Spectrum: A quantitative pipeline for optically cleared tissue validated in crescentic nephritis.

Background: Optical clearing and advanced light microscopy have revolutionised three-dimensional quantification in cell biology. Here, we present a Systematic Pipeline of Enhanced Clarification for Three-dimensional Rendering and Unbiased Morphometrics (SPECTRUM) as a unique tool for the comprehensive analysis of glomerular health and disease. Method(s): SPECTRUM is based on a combination of single cell identification (ie. lineage tracing), optical clearing, advanced light microscopy with single cell resolution, and 3D morphometrics. Podocytes and parietal epithelial cells (PECs) were genetically labelled with eGFP using inducible mouse systems (POD or PEC -rtTA/H2B-eGFP). Crescentic nephritis was used as a validation model. Several optical clearing protocols were optimized for kidney tissue, including aqueous, hydrogel, and solvent-based approaches. Image acquisition was based on whole structures (ie. glomeruli and lesions) using light sheet, confocal and two-photon microscopy. Subsequent tissue de-clarification with immunolabelling and classical histopathology allowed the combination of 3D and 2D analyses. Result(s): While IgG deposition showed a homogeneous distribution among all analysed glomeruli, only 80 showed signs of podocyte loss (podocyte loss per glomerulus of about 60). Only glomerular lesions showed significant increases in numbers of PECs (sign of PEC activation) in close association with reductions in glomerular capillary volume (sign of capillary injury). PEC activation was confirmed via immunofluorescence in recycled tissue samples (ie. de-novo CD44 expression). Based on 3D analysis of intraglomerular lesion location, we identified a progressive pattern from localised tubular lesions to segmental lesions and development of atubular glomeruli. Conclusion(s): SPECTRUM provides new roadmap for morphometrics in the kidney. In crescentic nephritis, SPECTRUM revealed that despite of uniform IgG deposition, there was focal lesion development and podocyte