小鼠致密斑后肾小管与入球微动脉毗邻关系的三维可视化

Three-dimensional visualization on the relationship between tubules after macula densa and afferent arterioles in mouse kidney

目的采用微细结构三维可视化技术,建立致密斑后远端小管与入球微动脉的空间走行,定位检测毗邻关系部位的蛋白。方法C57 BL/6小鼠灌流固定取肾,垂直于肾长轴切取组织块,树脂812包埋,从肾被膜到外髓外带,获得720张2.5μm厚的连续切片,通过计算机配准程序将数字化显微图像进行对齐,并采用C语言编辑的追踪程序对小管及血管走行进行三维重建。选取接触部位的组织切片,应用改良的免疫过氧化物酶染色方法检测H+-ATPase和Pendrin。结果通过重建3只小鼠90个肾单位的入球微动脉、远端小管及其延续的连接小管的走行,分析毗邻关系并测量长度,发现小管-入球微动脉密切接触多发生在浅表及中间皮质肾单位,其接触处的小管细胞表达H+-ATPase和Pendrin,是非A-非B型闰细胞。结论致密斑后的小管节段与入球微动脉的接触并非偶然,而是发生于远曲小管末端或(和)连接小管的非A-非B型闰细胞处,提示该部位的接触对致密斑处的管球反馈可能起拮抗作用,从而恢复肾小球滤过率和肾血流量。

Objective To establish the spatial course of distal tubule and afferent arterioles after macula densa,and to locate and detect the proteins in the adjacent parts by using three-dimensional visualization technology of microstructure.Methods C57 BL/6J mice were fixed by perfusion and embedded in epon 812.Tissue blocks were cut perpendicular to the longitudinal axis of the kidney.And a total of 720,2.5μm-thick consecutive sections were obtained from the renal capsule to the outer stripe of the renal outer medulla.After aligning the digital microscopic images through computer registration procedures,the tubules and vessels were traced by 3D reconstruction program edited by C Language.Selecting the tissue sections of the contact site and applying the improved immunoperoxidase staining method to detect H+-ATPase and Pendrin.Results By reconstructing the course of the afferent arterioles,distal tubules and their extended connecting tubules of 90 nephrons in three mice,analyzing the adjacent relationship and measuring the length,it was found that the close contact between tubules and afferent arterioles mostly occurred in superficial and midcortical nephrons,and the cells at the contact expressed H+-ATPase and Pendrin,which was non-A-non-B intercalated cells.Conclusion The contact between the tubules after macula densa and the afferent arterioles is not accidental,but is occurred at the non-A-non-B intercalated cells in the end of the distal convoluted tubules or(and)the connecting tubules,suggesting that the contact at this site may antagonize the tubuloglomerular feedback at the macula densa,thereby restoring glomerular filtration rate and renal blood flow.