重组如何共同促进肾小球病理生理学和进展为FSGS的开创性问题。因此，我们将采用最先进的成像方法，包括活体内多光子显微镜、创新的报告鼠模型、系列多光子成像和超分辨率技术。这项研究的主要目标是了解钙信号的致病作用及其对足细胞肌动蛋白细胞骨架变化的影响。具体地说，我们将(1)表征足细胞肥大过程中细胞内钙水平和肌动蛋白重塑的相互联系，(2)研究血管紧张素II 1型受体对足细胞钙稳态和信号的影响，以及(3)使用光遗传学工具触发足细胞结构性钙进入，表征所涉及的离子通道，并在体内可视化对肌动蛋白细胞骨架的影响。

Loss of podocytes is central to the development of focal segmental glomerulosclerosis (FSGS) and results in hypertrophy of neighboring podocytes to maintain the filtration barrier. However, the effectiveness of hypertrophy is limited and once podocyte loss exceeds the threshold of 20 %, this will result in glomerular sclerosis. For the adjustment of podocytes to hypertrophy, struggling to maintain the elaborate podocyte architecture and to prevent detachment from the basement membrane, the dynamic capacities of the actin cytoskeleton are of prime importance. Hence, in this research proposal we will address the seminal question of how the interplay of altered calcium signaling and the reorganization of the actin cytoskeleton jointly contribute to glomerular pathophysiology and to progression towards FSGS. Therefore, we will employ a state-of-the-art imaging approach including intravital multiphoton microscopy, innovative reporter mouse models, serial multiphoton imaging and super-resolution technologies. The overarching goal of this research proposal is to understand the pathogenetic effect of calcium signaling and its impact on alterations of the actin cytoskeleton in podocytes. Specifically, we will (1) characterize the interconnection of intracellular calcium levels and actin remodeling during podocyte hypertrophy, (2) study the impact of Angiotensin II type 1 receptors on calcium homeostasis and signaling in podocytes, and (3) use optogenetic tools to trigger a constitutive calcium entry in podocytes, characterize the involved ion channels and visualize the effect on the actin cytoskeleton in vivo.