Antibody-Based Imaging of Lymphatic Architecture in Murine Kidneys

Key Points Optimization of an antibody-based method for imaging murine kidney lymphatics. Accessible approach to studying difficult to visualize lymphatic vessels. Novel tools that can be used to investigate kidney-specific disease states. Background The lymphatic system has historically been understudied due to the difficulty of distinguishing it from the blood vasculature. In AKI, the lymphatic system undergoes a process of expansion termed lymphangiogenesis. Current mainstay treatments inadequately treat the clinical consequences of AKI. Understanding kidney endothelial architecture and modifying its functional properties can serve as a promising therapeutic strategy for ameliorating AKI of multiple etiologies. A current barrier in lymphatic-modulating therapies is our inability to comprehensively examine the structural and functional integrity of kidney lymphatics. Current methodology to detect lymphatics includes traditional sectioning and immunolabeling for lymphatic markers. Because of the sparsity of kidney lymphatics, there may be significant variability in observed vessel density when using traditional sectioning and immunolabeling. Methods Here, we describe a methodology adapted from the immunolabeling-enabled three-dimensional imaging of solvent cleared organs tissue clearing pipeline for visualizing intact lymphatic architecture at the organ level, providing a comprehensive analysis of the kidney lymphatic system. More specifically, we describe a detailed protocol outlining key steps in kidney tissue preparation, immunolabeling with two canonical lymphatic markers (vascular endothelial growth factor receptor-3 and lymphatic vessel endothelial hyaluronan receptor 1), optical clearing, imaging, and quantifying important lymphatic features such as vessel diameter, branch points, total volume, total length, and average segment length to assess the extent of lymphangiogenesis in injured kidneys. We use the software program Imaris to analyze three-dimensional kidney images and quantify their lymphatic features. Results We have optimized an antibody-based approach for visualizing kidney lymphatics using confocal microscopy, offering a new approach to study lymphatics in the kidney. Quantification of lymphatic features reveals that injured kidneys have increased numbers of branch points, total volume, and filament length suggesting ischemia-induced kidney injury results in lymphangiogenesis. Conclusions We have optimized an antibody-based method for imaging historically hard-to-visualize adult murine kidney lymphatics and developed a user-friendly approach for quantifying key lymphatic features that define lymphangiogenesis.