Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature.

Tissue macrophages play an important role in organ homeostasis, immunity and the pathogenesis of various inflammation-driven diseases. One major challenge has been to selectively study resident macrophages in highly heterogeneous organs such as kidney. To address this problem, we adopted a Translational Ribosome Affinity Purification (TRAP)- approach and designed a transgene that expresses an eGFP-tagged ribosomal protein (L10a) under the control of the macrophage-specific c-fms promoter to generate c-fms-eGFP-L10a transgenic mice (MacTRAP). Rigorous characterization found no gross abnormalities in MacTRAP mice and confirmed transgene expression across various organs. Immunohistological analyses of MacTRAP kidneys identified eGFP-L10a expressing cells in the tubulointerstitial compartment which stained positive for macrophage marker F4/80. Inflammatory challenge led to robust eGFP-L10a upregulation in kidney, confirming MacTRAP responsiveness in vivo. We successfully extracted macrophage-specific polysomal RNA from MacTRAP kidneys and conducted RNA sequencing followed by bioinformatical analyses, hereby establishing a comprehensive and unique in vivo gene expression and pathway signature of resident renal macrophages. In summary, we created, validated and applied a new, responsive macrophage-specific TRAP mouse line, defining the translational profile of renal macrophages and dendritic cells. This new tool may be of great value for the study of macrophage biology in different organs and various models of injury and disease.

Evidence for functional and dynamic microcompartmentation of Cav-1/TRPV4/K(Ca) in caveolae of endothelial cells.

Ca(2+)-activated K(+) channels (KCa) play a pivotal role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, KCa depend on an increase of intracellular calcium which is substantially mediated by Ca(2+)-permeable cation channels including the transient receptor potential V4 (TRPV4). It has been proposed that KCa and Ca(2+)-permeable cation channels may be clustered in localized positions within the cell membrane to form functional units and that caveolae may constitute the scaffolding for such microcompartmental organization. Here, we sought to elucidate the composition and functional relevance of these microcompartments in vitro and in vivo. We show that TRPV4 and small-conductance KCa2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found a caveolae-dependent association between caveolin-1, TRPV4 and small conductance KCa2.3, but not intermediate conductance KCa3.1, in endothelial cells under static condition. Mechanical stimulation of cells via exposure to shear stress led to a partial de-novo colocalization of KCa3.1 with Cav-1 and TRPV4. In a mouse model of genetic Cav-1 deficiency, we found significantly reduced KCa-mediated currents as determined by patch-clamping in carotid artery endothelial cells (CAEC) from Cav-1(-/-) mice compared to wildtype. Functionally, Cav-1 deficiency was associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in response to shear stress and acetylcholine. In summary, our findings provide evidence for a dynamic microcompartmentation of TRPV4/KCa in caveolae of endothelial cells and highlight the importance of Cav-1 for endothelial KCa functions and flow-induced vasodilation.