Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging, a powerful tool for analyzing the 3D cellular ultrastructure, to precisely reveal the morphological process of podocyte development, such as the formation of foot processes. Development of foot processes gives rise to three morphological states: the primitive, immature and mature foot processes. Immature podocytes were columnar in shape and connected to each other by the junctional complex, which migrated toward the basal side of the cell. When the junctional complex was close to the basement membrane, immature podocytes started to interdigitate with primitive foot processes under the level of junctional complex. As primitive foot processes lengthened, the junctional complex moved between primitive foot processes to form immature foot processes. Finally, the junctional complex was gradually replaced by the slit diaphragm, resulting in the maturation of immature foot processes into mature foot processes. In conclusion, the developmental process of podocytes is now clearly visualized by block-face SEM imaging.

Background Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear.Methods To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images.Results Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridgelike prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. ConclusionsThe three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focusedion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Anatomic characterization of the humeral nutrient artery varies among the several textbooks on human anatomy. To clarify the anatomic characteristics of the humeral nutrient artery, we reexamined its origin and course by cadaveric dissection. In typical cases, one prominent nutrient foramen was situated on the anteromedial surface of the humeral shaft, and the nutrient canal distally penetrated the cortical bone layer. The humeral nutrient artery originated from the brachial artery below the level of the nutrient foramen as a short ascending branch. On reaching near the nutrient foramen, the humeral nutrient artery formed a hairpin loop on the periosteum to enter into the nutrient foramen. In some cases, an accessory nutrient foramen was also found near the groove for the radial nerve on the posterior surface of the humerus. This accessory nutrient foramen received an accessory humeral nutrient artery that originated from the radial collateral artery. The present findings corresponded well with the descriptions in the anatomy textbooks published in English-speaking countries. However, textbooks published in German-speaking countries describe only one type of humeral nutrient artery, the branch of the profunda brachii artery. Terminologia Anatomica, the international standard in human anatomic terminology, most likely adopted the description in the German anatomy textbooks, and thus, it is necessary to correct the position of the humeral nutrient artery in the hierarchy of Terminologia Anatomica for accurate morphological description. Clin. Anat. 30:978-987, 2017. © 2017 Wiley Periodicals, Inc.

Nephrocytes are similar in structure to podocytes and play a role in the isolation of toxic substances from hemolymph in insects. Drosophila melanogaster nephrocytes have recently been used to study podocyte function and disease. However, the threedimensional ultrastructure of nephrocytes is not clearly understood because their surrounding basement membrane makes it difficult to observe using conventional scanning electron microscopy. We reconstructed the three-dimensional ultrastructure of Drosophila pericardial nephrocytes using serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. The basal surfaces were occupied by foot processes and slit-like spaces between them. The slit-like spaces corresponded to the podocyte filtration slits and were formed by longitudinal infolding/invagination of the basal plasma membrane. The basal surface between the slit-like spaces became the foot processes, which ran almost linearly, and had a Bwashboard-like^appearance. Both ends of the foot processes were usually anastomosed to neighboring foot processes and thus free ends were rarely observed. We demonstrated that FIB/SEM is a powerful tool to better understand the three-dimensional architecture of nephrocytes.

The localization of nutrient foramina and direction of nutrient canals have been studied. However, information about the origin and extraosseous course of nutrient arteries is lacking in some types of long tubular and irregular bones. Thus, we aimed to reexamine the origin and course of the femoral nutrient artery (FNA) through cadaveric dissection to clarify its anatomic characteristics. Sixty thighs were collected from 57 cadavers. To fix the cadavers and visualize the small arterial branches, 10% formalin was injected from the femoral artery, followed by an injection of acrylic ink. The arterial tree in the posterior part of the thigh was recorded by line drawings. The femur received single or double FNAs via the femoral nutrient foramina, which were on and along the linea aspera. In cases with single FNA (41 of the 60 thighs), it typically arose from the four parts of the profunda femoris system: profunda femoris artery between the origins of the third and fourth perforating arteries; second perforating artery; third perforating artery; and terminal branch. In cases with double FNAs (remaining 19 thighs), the superior FNA typically arose from the second perforating artery, and the inferior FNA arose from the terminal branch of the profunda femoris artery and popliteal system. FNAs are described as branches of the perforating arteries in Terminologia Anatomica and anatomy textbooks. However, we found that FNAs also frequently arose from the profunda femoris artery and popliteal system, in addition to the perforating arteries. Clin. Anat. 33:479-487, 2020.

The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. In this study, using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, we revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy—they filled an enormous gap, which can be called “missing link,” in the evolutionary diversity of podocytes and nephrocytes. Thus, we concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.

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