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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. 5 μm-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
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[Abstract] Objective To investigate the branching pattern of the ureteric bud and the number of the nephron induced by each ureteric bud tip, through the three-dimensional tracing of the ureteric tree, combined with the morphological analysis and measurement of the ureteric tree. Methods The kidneys were obtained from three mice at various developing time points and prepared for paraffin and epoxy sections. Then the microscopic images were digitized and aligned from these sections. Based on the computer-assisted tracing and visualization of ureteric tree, the number of branches and the nephron induced by each ureteric bud tip were obtained by counting. In addition, paraffin sections were stained with HE staining for morphological observation of nephrogenic zone and ureteric bud, while in order to reflect the density of the ureteric bud tips at nephrogenic zone, the distance between two neighboring ureteric bud tips was measured aided with the Claudin-7 immunohistochemical staining. Results The ureteric bud branching tree revealed that the initial bifid iterative branching formed the framework of renal medulla, the branching became complicated and dense in cortex and nephrogenic zone, while the distance between ureteric bud tips were also decreasing. The number of the nephron induced by each ureteric bud tip increased from one (E14. 5) to two (E17. 5), and occasionally to three. Conclusion Threedimeasional Visualization of ureteric bud branching tree reveals regional complication, suggesting molecules in different regions drive different branching patterns; While the density of the ureteric bud tips at nephrogenic zone increases corresponding to decreasing of thickness of the nephrogenic zone, and the disappearance of the ureteric bud tips after birth is also consistent with the gradual consumption of nephron progenitor cells.
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Objective To describe quantitatively the development of the capillary loop stage glomerulus (capG) with respect to the volume density of capillaries in the glomerulus based on the morphogenesis of the kidney. Methods The kidneys were obtained from mice at various developing time points and prepared for paraffin sections. The volume density of CD34 positive endothelial cells and surrounded capillary lumen in glomeruli was measured using a combination of immunohistochemical staining and the stereological grid system. Results The capG was divided into early, middle, and late phases, and middle phase capG was subdivided into early-middle and late-middle phases, according to the morphology of developing glomeruli and the arrangement of podocytes. As result, the volume density of capillary loops in early phase capG could not be measured due to the complex "glomerular" shape. The volume density of capillary loops increased from (35.95±6.45)% in the early-middle phase capG, to (58.36±6. 30) % in the late-middle phase capG, and to (79.89± 5.21) % in the late phase capG, compared to (93.61 ±1.96) % in the mature glomerulus. Furthermore, the volume density of capillary loops remained constant at same stage even though at different developmental time points. Conclusion This study demonstrated a significantly increased volume density of capillary loops with the kidney development. In addition, the results provide a descriptive and reliable parameter for the evaluation of glomerular development.
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Objective Adult proximal tubule ( PT ) is not only the segment most frequently involved in acute renal tubule injury, but also the easiest to repair. It may be consistent with the rapid growth and differentiation mechanism of this segment during the development of the kidney, while the developing information is insufficient. Therefore, we three- dimensional visualized the developing PT to analysis its spatiotemporal morphogenesis. Methods The kidneys were obtained from mice at various developing time point, embryonic day ( E ), postnatal day ( P ). The volume density of Claudin-2 positive PT in the cortex was measured using a stereological method in paraffin sections. After image recording and alignment of the serial sections, the spatial courses of the developing PT were traced and visualized in three dimensions using computer-assisted program. The length of the developing PT was calculated at the same time. Results The volume density of PT in the cortex of PI mice was significantly higher than that in the embryonic stage. Then it experienced a decline ( P3, P5 ), an increase ( start at P7 ) to a stable adult level ( P28 ). The tubular tracing showed that the lengths of developing PT and the number of convolutions of their convoluted part increased with the maturation, but lower than that of adultin E14. 5, E17. 5 and P5 PT in E14. 5 and E17. 5 mice were similar to that of adult with respect to general spatial courses. They were, however, significantly different from adult in the initial direction of PT and the arrangement of the straight part of PT in the medullary rays. While, it was in P5 that the spatial pattern of some PT was gradually approaching to the adult model. Conclusion This study demonstrated that the development of PT was consistent with the kidney development in terms of its volume density in cortex, length and spatial course. It started at the S-shaped body, kept throughout the embryonic period and continued to postnatal, ended at kidney maturation ( P28 ).