应用活体冷冻技术分析急性高血压时免疫球蛋白G在小鼠近端肾小管中的分布变化

Examination of serum immunoglobulin G distribution in renal proximal tubules during acute hypertension by in vivo cryotechnique

目的应用活体冷冻技术结合免疫组织化学方法形象化地研究急性高血压血流动力学条件下,小鼠免疫球蛋白G(IgG)及其轻、重链经肾小球毛细血管袢滤过后,在近端小管再吸收的病理过程。方法分别在正常血流、急性高血压和心脏骤停等血流动力学条件下,进行小鼠肾脏活体冷冻及冷冻置换。连续的石蜡切片后,分别进行苏木精-伊红(H-E)染色和IgG、kappa轻链和IgG1重链免疫组织化学染色。结果正常血压和心脏骤停情况下,IgG在血管内清晰定位,只有极少量出现在近端小管顶端的细胞质内。而在急性高血压情况下,kappa轻链几乎免疫定位于所有近端小管,无IgG1重链表达。并首次展示了小鼠活体内血清IgG的轻链而非重链通过肾小球滤过后,迁移至近端肾小管重吸收的过程。结论活体冷冻技术结合冷冻置换方法能够原位瞬间捕获不同血流动力学条件下血清IgG在活体肾组织中的分布状态,可视化活体组织细胞的功能形态学特征。

Objective To visualize the topographical changes of serum immunoglobulin G （IgG） and its light chain and heavy chain passing through mouse glomerular capillary loops and their re-absorption in renal proximal tubules by immunohistochemistry combined with in vivo cryotechnique. Methods The in vivo cryotechnique was performed on mouse left kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The kidneys were then routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse IgG, kappa light chain or IgG1 heavy chain antibodies. Results Under normotensive and heart-arrest conditions, IgG was clearly detected within the blood vessels and slightly in the apical cytoplasm of some proximal tubules. Under acute hypertensive condition, the kappa light chain, but not IgG｜ heavy chain, was densely immunolocalized in the apical cytoplasm of almost all the proximal tubules. Our study was the first in vivo visualization of glomerular passage of the light chain, not heavy chain, and its transtubular absorption in renal proximal tubules. Conclusion The in vivo cryotechnique with freeze-substitution can transiently capture the distributions of IgG under various hemodynamic conditions and functional morphology of cells and tissues of living animal.