Molecular mechanisms in Notch3-dependent immune cell infiltration, fibrogenic niche formation and kidney fibrosis

慢性肾脏疾病影响约10%的总人口，并由于高成本和慢性性而构成卫生保健系统的主要负担。在大多数受影响的个体中，发生肾功能的无情下降。潜在的原因是多种多样的，从动脉高血压，高血糖症到自身免疫性疾病。肾组织损伤与肾小管间质细胞浸润和“激活的”纤维化小生境的时间和空间密切相关。我们小组的研究结果确定了Notch家族的受体是肾损伤的关键调节因子。Notch 3受体基因切除的小鼠免受实验性肾脏疾病的影响。受体Notch 3对于组织损伤应答的基本作用通过钝化的整联蛋白活化、较少的白细胞迁移和缺乏NF-κ B B信号传导的发现而被强调。该项目的大纲将解决以下问题：（一）在何种程度上受体Notch 3执行白细胞迁移和激活骨髓嵌合动物的纤维化生态位的精致任务？β1整合素激活和Notch 3表达与中性粒细胞迁移功能相关吗？将阐明Notch 3对肾损伤的细胞特异性贡献。将建立新的嵌合动物模型，以在Notch 3敲除背景下重建特定细胞亚群中的细胞内Notch 3信号传导。在疾病诱导（UUO、I/R）后，对动物进行炎症反应和器官纤维化的表型分析。表达生腱蛋白-C或Notch 3受体的细胞的串扰的纤维化小生境的形成将被可视化用于时间变化和诱导肾小管细胞中的部分上皮向间充质转化。(ii)为什么循环Notch 3敲除免疫细胞不会迁移到“发炎”组织中？实验将仔细检查粘附于内皮细胞的免疫细胞的Notch 3依赖性相互作用。(iii)与Notch 3受体敲除动物相比，肾小管或Gli 1阳性细胞中Notch 3受体的细胞特异性阻断是否改善了实验性肾病的病程？一种干预策略是设想与交付的腺相关病毒以及慢病毒。将引入小干扰RNA以特异性下调肾小管和Gli-1阳性细胞中的受体Notch 3表达。同时，将建立可溶性Notch 3诱饵受体的表达系统。总的来说，这项拨款申请将加深我们对慢性肾脏疾病病理生理学的理解，围绕一个关键的受体分子。最终，这些结果将使干预策略能够在细胞水平上对抗炎症反应和器官纤维化。

Chronic kidney diseases affect about 10% of the general population and constitute a major burden to the health care system due to high costs and chronicity. In the majority of affected individuals a relentless decline of kidney function takes place. Underlying causes are diverse, ranging from arterial hypertension, hyperglycemia to autoimmune diseases. A close temporal and spatial link of kidney tissue damage with tubulointerstitial cell infiltration and an “activated” fibrogenic niche is frequently detected. The findings from our group identified receptors of the Notch family as pivotal regulators of kidney damage. Mice with genetic ablation of receptor Notch3 are protected from experimental kidney diseases. The fundamental role of receptor Notch3 for the tissue damage response is underscored by the findings of blunted integrin activation, less leukocyte transmigration and absent NF-B signaling. The project outline will address the following questions: (i) To which extent does receptor Notch3 perform exquisite tasks in leukocyte transmigration and activation of the fibrogenic niche in bone marrow chimeric animals? Are β1-integrin activation and Notch3 expression functionally linked with neutrophil transmigration? The cell-specific contribution of Notch3 to kidney damage will be clarified. Novel chimeric animal models will be established to reconstitute intracellular Notch3 signaling in specific cell subsets on an otherwise Notch3 knockout background. Phenotyping of animals for the inflammatory response and organ fibrosis will be performed following disease induction (UUO, I/R). The formation of the fibrogenic niche with crosstalk of cells that express tenascin-C or receptor Notch3 will be visualized for temporal changes and induction of partial epithelial-to-mesenchymal transition in tubular cells. (ii) Why do circulating Notch3 knockout immune cells not transmigrate into “inflamed” tissue? Experiments will scrutinize the Notch3-dependent interaction of immune cells adherent to endothelial cells. (iii) Does cell-specific blockade of receptor Notch3 in tubular or Gli1-positive cells improve the course of experimental kidney disease comparable to receptor Notch3 knockout animals? An intervention strategy is envisioned with delivery of adeno-associated as well as lentiviruses. Small interfering RNA will be introduced to specifically down-regulate receptor Notch3 expression in tubular and Gli-1 positive cells. In parallel, an expression system for soluble Notch3 decoy receptors will be set up.Overall, this grant application will deepen our understanding of chronic kidney disease pathophysiology centered around a key receptor molecule. Ultimately, the results will enable intervention strategies to combat inflammatory responses as well as organ fibrosis at the cellular level.