小鼠肾远曲小管三维可视化研究

Three-dimensional visualization of the mouse renal distal convoluted tubule

目的肾远曲小管(DCT)是肾单位最后一段小管,其与相邻小管的分界及走行的毗邻关系是理解肾形态发生中集合管与肾单位连接方式,以及该段小管参与水盐代谢调节机制的重要结构基础。本实验在肾组织连续切片基础上,采用微细结构三维可视化技术,建立小鼠肾远曲小管的空间走行。方法 C57/BL/6 J小鼠灌流固定后取肾,垂直于肾长轴切取组织块,树脂812包埋,从肾被膜到肾外髓外带获得2. 5μm厚连续切片720张,获取数字化显微镜图像并通过计算机程序进行配准,追踪来自3只小鼠共90个肾单位远曲小管,观察其空间走行并测量其长度。结果肾远曲小管起始于远端小管致密斑后40~180μm处,上皮由矮柱状或立方陡然变为高柱状,细胞核靠近腔面,走行在皮质迷路其自身的肾小球周围,区域相对独立,末端向被膜方向逐渐移行为连接小管;此处上皮再次变矮,细胞核排列不局限在近腔面。浅层皮质肾单位的远曲小管在回旋返折最高处接触肾被膜1次。远曲小管长度为500~900μm。结论远曲小管较短且盘曲走行,较少与其他小管交错,所以区域较小而独立,可能有助于该节段的吸收功能受激素的精准调节。

Objective The renal distal convoluted tubule（ DCT） is the last segment of the nephron. The study on the demarcation,as well as the spatial course and relationship between the DCT and its adjacent tubules,may lay an important structural basis for understanding the mechanism on the morphogenesis of the connection between the collecting duct and the nephrons,and contribute to the understanding on the regulation mechanism of water and salt metabolism in the DCT. The aim of this study is to visualize the course of the mouse distal convoluted tubules with the aid of the threedimensional reconstruction technology based on the consecutive renal tissue sections. Methods Three C57/BL/6 J mice were fixed via perfusion and embedded in Epon 812. Tissue blocks were cut perpendicularly to the longitudinal axis of the kidney. A total of 720 consecutive sections with a thickness of 2. 5 μm were obtained from the renal capsule to the outer stripe of the renal outer medulla. After image recordings and alignment,distal convoluted tubules from 90 nephrons were traced with the custom-made computer program. Based on the recorded data,the courses and the spatial arrangement of the DCT were visualized,and the length was also measured. Results The DCT commenced downstream from the macula densa at an average length of 40-180 μm. The shape of the cell of DCT changed abruptly from simple cubical to high columnar,and the cell nucleus was located near the apical membrane of the cell. The DCT surrounded their parent glomerulus which were located at cortical labyrinth. Virtually,the DCT did not intermingle and each tubule occupied its own separate territory in the cortex. The end of the DCT gradually transited to the connecting tubules whose cells turned short and the nuclei were not limited to the apical membrane. Usually,the DCT of superficial nephron returned and made coils on the way to touch the capsule just for one time. The length of distal convoluted tubule was 500-900 μm. Conclusion Because of the short and convoluted length,the DCT occupied a relatively small and independent space,which may facilitate precise regulation by hormone along the segment.