Charakterisierung und Validierung einer neuartigen makrophagenspezifischen TRAP-Mauslinie (MacTRAP) für die Analyse von Translatomsignaturen renaler Makrophagen

Tissue macrophages play critical roles in organ homeostasis, immunity, and the pathogenesis of various inflammatory-driven diseases. A particular challenge is the selective investigation of resident macrophages in highly heterogeneous organs such as the kidney. To address this problem, the TRAP (Translational Ribosome Affinity Purification) approach was recently established, and a novel macrophage-specific TRAP mouse line was generated. In these transgenic mice (c-fms-eGFP-L10a, MacTRAP), an eGFP-tagged ribosomal protein (L10a) is expressed under the control of the macrophage- specific promoter c-fms. The aim of the dissertation was a first scientific characterization and comprehensive validation of this recently created TRAP mouse line using biometric, molecular biological, biochemical and immunohistological methods. First, observational and biometric studies found no gross abnormalities in the development, behavior and reproduction of MacTRAP. In the next step, transgene expression was examined and successfully detected in the kidney tissue using RT-PCR and Western blotting. Immunohistological analyses of kidney sections from MacTRAP mice clearly identified eGFP-L10a expressing cells in the tubulointerstitial compartment, which were predominantly also positive for the macrophage surface marker F4/80. By contrast, no significant co-staining was detected with classical markers of other cell types located in the tubulointerstitium, such as CD31 (endothelial cells), PDGFRβ (pericytes, perivascular fibroblasts), αSMA (myofibroblasts, VSMC) and CD3 (lymphocytes). Proinflammatory and fibrosis-inducing stimulation by unilateral ureteral obstruction (UUO maneuver) resulted in significant eGFP-L10a upregulation in fibrotic UUO kidneys, indicating adequate responsiveness of the model in vivo and thus illustrating and corroborating the usefulness of a potential application of MacTRAP in experimental disease models. Based on these promising validation results, MacTRAP could well be used in follow-up projects for the extraction of macrophage-specific, polysomal mRNA from kidney tissue and subsequent RNA sequencing combined with bioinformatic analyses. This innovative approach may contribute to the definition of the hitherto poorly understood in vivo gene expression dynamics and signaling pathway signatures of resident renal macrophages. In summary, this new mouse line provides a new tool to help better understand renal macrophage biology during homeostasis and in various inflammation-driven disease models of the kidney including renal fibrosis. Given the universal applicability, MacTRAP may also be used for the investigation of tissue macrophages in various other organs and different disease models in the future.