Antibody-based Imaging of Lymphatic Architecture in Murine Kidneys.

The lymphatic system has historically been understudied due to the difficulty of distinguishing it from the blood vasculature. In acute kidney injury (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. Here, we describe a methodology adapted from the iDISCO 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 (VEGFR-3 and LYVE-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 3D kidney images and quantify their lymphatic features. 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. 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.