EBDE clearing: A strategy for simultaneous 3D visualization and quantitative analysis of both renal arteries and glomeruli.

Background: Three-dimensional (3D) visualization of the renal vasculature plays a crucial role in the pathological assessment and mechanistic investigation of kidney diseases. However, existing 3D imaging approaches remain unable to rapidly and accurately achieve continuous visualization spanning from millimeter-scale arteries to micrometer-scale glomerular capillaries. Here, we developed an innovative strategy, termed EBDE clearing, which enables continuous 3D visualization from the renal artery to the glomerulus, systematically characterizes the spatiotemporal architecture of the entire renal arterial tree and glomeruli in unilateral ureteral obstruction (UUO) and diabetic nephropathy (DN) mouse models. Methods: We combined the vascular tracers of Evans blue and dextran with Ethanol-ECi tissue clearing (EBDE clearing) to achieve the improved labeling of all renal arteries and glomeruli. By further integrating light-sheet microscopy-based 3D imaging with reconstruction and segmentation, we performed high-resolution 3D visualization and quantitative analysis of both renal vascular and glomeruli in normal and diseased kidneys. Furthermore, we investigated, classified, and statistically analyzed the spatiotemporal architecture of the arteries and glomeruli in the kidneys of mice with UUO and DN. Results: Firstly, through quantitative analysis of renal artery diameter, vascular branching, and vascular segment straightness, we found that the arteries of both UUO and DN mice exhibited varying degrees of damage. The renal vascular damage in UUO mice was more severe, manifested by a significant reduction in vessel diameter and the number of vascular branches, as well as an increased in the segment straightness of some arteries; in contrast, the pathological changes in the renal arteries of DN mice were mainly concentrated in vessel diameter and the number of vascular branches. We further classified the glomeruli into three distinct types (M1, M2, and M3) based on their origins, and conducted a quantitative analysis of their 3D spatial distribution within the kidneys of UUO and DN mice, and discovered that in UUO mice, there was a severe reduction in the number and the average volume of the both total and the individual M1-M3 glomeruli. However, in the DN mice, apart from no changes in the total and M1 glomerular number, the other relevant pathological parameters of glomeruli also showed a decrease, although the extent of this decrease was much milder compared to that in the UUO mice. Conclusion: EBDE clearing enables continuous, high-resolution 3D visualization and quantitative analysis from the renal artery to the glomerulus in both normal and diseased intact kidneys. The study not only provides important insights into the renal pathophysiology of UUO and DN, but also establishes a rapid and comprehensive strategy for visualizing structural alterations during disease progression in the kidney and potentially other organs.