Mechanical stress protection at the kidney filtration barrier

肾脏的功能依赖于大量水和少量溶质的大量过滤，以清除来自细胞和肠道微生物代谢的潜在毒素，并维持有机体中盐、水和酸碱的动态平衡。值得注意的是，健康成年人每天进行多达180升的肾小球滤过。肾小球滤过是由大约40毫米汞柱的静水压力梯度驱动的。这种显著的管腔压力对毛细血管壁施加物理力量，而这种力量被足细胞--特化的终末分化的上皮细胞--所抵消。这些细胞以交错的初级和次级突起包裹肾小球毛细血管，并由一个特殊的膜状细胞连接连接，称为裂隙横隔膜。复杂的足细胞结构依赖于严格调控的肌动蛋白细胞骨架机制，使它们能够通过细胞特异性的焦点黏附复合体和缝隙横隔膜附着在肾小球基底膜上，裂隙横隔膜连接足细胞并包含机械感觉蛋白，感知细胞连接处施加的机械力。狭缝横隔膜蛋白复合体或粘着斑/细胞骨架连接的突变会导致人类滤过屏障功能障碍和进行性肾脏疾病。在这个项目的第一阶段，我们使用超分辨受激辐射耗竭(STED)显微镜结合数学模型来了解肾脏超滤的生物物理特性(Butt等人，2020)。该模型显示足细胞在抵消过滤压力以维持过滤屏障方面发挥了积极作用。我们在足细胞中展示了机械保护反应，涉及细丝蛋白表达和内吞活性的适应(Koehler等人，2020)和蛋白分解系统。此外，我们还发现Bag3和伴侣辅助的选择性自噬(CASA)复合体富含足细胞。Bag3定位于裂隙横隔膜，提示Bag3及其相关蛋白可能参与足细胞的机械保护。此外，我们还可以证明Bag3与肌动蛋白细胞骨架的调节因子相互作用，包括Rho A、Dynamin 2和Arpc2。在小鼠模型中，Bag3的干扰导致了滤过功能的改变和迟发性蛋白尿。在该项目的下一阶段，我们将(1)研究伴侣机制和降解途径调节对机械应激的抵抗的作用，(2)表征调节足细胞机械敏感性和机械应激保护的代谢信号途径，以及(3)了解应激保护系统与维持体内完整的肾滤过屏障的相关性。

Kidney function depends on the bulk filtration of large volumes of water and small solutes to clear potential toxins derived from cellular and gut microbial metabolism, and maintain salt and water and acid-base homeostasis in the organism. Remarkably, as much as 180 liters/day of glomerular filtration takes place in healthy adults. Glomerular filtration is driven by a hydrostatic pressure gradient of about 40 mmHg. This remarkable lumenal pressure exerts physical forces on the capillary wall that are counteracted by podocytes, specialized terminally differentiated epithelial cells. These cells enwrap the glomerular capillaries with interdigitating primary and secondary processes and are connected by a specialized membrane-like cell junction called the slit diaphragm. The complex podocyte architecture depends on a tightly regulated actin cytoskeletal machinery that enables them to adhere to the underlying glomerular basement membrane by cell-specific focal adhesion complexes and the slit diaphragm, which connects podocytes and contains mechanosensory proteins that sense mechanical forces exerted at the cell junctions. Mutations in either the slit diaphragm protein complex or the focal adhesion/cytoskeleton connection cause dysfunction of the filtration barrier and progressive renal disease in humans. In the first phase of this project, we used ultraresolution stimulated emission depletion (STED) microscopy in combination with mathematical modelling to understand the biophysical properties of kidney ultrafiltration (Butt et al., 2020). The model shows an active role of the podocyte in counteracting filtration pressure to sustain the filtration barrier. We demonstrated mechano-protective responses in podocytes to involve an adaptation of filamin expression and endocytic activity (Koehler et al., 2020) and proteolytic systems. Moreover, we showed that Bag3 and the chaperone-assisted selective autophagy (CASA) complex are enriched in podocytes. Bag3 localizes to the slit diaphragm suggesting that Bag3 and associated proteins may be involved in mechanoprotection in podocytes. Moreover, we could show that Bag3 interacts with regulators of the actin cytoskeleton including Rho A, Dynamin 2 and Arpc2. Perturbation of Bag3 induced a functional alteration of filtration and a late on-set proteinuria in the mouse model. In the next phase of the project we will now (1) study the role of regulators of chaperone machineries and degradation pathways mediating resistance to mechanical stress ,(2) characterize metabolic signalling pathways regulating mechanosensitivity and mechanical stress protection in podocytes, and (3) understand the relevance of stress protection systems for the maintenance of an intact renal filtration barrier in vivo.