Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.