Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy

Abstract

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

Figure 1. Podocyte subcellular compartments shown by conventional SEM and TEM.

Podocyte are traditionally divided into three kinds of subcellular compartment, cell body (CB), primary process (P) and foot process. (a) Conventional SEM image shows the luminal surface of all three kinds of podocyte subcellular compartments. Three neighboring podocytes are individually colored with blue, green, and red. (b, c) Conventional TEM images also show the three subcellular compartments. The foot processes are predominantly protruding from the primary processes, but some of them are from the undersurface of cell body (yellow arrows in b). The foot processes possess the electron-dense actin bundle within their luminal cytoplasm (yellow arrowheads in c). Directions of luminal and basal views are indicated by black arrows. (d) Alkaline-maceration SEM images clearly show the basal surface of neighboring podocytes. The region in contact with the glomerular basement membrane consists almost entirely of the interdigitating foot processes. Two neighboring podocytes are individually colored with green and red. Cap, capillary lumen. US, urinary space of the Bowman's capsule. Bar scales, 1 μm in (a), (b); 200 nm in (c), (d).

Figure 2. Comparison with block-face SEM and conventional TEM images of podocytes.

The block-face images obtained with SBF-SEM (a1, a2) and FIB/SEM (b1, b2) are similar to the conventional TEM images (c1, c2). Due to the combinatorial heavy metal en bloc staining, the Golgi apparatus and endoplasmic reticulum in the cell bodies of podocytes are electron-densely depicted (pink arrowheads in a1, b1, c1). The electron-dense actin bundles, which are visualized at the luminal cytoplasm of the foot processes in the conventional TEM image (yellow arrowheads in c2), are difficult to see in the FIB/SEM and SBF-SEM images. Cap, capillary lumen. Bar scales, 500 nm in (a1), (b1), (c1); 200 nm in (a2), (b2), (c2).

Figure 3. Luminal surface of reconstructed podocytes based on serial FIB/SEM images.

Individual podocytes are represented with different colors. (a) The reconstructed image is quite similar to the conventional SEM images as shown in Figure 1a. (b, b′) These two reconstructed images are shown in the same region, and the green-colored podocyte is displayed semi-transparently in b′. The distal ends of foot processes are situated under the primary process (asterisks in b′), and both lateral surfaces of the ridge face the distal ends of foot processes. Bar scales, 1 μm in (a), 200 nm in (b′). These three-dimensionally reconstructed podocytes can be observed from any directions on the reconstruction software. CB, cell body of podocyte; P, primary process. (See also Supplementary Movies S1, S2).

Figure 4. Basal surface of reconstructed podocytes based on serial SBF-SEM (a1, a2) and FIB/SEM (b1–b3) images.

Individual podocytes are represented with different colors. (a1, a2, b1) These reconstructed images are quite similar to those of alkaline-maceration SEM as shown in Figure 1d, d′. The region where podocytes are in contact with the GBM consisted almost entirely of the interdigitated foot processes. (b2, b3) When the separate podocytes are individually observed from the basal side, the foot processes from the green-colored podocyte mainly protrude from the primary processes (b2), and the foot processes from the purple-colored one from the cell body (b3). The nucleus (N) in the cell body (CB) is visualized by clearing the surface and cytoplasm of cell body (a1′, b3′). Bar scales, 1 μm in (a1); 200 nm in (a2), (b3).

Figure 5. Three-dimensional structure of podocyte foot processes.

A single reconstructed podocyte based on serial FIB/SEM images is observed from luminal (a1, a2) and basal (b1, b2) sides. (a2 and b2) are the magnification of the area indicated by rectangles in (a1 and b1), respectively. In the luminal surface view, the foot processes appeared to be simply branched from the lateral aspects of primary process, as found in a plant leaf vein (a2). In the basal surface view, the most proximal portions of foot processes are connected each other via a tortuous ridge-like prominence, which was formed on the basal surface of the primary process (asterisks in b2). The ridge-like prominences was quite difficult to predict their existence only by the conventional SEM observation from the luminal side, and was structurally similar to the usual foot processes. CB, cell body; P, primary process. Bar scales, 1 μm in (b1); 200 nm in (b2). The three-dimensionally reconstructed podocyte can be observed from any direction on the reconstruction software. (See also Supplementary Movies S1, S2).

Figure 6. Structural relation of ridge-like prominence and foot processes.

(a) Luminal surface view of podocytes shows primary and foot processes. Cross sections at the sites indicated by arrows (Sec. b, c, d) are shown in (b, c, d), respectively. (b) Podocytes adhere to the glomerular basement membrane with their foot processes. (c, d) The basal portion of primary process form the ridge-like prominence, and the primary process adheres to the glomerular basement membrane via the ridge-like prominence (RLP). The foot processes also protrude from the primary process via the ridge-like prominence. F, foot process; GBM, glomerular basement membrane; P, primary process.

Figure 7. Structural hierarchy of podocyte subcellular compartments.

(a) Previous simple configuration. The three subcellular compartments of podocyte are simply connected in this order. (b) Newly proposed configuration based on the present three-dimensional analysis. The ridge-like prominences, which are formed at the basal surface of the cell body and primary processes, serve as an adhesion apparatus for the attachment of cell body and primary processes to the glomerular basement membrane, and as a connecting apparatus of foot processes to cell body and primary processes.