Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis

Abstract

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new highthroughput endpoints for the analysis of complex tissues with single-cell resolution.

A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales

“…There have recently been demonstrations that pathological parameters can be extracted from cleared kidney tissue using both non-labelled (autofluorescence) and fluorescently labelled samples 20,21 . Furthermore, a recent study shows that "virtual histology" can be performed using deep learning algorithms to auto-fluorescence images 22 , and neural network-based segmentation and classification of kidney pathologies has also been shown 23,24 . Merging novel sample-preparation methods (as the ones shown in this study) with new image analysis tools poses many interesting digital possibilities in the future, and we will likely see changes…”

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

“…The 3D approach enabled visualization of normal and damaged glomerular capsules and revealed changes in the distribution and morphology of parietal epithelial cells. Recently, comparative studies have been carried out using several reagents and protocols, including Scale and ECI, CLARITY, CUBIC1, and uDISCO, to study crescentic diseases that may include podocyte depletion ( Puelles et al., 2019 ; Masselink et al., 2019 ). Related to pathologies in the kidney, potassium-mediated tubule remodeling has also been revealed by optical clearing ( Saritas et al., 2018 ) and ethanol-ECi (ethyl-cinnamate) cleared kidneys have enabled the study of renal nephrosis.…”

A practical guide to the stereological assessment of glomerular number, size, and cellular composition

“…Other modelbased methods are also available; they are less accurate, but have some advantages given they are considerably less time-consuming (and therefore a wider sampling of glomeruli can be assessed) and require less specialized imaging equipment. Although not detailed in this article, other feasible methodologies for the estimation of podocyte number have also been developed recently for use in cleared tissue (Puelles et al, 2016;Puelles et al, 2019), and single histological sections (Venkatareddy et al, 2014).…”