Division of Nephrology and Endocrinology, The University of Tokyo;
Division of Nephrology and Endocrinology, The University of Tokyo.
J Vis Exp. 2022 Jul 18(185). doi: 10.3791/63986.
Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.
尽管传统病理学提供了大量关于肾脏微观结构的信息，但由于缺乏三维（3D）信息，很难了解肾脏中血管、近端肾小管、收集管、肾小球和交感神经的精确结构。光学透明化是克服这一难题的好策略。通过结合组织透明化和 3D 成像技术，可以在单细胞分辨率下分析整个器官中的多个细胞。然而，用于整个器官成像的细胞标记方法仍不发达。特别是，由于抗体渗透困难，整个器官的染色仍然具有挑战性。本方案使用 CUBIC（透明、无阻碍的大脑/身体成像鸡尾酒和计算分析）组织透明化方法，开发了一种用于 3D 成像的全肾染色的方案。该方案使我们能够从全面的角度观察到缺血再灌注损伤后的肾脏交感神经去神经和糖尿病肾病早期的肾小球肿大。因此，通过提供宏观视角，该技术可以为肾脏研究带来新的发现。
