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

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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.