Three-dimensional architecture of pericardial nephrocytes in Drosophila melanogaster revealed by FIB/SEM tomography

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 three-dimensional 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 “washboard-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.


Introduction
Excretion is crucial to maintain the homeostasis of body fluid in metazoans. The metazoan excretory system generally produces primary urine by ultrafiltration of body fluid via filtration epithelia. The primary urine is subsequently transferred to the modulating tubule, where it is modified by epithelial secretion and reabsorption and discharged outside the body as the final urine Ruppert et al. 2003). Filtration epithelia are formed by podocytes in eucoelomates, which include vertebrates (Ruppert and Smith 1988). In vertebrate kidneys, the glomeruli include the podocyte-based filtration epithelia and the renal tubule plays a role as the modulating tubule   (Ruppert 1994).
Podocytes exhibit a characteristic architecture that is specialized for the ultrafiltration of body fluid (Kriz and Kaissling 2007). The podocyte cell body possesses several thick primary processes, each of which subsequently projects a number of fine foot processes (Fig. 2c). Adjacent podocytes are interdigitated with each other at their foot processes (Fig. 2a-c), which are separated from each other by filtration slits and bridged with a specialized intercellular junction, slit diaphragm, which serves as a filtration barrier apparatus (Figs 1d and 2c) (Pavenstadt et al. 2003).
Pterygote insects, including the fruit fly (Drosophila melanogaster), are the exception in that they do not possess filtration epithelia; instead, they produce urine via epithelial transport of the modulating tubules that are called Malpighian tubules ( Fig. 1e) (Kolosov et al. 2018;Sun et al. 2018).
Nephrocytes, which are similar in structure to podocytes, exist in the hemolymph space independently from the Malpighian tubules ( Fig. 1e-g) and play a role in the isolation of toxic substances, such as heavy metals, from insect body fluid, hemolymph (Crossley 1984). Nephrocytes form numerous foot processes that adhere to the basement membrane and possess slit diaphragms between them (Fig. 1h). The molecular components of the slit diaphragm are highly conserved between the nephrocytes in D. melanogaster and podocytes in vertebrates (Weavers et al. 2009;Zhuang et al. 2009) and Drosophila nephrocytes have recently been used as a model system to study podocyte function and disease (Fu et al. 2017;Helmstädter and Simons 2017;Na et al. 2015;Tutor et al. 2014;Weide et al. 2017). Podocytes are localized at the glomerulus (G), a primary urine-producing apparatus. The renal tubule (RT) subsequently receives and modifies the primary urine by epithelial secretion and reabsorption. (b) Conventional scanning electron microscope (SEM) image of a glomerulus. Podocytes form the most outer epithelial layer of the glomerulus (G). RT, renal tubule; arrows, parietal layer of Bowman's capsule. (c) Schematic drawing of a glomerulus. The epithelial layer of podocytes (P) covers the glomerular capillaries and continues to the renal tubule (RT) via a thin parietal layer of the Bowman's capsule (arrows). AA, afferent arteriole; EA, efferent arteriole. (d) Conventional transmission electron microscope (TEM) image of the glomerular wall. Podocytes cover the glomerular basement membrane (GBM) by the foot processes, which are bridged by the slit diaphragm (arrows). (a) and (c) are based on Kriz and Kaissling (2007). (a-e) Podocytes in rat kidney. (f-j) Pericardial nephrocytes (PCNs) in Drosophila melanogaster. Two neighboring podocytes or PCNs are indicated by different coloring (purple and green). (a, b, f, g) Schematic drawings of two neighboring podocytes (P) or PCNs (N). Podocytes interdigitate each other via the foot processes (a, b); PCNs exist independently from each other (f) and form the foot processes by basal infolding/ invagination (g). The slit diaphragms are formed between foot processes in podocytes and PCNs (b, g). The podocyte slit diaphragm is regarded as an intercellular junction; the slit diaphragm of PCNs is regarded as an autocellular junction. The arrowheads indicate the directions of observation in (c)-(e), (h) and (i). E, glomerular endothelial cell; M, mesangial cell. (c, h) Conventional scanning electron microscope (cSEM) image. cSEM is useful to observe the characteristic architecture of podocytes (P) (c); however, it is not impossible to observe the foot processes of PCNs (N) because PCNs are enwrapped by the basement membrane (h). (d, e, i) Reconstructed podocytes or PCN based on serial focused-ion beam/ scanning electron microscopy (FIB/SEM) images. The basal surface structure of podocytes (e) and PCN (i), which cannot be observed by cSEM, is clearly visible in the reconstructed images. (j) Schematic drawing for the orientation of the reconstructed image. The reconstructed podocytes and PCN shown here are also shown in S1 and S2. Scale bar: 1 μm Although the foot processes are similar in structure between Drosophila nephrocytes and vertebrate podocytes, the formation of the foot processes differs greatly between them. Podocyte foot processes are formed by the interdigitation between adjacent podocytes ( Fig. 2a-c); thus, the podocyte slit diaphragm is regarded as an intercellular junction. In D. melanogaster, individual nephrocytes exist independently without intercellular junctions forming between them (Fig. 2e) and the nephrocyte foot processes are formed by the infolding/invagination of the basal plasma membrane (Fig. 2g). The slit diaphragms of nephrocytes are regarded as autocellular junctions.
Recently, we revealed the three-dimensional (3D) ultrastructural architecture of podocytes using focused ion beam/ scanning electron microscopy (FIB/SEM) tomography in combination with a reconstruction technique (Ichimura et al. 2015(Ichimura et al. , 2019) (see Fig. S1 for further details of this technique). The reconstructed images of podocytes were similar to the images acquired by conventional SEM (Fig. 2c, d). Furthermore, the reconstructed images clearly visualized the basal surfaces of the podocytes, which are difficult to observe by conventional SEM due to their surrounding basement membrane ( Fig. 2e; Movie S1).
In the present study, we reconstructed the ultrastructure of nephrocytes in D. melanogaster using FIB/SEM tomography ( Fig. 2i, j) and thus successfully described their 3D architecture using this approach.

Drosophila melanogaster
Drosophila melanogaster cultures and crosses were performed using standard fly food that contained yeast, cornmeal and glucose and the flies were raised at 25°C. The migrating larvae and 4-week-old adults of Canton-S strain were used to analyze the normal 3D structure of the PCNs. The nephrocyte-specific snsknockdown flies were generated by mating male sns-UAS-RNAi (P{KK108577}VIE-260B established at the Vienna Drosophila RNAi Center) and female Dot-GAL4 (Kimbrell et al. 2002) from the Bloomington Drosophila Stock Center. Migrating larvae and adults were fixed in 2.5% glutaraldehyde solution buffered with 0.1 M phosphate buffer. The fixed samples were further immersed in the same fixative solution and stored at 4°C.

Rats
The ultrastructure of podocytes from adult (10-week-old, male) Wistar rats (Charles River Japan, Yokohama, Japan) was compared with that of the nephrocytes. The rats were perfused (under anesthesia using pentobarbital) with physiological saline and subsequently 2.5% glutaraldehyde fixative that was buffered with 0.1 M phosphate buffer. The fixed kidneys were further immersed in the same fixative and stored at 4°C.

Conventional SEM
Conventional SEM was performed as previously described (Dong et al. 2010). In brief, fixed samples were immersed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h. After dehydration using a graded series of ethanol, specimens were transferred to t-butyl alcohol and freeze-dried with an ES-2030 freeze dryer (Hitachi High-Technologies, Tokyo, Japan). After mounting on aluminum stubs with carbon paste, the dried specimens were coated with osmium using an OPC80T osmium plasma coater (Filgen, Nagoya, Japan) and observed using an S-4800 field-emission SEM (Hitachi High-Technologies).

Sample preparation for FIB/SEM
The fixed samples were processed using a combinatorial heavy metal staining protocol. This protocol was designed to enhance the signal for the backscatter electron imaging of epoxy-resin-embedded mammalian tissue at low accelerating voltages. In brief, the samples were successively immersed in 1% osmium tetroxide that contained 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 1 h on ice, 1% low molecular weight tannic acid (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M cacodylate buffer for 4 h at 25°C, 2% aqueous osmium tetroxide for 30 min at 25°C and 1% aqueous uranyl acetate overnight at 25°C. The samples were then dehydrated with a graded series of ethanol and were embedded in epoxy resin, Oken Epok 812 (Okenshoji, Tokyo, Japan).

FIB/SEM tomography
Serial FIB/SEM images were obtained at 30-nm increments with a backscattered electron detector at a 2.0-kV acceleration voltage using a Helios Nanolab 660 FIB/SEM (Thermo Fisher Scientific, Waltham, MA, USA). The pixel size of each FIB/ SEM image was 13.5 × 17.1 × 50 nm/pixel (width × height × depth). The pixel dimensions for a recorded image was 3072 × 2048 pixels. Thus, the dimension of serial imaging by FIB/SEM was 41.5 × 35.0 × 20.0-35.0 μm (width × height × depth). The new surface for serial FIB/SEM imaging was generated by FIB milling using a 0.77-nA beam current, where gallium ions were accelerated with a voltage of 30 kV.

Data processing for 3D reconstruction
The 3D reconstruction of podocytes was performed using AMIRA 6.1 Software (Thermo Fisher Scientific) on a MousePro-W994DQP5X professional workstation (Mouse Computer Co. Ltd., Tokyo, Japan). We used a Cintiq 27QHD interactive pen display for the segmentation procedure (Wacom, Tokyo, Japan).

Statistical analysis
Width of foot processes and slit diaphragms were measured on FIB/SEM images. All measurements were shown as mean ± SEM. Statistical analyses were performed using a two-tailed Student's t test. P values < 0.05 were considered statistically significant.

Results
Drosophila melanogaster possesses two kinds of nephrocytes: garland and pericardial, in both its larval and adult stages (Zhuang et al. 2009). The garland and pericardial nephrocytes are located around the junction of the esophagus and proventriculus and along both sides of the heart tube, respectively. In the present study, we examined the 3D ultrastructure of the pericardial nephrocytes (PCNs) in D. melanogaster.

Sectional (FIB/SEM) images of PCNs
We obtained serial FIB/SEM images of PCNs in migrating larvae and adults. The contrast-inverted FIB/SEM images achieved a quality that was comparable to conventional transmission electron microscopy (TEM) images (Figs 3 and S1). The FIB/SEM images showed PCNs located near the heart tube ( Fig. 3a) and possessed numerous electron-dense endosomes and lysosomes (Fig. 3b), which corresponded to the separation function of toxic materials from hemolymph in PCNs. The FIB/ SEM images are also able to visualize numerous fine foot processes and slit diaphragms bridging between them, as demonstrated by previous TEM studies (Crossley 1972) (Fig. 3c).

Reconstructed images of normal PCNs
The basal surfaces of individual PCNs were completely covered by a basement membrane (arrowheads in Fig. 3b,c); thus, conventional SEM was not suitable to observe their foot processes (Fig. 2h). To overcome this problem and reveal the 3D structure of PCNs in migrating larvae and adults, we reconstructed the basal surface of PCNs using their serial FIB/SEM images (Figs 4-6; Movies S2 and S3).

PCNs in migrating larvae
The basal surfaces of reconstructed PCNs were observed to be occupied by the foot processes and the slit-like spaces between neighboring foot processes (Fig. 4a,d,e; Movie S2).
The slit-like spaces, which corresponded to the filtration slits in podocytes, were formed by the longitudinal infolding/ invagination of the basal plasma membrane (Fig. 4d). The remaining regions of the basal surface between the slit-like spaces became the foot processes. Both ends of the foot processes were usually anastomosed to the neighboring foot processes (green arrows in Fig. 4d,e) and thus free ends were rarely observed. Furthermore, many tiny cytoplasmic bridges existed between the neighboring foot processes (yellow arrowheads in Fig. 4d,e; green lines in Fig. 5a). The slit diaphragm spanned the space surrounded by the foot processes and tiny cytoplasmic bridges. The foot processes almost continued to the main body of the PCN along their length and looked like Bridges^that protruded from the main body (Fig. 6a-c).

PCNs in adults
The foot processes of PCNs became significantly narrower in width in the adults than in the migrating larvae (Fig. 4b,f,g; green bars in Fig. 4c; Movie S3); however, the width of the slit diaphragms remained unchanged (blue bars in Fig. 4c). Moreover, the width of the foot processes was narrower in adult Drosophila PCNs than in the adult rat podocytes (Fig. 7a); however, the width of the slit diaphragm was similar between adult Drosophila PCNs and adult rat podocytes (Fig.  7b).
Most of the tiny cytoplasmic bridges disappeared in adult PCNs; however, some of them remained (Figs 4f and 5b). The foot processes continued to the main body of the PCN only by several regions (Fig. 6d-h; Movie S4) and became thinner in comparison to those of larval PCNs as described above; they looked like Bbars^ (Fig. 6d-h; Movie S4). The basal surface, which has a Bwashboard-like^appearance, is clearly visible in the larva and adult reconstructed images. (d-g) Magnification of reconstructed images shown in (a) and (b). The basal surface membrane of foot processes, which adhere to the basement membrane, is shown in whitish purple. The foot processes run parallel to each other and their ends anastomose to the neighboring foot processes (green arrows in d-g). The thin cytoplasmic bridges are found between the neighboring foot processes in the larva (yellow arrowheads in d, e) but most of them were lost in the adult (f, g). The reconstructed PCNs shown here are also shown in Movies S2 and S3. Scale bars : 1 μm (a, b) and 500 nm (d, f). (c) Width of foot processes and slit diaphragms. The foot processes became significantly narrower in the adult than larval PCNs (green bars) but the width of the slit diaphragms was unchanged between the larva and adult (blue bars). Values represent means ± standard errors of the means. Differences were tested using two-tailed Student's t test. *P < 0.05 was considered statistically significant Reconstructed images of sns-knockdown PCNs FIB/SEM tomography was useful in revealing the normal 3D ultrastructure of PCNs. To confirm the usefulness of FIB/SEM tomography in studying pathologic alterations of nephrocytes, we examined the 3D ultrastructure of sticks and stones (sns)knockdown PCNs using this method. Sns is a Drosophila ortholog of vertebrate NPHS1, which encodes Nephrin, an  (Gerth et al. 2005;Ichimura et al. 2013;Kestila et al. 1998;Kramer-Zucker et al. 2005).
The reconstructed images clearly showed the 3D structural alterations in sns-knockdown PCNs ( Fig. 8; Movie S5). Compared to the control PCNs (Fig. 8c), the foot processes were irregular in width and shape and the slit-like spaces were predominantly reduced in sns-knockdown PCNs (Fig. 8a). Similar to control PCNs, the slit diaphragms were formed at the reduced slit-like spaces in sns-knockdown PCNs (Fig. 8b, d).

Discussion
The two-dimensional ultrastructure of nephrocytes has been investigated in numerous species of insects by using conventional TEM (Crossley 1984). However, the 3D ultrastructure of nephrocytes has not been precisely elucidated because the basement membrane that completely surrounds the individual nephrocytes makes it difficult to observe their 3D ultrastructure using conventional SEM. In the present study, FIB/SEM tomography, including a reconstruction technique, presented a viable solution to this problem and enabled the 3D ultrastructure of Drosophila PCNs to be analyzed, which has positive implications for further study of health and disease.

Similarity between PCNs and renal epithelial cells
Drosophila PCNs possess structural or functional similarities to some kinds of epithelial cells of the vertebrate nephron. Structurally, PCNs possess foot processes that link with the slit diaphragms like podocytes and have basal infoldings/ invaginations like the principal cells of the cortical collecting duct (Kaissling and Kriz 1979;Fukudome 2001). Functionally, PCNs absorb hemolymph proteins via the Drosophila orthologs of mammalian Cubilin and Amnionless (major receptors for protein reabsorption), like the proximal tubular epithelial cells (Zhang et al. 2013).
In invertebrate eucoelomates, the podocytes possess numerous lysosomes, including inclusion bodies , which indicates that the invertebrate podocytes serve to maintain the quality control of plasma proteins and to remove toxic substances from the blood, like insect nephrocytes. In other words, it can be argued that insect nephrocytes have retained some functional features of invertebrate podocytes.

Nephrocytes in other invertebrate species
Nephrocytes are recognized in three invertebrate phyla; Arthropoda, Onycophora and Mollusca (Crossley 1984;Haszprunar 1996;Seifert and Rosenberg 1977). The twodimensional ultrastructure of nephrocytes has been reported in a variety of species belonging to these phyla (Boer and Sminia 1976;Goodman and Cavey 1990;Kokkinopoulou et al. 2014). In several kinds of insects and decapods, nephrocytes have been reported to possess multiple nuclei (Crossley 1984). However, it is unclear whether such nephrocytes possess multiple nuclei per cell, as is the case in the garland nephrocytes found in D. melanogaster (Zhuang et al. 2009), or if multiple nephrocytes are connected to form an epithelioid mass. Our research group has been revealing a great diversity in the 3D ultrastructure of nephrocytes among arthropod species, including insects, using FIB/SEM tomography and these data will be published in a subsequent research paper.

Adaptation of FIB/SEM tomography to podocyte-related cells
Almost all metazoans produce primary urine by ultrafiltration of body fluid via the filtration epithelium Ruppert and Smith 1988;Ruppert et al. 2003;Schmidt-Rhaesa 2007). The acoelomates and pseudocoelomates, which do not possess a body cavity lined with mesothelium, generally use the terminal cells as the filtration epithelium (Wilson and Webster 1974). As is the case with nephrocytes, it is difficult to evaluate the 3D ultrastructure of terminal cells by conventional SEM because the terminal cells are almost completely enwrapped by the basement membrane. For Values are means ± standard error of the mean. Differences were tested using two-tailed Student's t test. *P < 0.05 was considered statistically significant instance, in freshwater planarians, Ishii (1980) suggested that the filtration slits are formed between terminal cells, whereas another study reported that the filtration slits are formed as numerous rectangular fenestrations that open on the tubular cytoplasmic wall of the terminal cells (McKanna 1968). FIB/ SEM tomography should be useful in revealing the precise and accurate 3D architecture of terminal cells.
Limitations of FIB/SEM tomography FIB/SEM tomography is a powerful tool for precisely analyzing the 3D ultrastructure of nephrocytes and podocytes; however, there are several technical disadvantages to this method. One of the major technical difficulties is that the observational volume is restricted; i.e., to smaller than approximately 40 × 40 × 40 μm (width × height × depth). Therefore, it is impossible to obtain serial FIB/SEM images that contain the entire volume of one nephrocyte, podocyte, or glomerulus. Array tomography, which is used to obtain serial images from serial ultrathin sections mounted on silicon or glass bases, is a potential solution to this problem (Koga et al. 2018;Koike et al. 2017;Micheva and Smith 2007;Wacker and Schroeder 2013). This method enables serial images of larger volumes than FIB/SEM tomography to be obtained, which may enable the entire volume of the murine glomerular capillary to be reconstructed by array tomography (Dittmayer et al. 2018). Reconstructed images. The snsknockdown PCNs were produced by mating male sns-UAS-RNAi (P{KK108577}VIE-260B) and female Dot-GAL4. The foot processes were irregular in shape and width in the sns-knockdown larval PCN (a), in comparison to the control Dot-GAL4 larval PCN (c). The basal surface membrane attached to the basement membrane is shown in whitish purple. The reconstructed PCNs shown here are also shown in Movie S5. (b, d) FIB/SEM images. In the snsknockdown PCN, the labyrinths were simpler, although the slit diaphragms (arrowheads) were formed at the slit-like spaces similar to the control Dot-GAL4 PCN. Scale bars: 500 nm (a, c); 100 nm (b, d)

Conclusion
FIB/SEM tomography is a powerful tool that can be used to reveal the 3D architecture of nephrocytes in more detail than previously possible using conventional TEM and SEM.