High-Throughput Nephron Tracking Reveals Hierarchical Organization in Mouse Kidneys

Background: The kidney’s essential roles are carried out by an elaborate network of thousands of nephrons functioning in parallel. Although nephrons play a central role in all of the kidney’s functions, they are difficult to study individually due to their small diameter, high density, and convoluted paths spanning nearly the full width of the kidney. These challenges have created a blind spot in our understanding of kidney physiology at the level of whole nephrons and their collective organization. Methods: We have developed a pipeline for high-throughput, organ-scale mapping of nephrons in the mouse kidney. Our approach combines a novel tri-color stain with tissue clearing and light-sheet fluorescence microscopy, followed by tracking and segmentation. We have also developed a suite of analytical tools to study individual nephrons (by morphometry, cytometry, sub-segment identification, cortical vs juxtamedullary) and networks of nephrons (by nephron-nephron proximity analysis, clustering). Results: We mapped over 1,000 complete nephrons in a ~1mm slab of healthy adult mouse kidney. The resulting spatial maps reveal a hierarchical structural organization that approximately recapitulates traditional concepts of layers of the kidney (cortex, outer medulla, inner medulla) as well as lobules that are identified from clustering of nephrons within medial rays. However, we also observe substantial overlap across these traditional boundaries, suggesting a more continuous and interconnected spatial architecture than previously appreciated. Conclusion: High-throughput nephron tracking enables whole-nephron reconstruction and spatial network analysis at organ scale. It has revealed the limits of zonation and substantial overlap within traditional domains of the kidney, with broad implications for the understanding of kidney diseases. Funding: NIDDK SupportFigure 1. Rendering of ~1000 nephrons tracked within a 1mm thick mouse kidney tissue section, with nephron color determined by distance of glomerulus to cortex.