X-Ray-Based 3D Histopathology of the Kidney Using Cryogenic Contrast-Enhanced MicroCT

The kidney's microstructure, which comprises a highly convoluted tubular and vascular network, can only be partially revealed using classical 2D histology. Considering that the kidney's microstructure is closely related to its function and is often affected by pathologies, there is a need for powerful and high-resolution 3D imaging techniques to visualize the microstructure. Here, we present how cryogenic contrast-enhanced microCT (cryo-CECT) allowed 3D visualization of glomeruli, tubuli, and vasculature. By comparing different contrast-enhancing staining agents and freezing protocols, we found that the preferred sample preparation protocol was the combination of staining with 1:2 hafnium(IV)-substituted Wells-Dawson polyoxometalate and freezing by submersion in isopentane at -78°C. This optimized protocol showed to be highly sensitive, allowing to detect small pathology-induced microstructural changes in a mouse model of mild trauma-related acute kidney injury after thorax trauma and hemorrhagic shock. In summary, we demonstrated that cryo-CECT is an effective 3D histopathological tool that allows to enhance our understanding of kidney tissue microstructure and their related function.