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

慢性肾脏疾病影响了大约10%的普通人群，由于高成本和慢性，对卫生保健系统构成了主要负担。在大多数受影响的个体中，肾功能会持续下降。潜在的病因多种多样，从动脉高血压、高血糖到自身免疫性疾病。肾组织损伤与小管间质细胞浸润和“活化”的纤维化生态位之间存在密切的时间和空间联系。我们小组的发现确定了Notch家族的受体是肾损伤的关键调节因子。基因消融受体Notch3可保护小鼠免受实验性肾脏疾病的侵袭。整合素激活减弱、白细胞迁移减少和NF-B信号缺失的发现强调了受体Notch3在组织损伤反应中的基本作用。该项目大纲将解决以下问题：(i)受体Notch3在骨髓嵌合动物的白细胞转运和纤维化生态位激活中执行精细任务的程度？β1-整合素激活和Notch3表达是否与中性粒细胞转运功能相关？Notch3对肾损伤的细胞特异性贡献将被澄清。将建立新的嵌合动物模型，以在Notch3敲除背景下重建特定细胞亚群的细胞内Notch3信号。将在疾病诱导后对动物进行炎症反应和器官纤维化的表型分析（UUO, I/R）。表达tenascin-C或受体Notch3的细胞串扰形成的纤维原性生态位将在小管细胞的时间变化和诱导部分上皮到间质转化中可视化。（ii）为什么循环的Notch3敲除免疫细胞不转移到“炎症”组织？实验将仔细检查免疫细胞粘附内皮细胞的notch3依赖性相互作用。（iii）与Notch3受体敲除动物相比，细胞特异性阻断小管细胞或gli1阳性细胞中的Notch3受体是否能改善实验性肾脏疾病的病程？设想了一种干预策略，即递送腺相关病毒和慢病毒。在小管细胞和gli1阳性细胞中引入小干扰RNA特异性下调Notch3受体的表达。同时，将建立可溶性Notch3诱骗受体的表达系统。总的来说，这项拨款申请将加深我们对以关键受体分子为中心的慢性肾脏疾病病理生理学的理解。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.