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Ultrastructure of the nephron in the soft-shelled turtle, Pelodiscus sinensis (Reptilia, Chelonia, Trionychidae)
Micron 44 (2013) 451–462
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Ultrastructure of the nephron in the soft-shelled turtle, Pelodiscus sinensis (Reptilia, Chelonia, Trionychidae) Chun-Sheng Xu a,b , Ping Yang a , Hui-Jun Bao a , Xun-Guang Bian a , Qiu-Sheng Chen a,∗ a b
Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People’s Republic of China College of Animal Science and Technology, Shihezi University, Shihezi, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 September 2012 Accepted 4 October 2012 Keywords: Kidney Soft-shelled turtle Podocyte Nephron Ultrastructure
a b s t r a c t The structure of the nephron in adult soft-shelled turtles (Pelodiscus sinensis) was studied by light microscopy, transmission and scanning electron microscopy. The kidney contained 5–6 renal lobes. Nephrons of P. sinensis are composed of a renal corpuscle (RC) and of a renal tubule that appears divided morphologically into five distinct segments: neck segment (NS) (This segment is only present in approximately 10% of the nephrons), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The RCs and most of the convoluted DTs lie in the central zone, while the PTs and the CDs lie in the peripheral zone of the renal lobe. The renal corpuscle is relatively large with especial processes in podocytes and a thick basement membrane. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. The neck segment when presentis short and has a relatively narrow lumen, consisting of cuboidal or squamous cells. There is a well-developed endocytic-lysosomal apparatus in the apical cytoplasm of the PT. The proximal tubule and intermediate segment cells show some differences between male and female. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. Not previously reported in vertebrate kidneys. The IS is the narrowest nephron segment, formed by multiciliated as well as nonciliated cells. In DT cells, basolateral interdigitations and infoldings are particularly well-developed. The CD contains clear cells with numerous secretory granules and dark cells with dense mitochondria and an elaborate Golgi complex. This study was undertaken in order to disclose specific kidney features in P. sinensis that could be related to function. In addition, the ultrastructure of the nephrons in P. sinensis are discussed in relation to other turtles and vertebrates. © 2012 Published by Elsevier Ltd.
1. Introduction As holds for all vertebrates, the main function of the reptilian kidney is to eliminate waste products and maintain salt and water balance by regulating the composition of the body fluids (Solomon, 1985). In all vertebrates, the kidney has the nephron as the basic structural and functional unit. The reptilian nephron lacks a loop of Henle characteristic of kidneys that produce concentrated urine and hence reptiles cannot produce urine hyperosmotic to plasma. The basic components of the reptilian nephron are: a renal corpuscle and a renal tubule consisting of a short ciliated neck
∗ Corresponding author. Tel.: +86 25 84398669; fax: +86 25 84398669. E-mail address: [email protected] (Q.-S. Chen). 0968-4328/$ – see front matter © 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.micron.2012.10.001
segment, a proximal tubule, a short ciliated thin intermediate segment and a distal tubule, the latter opening into the collecting duct (Dantzler and Bradshaw, 2009). Urinary excretion of ions and water involves regulation of glomerular filtration as well as tubular reabsorption and secretion (Dantzler and Holmes, 1974). Studies made on different species among the orders of living reptiles have demonstrated structural variations among their nephrons (Meseguer et al., 1987; Solomon, 1985; Gabri and Butler, 1984; Gabri, 1983a,b; Schmidt-Nielsen and Davis, 1968) In the present study we investigate nephron structure in the Chinese Soft Shelled Turtle, Pelodiscus sinensis, which lives in a wide range of habitats in China and its kidneys have some structural differences when compared to those of other reptiles and vertebrates. Kidney structure has only been scantily documented in this species and considering previous physiological reports (Dantzler and Schmidt-Nielsen,
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1966; Schmidt-Nielsen and Skadhauge, 1967; Dantzler and Holmes, 1974), data on renal ultrastructure of reptiles, mainly Chelonia, lizards, and crocodiles could be morphological and physiological significant. In the present paper, we highlight the ultrastructure of nephrons in P. sinensis and discuss the functional significance of the findings. The morphological features have been compared to those of other vertebrates, mainly in other orders of reptiles. The present study, employing both light microscopy based reconstructions and electron microscopy, considerably improves the structural understanding of the reptilian kidney. This work is a contribution to the knowledge of the kidney of P. sinensis, frequently used as an experimental model for physiological and biochemical studies. 2. Materials and methods Twenty male and female adult soft-shelled turtles, Pelodiseus sinensi, weighing 850–950 g/each, were purchased from a wild breeding base in the Jiangsu province of China at the beginning of May. The animals were anesthetized with ethyl ether or chloroform and their ventral caparaces removed. Tissue was subsequently removed for light microscopy, transmission electron microscopy and scanning electron microscopy. All protocols were approved by the Chinese Committee for Animal Use for Research and Education.
Fig. 1. P. sinensis. Transverse sections of the renal lobe. Paraffin sections, 5 m, stained with hematoxylin–eosin. When viewed in transverse sections the kidney can be divided into a central zone (cz) and two peripheral zones (pz). The central zone is characterized by the presence of renal corpuscles, distal tubules, and intermediate segments. The peripheral zones are dominated by the presence of proximal tubules and the collecting duct. cd, collecting duct; dt, distal tubules; iv, intralobular vein; ra, renal artery; g, glomerulus; pt, proximal tubule; fc, fibrous capsule.
2.1. Light microscopy 3. Results Tissue fixed in 10% cold formalin PBS solution was rinsed for 1 h in tap water and then dehydrated by placing the tissue in a graded series of ethyl alcohol (70, 85, 95, and 100% for an hour each). After dehydration, the tissue was embedded in paraffin blocks for sectioning (5 m) with a Leica RM2015 microtome. Alternate slides were stained with hematoxylin–eosin (for general cytology). 2.2. Transmission electron microscopy (TEM) Blocks (1 mm3 ) of tissue were fixed by immersion for 5 h at 4 ◦ C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, then postfixed in 1% buffered osmium tetroxide and embedded in araldite. Ultrathin sections were obtained using a LKB III ultramicrotome, stained with uranyl acetate and lead citrate and examined in a JEM-1200EX electron microscope. 2.3. Scanning electron microscopy (SEM) Slices of tissue 1 mm thick were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate, bufferwashed (pH 7.2) and dehydrated through a series of acetones. The material was critically point-dried in CO2 and sputter-coated with gold/palladium for 3 min in an Emscope sputter coater. Blocks were viewed using a Philips 501B scanning electron microscope. 2.4. Reconstruction of nephrons Serial transverse and longitudinal sections of the araldite- and the paraffin-embedded kidney tissue were used for graphic reconstructions of nephrons. The reconstructions were performed using a light microscope to trace the course of the nephron segments in successive sections. The diameter of the different segments of the nephron was determined from TEM pictures of the specimens by using the TEM m markers as calibration. The magnification of the drawing was determined by means of a calibrated microscope slide. The thickness of the different components of the glomerular basement membrane (GBM) was measured on TEM micrographs.
3.1. Kidney zonation and nephron segmentation The paired kidneys of the soft-shelled turtle are hemicycle in shape, lobed bodies closely applied to the posterior wall of the pleuroperitoneal cavity. They are metanephric in origin and collecting ducts empty into the ureter in the ventral surface center of each kidney. When viewed in transverse sections the renal lobe can be divided into a central zone and two peripheral zones (Fig. 1). The central zone is characterized by the presence of renal corpuscles, distal tubules, and intermediate segments. The perpheral zones are dominated by the presence of proximal tubules and the collecting duct system. The TEM analysis of the specimens allowed identification of the following divisions within the nephron: the renal corpuscle, the neck segment, the proximal tubule, the intermediate segment, the distal tubule and the collecting duct. The renal corpuscle is large and consisted of a glomerulus (capillary endothelium, glomerular basement membrane and mesangium) and Bowman’s capsule (visceral layer and parietal layer). The main features of the P. sinensi nephron are summarized in Fig. 2. Reconstructions of the nephron, were made from serial transverse and longitudinal sections. The spatial location of the different nephron components allowed recognition of a renal lobe with three main divisions (Fig. 1): a central zone and two peripheral zones. The transverse sections ratio of proximal and distal tubules was 2:1 by counting a single female or male lobe. 3.2. Renal corpuscle and neck segment (Figs. 3–5) The P. sinensis renal corpuscle is roughly ovoid in shape, with a diameter of 120 m (n = 7) and a smooth outer surface. The renal corpuscle is formed by two structures: the glomerulus formed by blood capillaries and the capsule of Bowman, which surrounds the glomerulus. The latter is composed of a visceral layer and a parietal layer with a urinary space in between (Fig. 4B). The urinary pole of the RC is continuous with either a small the neck segment or the
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Fig. 2. Schematic representation of the nephron in P. sinensis in which the tubules are spread apart. The figure moreover provides a scheme of cell types from proximal tubule (A–C); intermediate segment (D and E); distal tubule (F–H) and collecting duct (I and J). Male proximal tubule (A); female proximal tubule clear cell (B) and dark cell (C); male intermediate segment (D); female intermediate segment (E); distal tubule initial (F), middle (G) and terminal (H) regions; collecting duct dark cell (I) and clear cell (J). ba, basal body; bm, basement membrane; ci, cilium; rer, rough endoplasmic reticulum; mc, mitochondria; mv, microvilli; nu, nucleus; no, nucleolus; ly, lysosome; sg, secretory granules; in, infold; va, vacuole; g, glomerulus; ns, neck segment; pt, proximal tubule; is, intermediate segment; dt, distal tubule; cd, collecting duct.
PT (Fig. 3). None of the usual components of the juxtaglomerular apparatus (juxtaglomerular cells and macula densa) are observed in our material. The epithelium of the parietal layer is composed of fusiform shaped squamous cells. The cells have a height of 2–4 m and bulge slightly into the urinary space (Fig. 4A). A large and irregular
Fig. 3. Paraffin section 5 m, stained with HE. In some nephrons a small neck segment (ns) is present, connecting the urinary pole to the proximal tubule. Central zone of a renal lobe showing renal corpuscles with glomerulus (g), neck segment (ns) andproximal tubule (pt), distal tubule (dt), intralobular vein (iv).
nucleus is situated centrally in the cell. The cytoplasm moreover contains a number of scattered rough endoplasmic reticulum, free ribosomes and a few rounded mitochondria. Lateral and basal intercellular spaces contain microvillar extensions of the lateral membrane. The intercellular spaces are narrow beneath the apical junctional complex. The cells constituting the parietal layer epithelium gradually become cuboidal towards the urinary pole, where they merge either with a small NS or directly with the PT (Fig. 3). NS is present in approximately 10% of the nephrons in male and female kidney (Fig. 3A). When present, the NS is the shortest segment of the renal tubule. Podocytes form the epithelium of the capsule’s visceral layer. They contain an indented nucleus and send out cell processes. The podycyte cell body sends out wide and flat primary processes, the latter stretch out to form secondary processes that eventually extend into a few tertiary processes. The finest branches of the cell, are the pedicels or foot processes (Fig. 4C). The alternating foot processes rest on a 300–600 nm thick basement membrane which separates the podocytes from the juxtaposed fenestrated capillary endothelium (Fig. 4B). Between the foot processes are narrow filtration slits which are bridges by a diaphragm (Fig. 4B insert). Transmission electron microscopy demonstrated a large indentation nucleus within the podocyte cell body, often with peripherally clumped chromatin. The cytoplasm contain small mitochondria, a Golgi complex, lysosomes, numerous free ribosomes and granular endoplasmic reticulum. These organelles extended into the terminal foot processes see Fig. 5. The glomerular basement membrane consists of a lamina rara beneath the podocyte processes, a central lamina densa (basal lamina), and a lamina rara bordering the endothelium of the
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Fig. 4. (A) Parietal layer of renal capsule. Cells contain lateral intercellular spaces and basal labyrinth (B). The filtration barrier of the renal corpuscle in the soft-shelled turtle. The podocytes consist of cell bodies containing the indented nucleus that send out cell processes with foot processes, all of which rest on the glomerular basement membrane. Insert: filtration slits with diaphragm. (C) The epithelium of the glomerular capillary is composed of cell bodies that bulge into the capillary lumen and send out cell processes that line the blood side of the glomerular basement membrane. (D) Few mesangial cell. TEM. Pl, parietal layer; bm, basement membrane; en, endothelial cell; fp, foot processes; fe, fenestration; nu, nucleus; no, nucleolus; po, podocyte; rer, rough endoplasmic reticulum; e, erythrocyte; ca, capillary space; us, urinary space; go, golgi apparatus; mc, mitochondria; jc, junctional complexes; lis, lateral intercellular space; bis, basal intercellular space; ecp, endothelial cell process; me, mesangial cell; pp, primary processes; sp, secondary processes; tp, third processes.
glomerular capillaries. The interna lamina rara and lamina densa together have a constant thickness of 200–350 nm (Fig. 4A–D). The capillary endothelium is composed of fenestrated endothelial cells with a large nucleus (Fig. 4C). The cytoplasm of these cells contain numerous free ribosomes sparse rough endoplasmic reticulum and vacuoles. This epithelium is composed of cell bodies that bulge into the capillary lumen and send out cell processes (Fig. 4C). These processes line the basement membrane and form the fenestrations of the endothelium (80–185 nm in size) (Fig. 4B). In the
soft-shelled turtle, this endothelium usually presented a reticulated appearance; fenestrated regions are occasionally found (Fig. 4B). Few thin mesangial cells are distinguishable at the base of the capillary loops (Fig. 4D). 3.3. Proximal tubule (Figs. 6 and 7) In the soft-shelled turtle, the proximal tubule showed some differences between specimens. In female soft-shelled turtles (n = 10)
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Fig. 5. (A) SEM micrograph showing a renal corpuscle. Partial elimination of Bowman’s capsule allows observation of the glomerular capillary tufts. The afferent arteriole ( ); the efferent arteriole ( ); the efferent arterioles often have a smaller diameter than the afferent arteriole. The parietal epithelium of Bowman’s capsule ( ); Bowman’s capsule ( ); podocyte cell body ( ). (B) Detailed SEM view of a glomerulus showing podocyte morphology with developed primary processes. pcb, podocyte cell body; pp, primary processes; sp, secondary processes; tp, tertiary processes.
the tubules are heterocellular, composed of large, clear and dark, higher columnar cells (Fig. 7A). They have long microvilli forming the brush border characteristic of this segment, and a round nucleus located towards the center area (Fig. 7A). In the females, the dark cells appeared as single intercalated cells between the clear cells. In males (n = 10), the proximal tubules contain tree cell type: cells with microvilli, bald-headed cells (Fig. 6C) and basal cells (Fig. 6D). The cytoplasm of proximale tubule cells in both males and females is dominated by a large numbers of rounded mitochondria, numerous lysosomes and scattered endoplasmic reticulum. Between adjacent cells, junctional complexes are found just beneath the free surface (Figs. 6A, B and 7A). In the females investigated, the lateral cell membranes are straight between neighboring cells and no intercellular spaces are observed (Fig. 7A). While, in the males, lateral intercellular spaces are present and the basal cell membranes formed a labyrinth (Fig. 6A). In the males clear basal cells and bald-headed cells can occasionally be observed. The former is restricted to the basal region of the epithelium; nuclei are located towards the center area of the cells. The latter appeares as single intercalated cells between the microvilli bearing cells. The apical surfaces of the bald-headed cell carried few microvilli. Endoplasmic reticulum, mitochondria andvesicles of various sizes are scattered throughout the cytoplasm of these cells. The average cell height of the proximal tubule cells is 20 m. The average external diameter of the proximal tubule is 60 m. SEM showed that the PT contain, in transverse sections, the PT cells are tall pyramidal or columnar in shape. The cells exhibit a brush border of microvilli and lateral and basal intercellular spaces are evident in the SEM preparation (Fig. 7B). 3.4. Intermediate segment (Fig. 8) The IS is a short, straight, well-defined segment. The transition from the proximal tubule is defined by a considerable decrease in the cell height (Fig. 8A and B). The intermediate segment contain numerous multiciliated cells and a few non-ciliated. The apical surface of non-ciliated cells are covered with dense short microvilli. The epithelium of this multiciliated segment consisted of 8–10 m high cuboidal cells with basal located nuclei (Fig. 8A–C). The cells have an irregular or indented nucleus and few organelles (Fig. 8A). The apical cytoplasm in the multiciliated cells is characterized by the presence of basal bodies. Some mitochondria, lysosomes and vacuoles are found in the cytoplasm (Fig. 8A and B).
The IS in male and female showed some differences. In male, the lateral cell membranes form interdigitating microvillar projections, whereas the basal cell membranes lack infoldings (Fig. 8A). In females the intermediate segment, consisted of the cuboidal cells. The cell membrane is without basal and lateral infoldings. One round, or oval nuclei, containing one nucleolus, is located towards the basal area. The apical junctional complex consists of a zonula occludens, a zonula adherens, and a desmosome (Fig. 8C). The SEM showed that the IS consisted of shorter cuboidal multiciliated cells and non-ciliated cells. The apical cytoplasm of the latter display a few microvilli (Fig. 8D). 3.5. Distal tubule (Fig. 9) The distal tubules begin near the renal corpuscles and ran mainly within the central zone, but also partly in the peripheral zone. Cell height is measured to 8–16 m. The transition from the distal tubule to collecting ducts is gradual and cannot be discerned as a definite junction. The distal tubule can be subdivided into initial, middle and terminal regions according to differences in ultrastructure of the epithelial cells. The initial region (8–9 m cell high) is composed of low cuboidal cells with a centrally located nucleus with relatively large amounts of heterochromatin, and tubule diameter is 37 m. The apical surface of the cells in this part of the distal tubule has small, irregular microvilli and the lateral and basal cell membranes have extensively well-developed infoldings (Fig. 9A). Elongated mitochondria are associated with the basal plasma membrane infoldings (Fig. 9A and B). In all parts of the distal tubule, the interdigitations of the lateral cell membranes often reach the apical cell membrane and are linked by apical cell junction (Fig. 9A–C). The cytoplasm contains numerous mitochondria and endoplasmic reticulum (Fig. 9A and C). A few lysosomes are often found in the apical cytoplasm. In the initial region, a single central cilium is present on each cell (Fig. 9D). The 11–14 m high cells forming the middle region’s epithelium are characterized by the presence of rod-shaped nuclei with a distinct nucleolus (Fig. 9B). Nuclei is located towards the apical area. The apical surface of the cells has sparse, short microvilli, while lateral and basal cell membranes are extensively infolded, and the region has a very narrow lumen. Elongated mitochondria are associated with the basal plasma membrane infoldings (Fig. 9B). The interdigitations of the lateral cell membranes often reach the apical cell membrane and are linked by numerous apical cell junctions. Mitochondria are mainly found in the apical and
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Fig. 6. (A–D) Proximal tubule cells from the kidney of male soft-shelled turtle. TEM. (A) The lateral intercellular space is narrow beneath the junctional complex and becomes wider towards the base of the cell. (B) The apical brush border consists of microvillar evaginations of the cell. The apical cytoplasm possesses a well-developed endocytoticlysosomal apparatus. Vesicles, absorptive vacuoles, and tubular inclusions are seen within the apical cytoplasm. The cytoplasm of the cells contains a large number of rounded mitochondria. (C) Bald-headed cells. (D) Basal cells. av, apical invagination; bb, brush border; bh, bald-headed cells; bis, basal intercellular space; jc, apical junctional complex; lis, lateral intercellular space; mc, mitochondria; mf, microfilament; nu, nucleus; rer, rough endoplasmic reticulum; va, vacuole; ve, vesicle.
basal regions, relatively rare in the middle region. Apical cytoplasm contains numerous lysosomes (Fig. 9B), but the Golgi apparatus and rough endoplasmic reticulum are poorly developed. The terminal region is composed of higher cuboidal cells with well-developed lateral and basal cell membrane infoldings, but they reach below the apical cell membrane. The cells in this region, are 11–16 m cell high and have a centrally located nucleus (Fig. 9C), and a very narrow lumen is observed. The apical cytoplasm bulged into the tubular lumen. Occasionally, microvilli are found on the apical surface. The lateral interdigitations nearly reach the
apical cell junctions. The cytoplasm of these cells contains numerous mitochondria, ribosomes, rough endoplasmic reticulum, a few vacuoles and lysosomes (Fig. 9C). 3.6. Collecting duct system (Fig. 10) The collecting ducts are formed by columnar cells with a height of 10–18 m. The epithelium of the collecting tubules is composed of clear and dark columnar cells. In the collecting tubule both cell types have basally situated nuclei, which are slightly
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Fig. 7. (A) TEM showing clear cell (c) and dark cell (d) of the proximal tubule in female. bb, brush border; jc, apical junctional complex; mc, mitochondria; nu, nucleus; rer, rough endoplasmic reticulum; ve, vesicle; ly, lysosome; c, clear cell; d, dark cell. (B) SEM micrographs of transverse fracture of the proximal tubule in male. bb, brush border; lis, lateral intercellular space; bis, basal intercellular space.
Fig. 8. The intermediate segment. A ciliated intermediate segment containing numerous multiciliated cells and a few non-ciliated is present in the nephrons of the soft shelled turtle. The cilia have a basal body. (A) TEM. Cells in male have lateral intercellular space. (B and C) TEM. Cells in female. A few mitochondria, lysosome and rough endoplasmatic reticulum is seen within the cytoplasm of the cells. ba, basal body; ci, cilia; nu, nucleus; rer, rough endoplasmic reticulum; no, nucleoli; mv, microvilli; mc, mitochondria; jc, junctional complexes; ly, lysosome; lis, lateral intercellular space; va, vacuole; za, zonula adherens; zo, zonula occludens; de, desmosome. (D) SEM micrograph showing the IS in transverse sections containing multiciliate cells and microvilli cells.
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Fig. 9. P. sinensis (A–C). TEM micrographs of initial (A), middle (B) and terminal regions (C) of the distal tubule illustrating the distal tubule cells. in, infoldings; mc, mitochondria; nu, nucleus; no, nucleolus; rer, rough endoplasmic reticulum; va, vacuole; jc, junctional complexes; ci, cilia; mv, microvilli; ly, lysosome. (D) SEM viewed in transverse sections the initial region of distal tubule.
heterochromatic (Fig. 10A). The clear cells have a round, or oval, indented euchromatinic nucleus with a nucleolus. The cytoplasm comprises a elaborate circularly arranges Golgi complex near the nucleus and scatter rough endoplasmic reticulum (Fig. 10B). The clear cells of the collecting tubule possess regular, sparse microvilli, numerous secretory granules and microtubules in the apical cytoplasm, and a basally situated nucleus (Fig. 10A). The dark cells have a condensed and lobated nucleus and an electron-dense cytoplasm. The nucleus of the dark cell is surrounded by variable numbers of dense elongated or rounded mitochondria with parallel cristae. The cytoplasm of the dark cells contain dense mitochondria, endoplasmic reticulum, polyribosomes and vesicles.
4. Discussion The nephron of the soft-shelled turtle is composed of a renal corpuscle and of a renal tubule that can be divided into five distinct segments: neck segment, proximal tubule, intermediate segment, distal tubule, and collecting duct, This is similar to the nephron composition found in freshwater turtles (Meseguer et al., 1987) and other reptiles (Moore et al., 2009; Solomon, 1985; Gabri, 1983a,b; Davis et al., 1976; Anderson, 1960; Soares and Fava-de-Moraes, 1984; Peek and McMillan, 1979a; Del Conte and Tamayo, 1973) (see Table 1). However, differences are also apparent as seen for example in the presence of the ciliated neck segment. Noticeably,
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Fig. 10. (A) P. sinensis, Collecting duct cell showing the presence of two cell types: clear cells and dark (mitochondria rich) cell type. (B) lateral cell walls interdigitate. The picture shows a circularly arranged Golgi complex in clear cell. TEM. c, clear cell; d, dark cell; jc, apical junctional complex; mc, mitochondria; mv, microvilli; nu, nucleus; no, nucleoli; ly, lysosome; rer, rough endoplasmic reticulum; go, golgi apparatus; ve, vesicle; lis, lateral intercellular space; sg, secretory granules.
the neck segment is not present in all nephrons of the soft-shelled turtle as the parietal layer of Bowman’s capsules in some nephrons connects directly to the proximal tubule. When compared to the nephrons of amphibians (Møbjerg et al., 1998, 2000, 2004; Carvalho and Junqueira, 1999; Sakai and Kawahara, 1983; Taugner et al., 1982; Clothier et al., 1978), birds (Casotti and Braun, 2000; Morild et al., 1985) and mammals (Tsujii et al., 1992; Bankir and de Rouffignac, 1985), there are also obvious differences, it is related to structural features and may make clear functional differences between segments. As discussed below. The renal corpuscle of the soft-shelled turtle is relatively large compared to that of other reptiles with a diameter of approximately 120 m. In freshwater turtles (Meseguer et al., 1987) and in juvenile alligators (Moore et al., 2009) a diameter of 50 m was reported and approximately 60 m in the Garter Snake (Peek and McMillan, 1979a) and lizard (Davis et al., 1976). The mesonephric renal corpuscles of amphibians are relatively large as compared to that of the reptilian metanephros. In the toad Bufo bufo renal corpuscle diameter was 150–200 m and in the Japanese newt a renal corpuscle diameter of 110–140 m was measured (Møbjerg et al., 1998; Sakai and Kawahara, 1983). In the gymnophione Siphonops annulatus the average renal corpuscle diameter was 95 m and in Geotrypetes seraphini a maximal size of 180 × 100 m was measured (Carvalho and Junqueira, 1999; Møbjerg et al., 2004). Exceptionally large renal corpuscles with sizes of to 270 × 370 m have been found in the freshwater gymnophione Typhlonectes compressicauda (see Wake,
1970; Sakai et al., 1986). In the African Lungfish, Protopterus dolloi the size was much smaller, approximately 74–92 m (Ojeda et al., 2006). And in mammals, approximate corpuscle diameters for platypus, rabbit and rat were 112, 150 and 120 m, respectively (Tsujii et al., 1992). When comparing nephron structure among reptiles some differences are seen among species. The parietal cells of Bowman’s capsule of freshwater turtles (Meseguer et al., 1987) may have a cilium. Toward the urinary pole, the parietal cells of the Garter Snake (Peek and McMillan, 1979a,b) bear numerous cilia, while the apical surface of parietal cell in the soft-shelled turtle is smooth and has no cilia. In the Garter Snake the parietal layer cells of the renal corpuscle became cuboidal toward the urinary pole (Peek and McMillan, 1979a). These cells represent transitional stages between the parietal epithelium and neck segment of the tubular nephron. We observed that the parietal layer cells of the soft-shelled turtle have a gradual transition from squamous cells to cuboidal cells. This may be a specific structural features of reptiles. The parietal layer cells of the soft-shelled turtle interdigitate with each other via conspicuous infoldings of the lateral and basal cell membranes, and the fluid movement across the parietal layer cells of the renal corpuscle of reptiles is correlated with the parietal layer intercellular spaces (Schmidt-Nielsen and Davis, 1968). This movement may role of reabsorb. As holds for microvilli that expand the absorptive surface area of cells, the infoldings of the parietal layer cells might be involved in reabsorption of selected elements of the
Table 1 The nephron construction of eight reptiles. Reptiles species
P. sinensis M. caspica1 Testudo graeca2 C. mydas L.3 Alligator mississippiensis4 Phrynosoma5 S. cyanogenys6 Thamnophis sirtalis7
Nephrons construction RC
NS
PT
IS
DT
CS
CT
CD
+ + + + + + + +
+or– + – + + – – +
+ + + + + + + +
+ 3 regions + 2 regions + + 2 parts
3 regions 3 regions + + + + 2 regions +
+ +
+ + +
+ + +
+, present; –, absent; , indicates no mention or no inclusion in literature cited. *present study; 1, Meseguer et al. (1987); 2, Zuasti et al. (1987); 3, Solomon (1985); 4, Moore et al. (2009); 5, Anderson (1960); 6, Davis et al. (1976); 7, Peek et al. (1977); CS, connecting segment; CT, collecting tubule.
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primary urine. This feature of parietal cells, have not previously been suggested in other vertebrate kidneys. A clear difference is observed between ciliated intermediate segments of different reptiles. Meseguer et al. (1987) studying two species of freshwater turtles, Pseudemys scripta elegans and Mauremys caspica, divided this segment into three regions; a middle region and two transition regions representing the gradual transition to respectively the proximal and distal tubules. In these species dark cells lacking cilia were observed among both light and dark cilia bearing cells. In the present study on the soft-shelled turtle, we found an intermediate segment consisting of multiciliated as well as a few non-ciliated cells and a well defined transition between this segment and the proximal and distal tubules. Similar observation have been made in other reptiles. The intermediate segment epithelium in the horned lizard, Phyrnosoma (Anderson, 1960) nephron has multiple ciliated cells and non-ciliated cells, while that of Lizard (Sceloporus cyanogenys) (Gabri, 1983) contains ciliated cells and brush border cells; the middle segment of freshwater turtle contains bright and dark cells with cilia; the middle segment of the green turtle (Chelonia mydas L.) (Solomon, 1985) comprise a initial ciliated region and a terminal mucous region. But cell numbers between bright and dark cells is different from other reptiles. In the present study we divided the distal tubule of P. sinensis into three segments, based primarily on the form and extent of the lateral and basal cell membrane infoldings, the form and location of the nucleus and on cell height (see Figs. 2 and 9). The transition between the three regions of the P. sinensis distal tubule is gradual. The cells forming the initial region have well developed lateral and basal plasma membrane infoldings, which extend to the apical zone of the cytoplasm. The membrane folds of the middle region are similar to that of the initial region, but in the middle region the lateral cell membranes interdigitations are more well-developed. In the terminal region the interdigitations of the lateral cell membrane occupy the cytoplasm under the nucleus. The initial and middle segments of the distal tubule bears the ultrastructural characteristics of the amphibian early distal tubule/diluting segment corresponding to the distal straight tubule/thick ascending limb of mammals (see e.g. Møbjerg et al., 2000; Hillyard et al., 2009). A similar subdivision of the distal tubule was found in the freshwater turtles P. scripta elegans and M. caspica (see Meseguer et al., 1987). The initial region cells have lateral interdigitated processes occupying a very narrow intercellular space. The middle region cells have more developed lateral interdigitated processes than those of the initial region, the intercellular spaces are filled by short and scarce interdigitated processes. In the terminal region of the distal tubule, the intercellular spaces are filled by short and scarce interdigitated processes. The cells of the distal tubule have short, irregular membrane infoldings, which are occasionally seen. The distal tubule of the green turtles (Solomon, 1985) was not divided into regions, the lateral and basal cell plasma membrane infoldings of that are also observed. The plasma membrane of the distal tubule of lizards (Anderson, 1960) is thrown into elaborate folds which interdigitate with similar structures in adjacent cells. The distal tubule of lizards (Gabri, 1983a) has been divided into two portions: a wide and a narrow segments. In the wide segments, the lateral surfaces have numerous projections which interdigitate with those of the adjacent cells, and in some sections these complicated intercellular spaces are located at the base of the cells. However, in narrow part of the distal tubule, the intercellular space has not complex as those of the wider region, and there is no basal plasma membrane infoldings in the distal tubule. The collecting duct of the soft-shelled turtle contains clear and dark cells, and only clear cell cytoplasm contains a number of mucus granules. Lateral intercellular space of collecting tube cells in soft-shelled turtle contains a number of finger-like projections
as lizards (Davis et al., 1976). The collecting tubule of the green turtles (Solomon, 1985) only contained the lateral plasma membrane folds, however it has no mucous granules. Mucous goblets and lateral interdigitated processes could be seen in P. scripta elegans and in M. caspica (Meseguer et al., 1987), but the extension of lateral interdigitated processes is less than that of soft-shelled turtle, and undeveloped than that of soft-shelled turtle. The mucous secretion pobably produce a luminal layer that protects the epithelium from precipitated inorganic salts and thereby facilitating the excretion of insoluble urates (Gabri, 1983b), which related to finger-like projections and lateral plasma membrane folds in the soft-shelled turtle are possess. Sever et al. (2005, 2008) and Del Conte and Tamayo (1973) reported on the renal sexual segment (RSS) in the Cottonmouth Snake (Agkistrodon piscivorous), the Ground Skink, (Scincella laterale) and lizards (Cnemidophorus l. lemniscatus). Mature squamates possess hypertrophied regions of the distal nephrons of male snakes and lizards, the renal sexual segment (RSS). The RSS is believed to provide seminal fluid that mixes with sperm and is released into the female cloaca during coitus (Sever and Hopkins, 2005a,b). The presence of a RSS can be considered a synapomorphy for Squamata and perhaps for Lepidosauria, but it is lacking in turtles (Sever et al., 2008). A long these lines we did not find evidence for such a segment in the soft-shelled turtle. However, in the middle segment and proximal tubule cells of soft-shelled turtle, there is some structure differences between male and female, which had not previously been reported in reptiles kidneys. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. The structure of intermediate segment and proximal tubule epithelial cell basal surface in softshelled turtle had some differences, whether these differences go with the reproductive activity needs further research. Additionally, the size of the intercellular spaces may be important for the material transport, the male proximal tubular reabsorption capacity may be stronger than the female. The glomerular filtration barrier, across which blood is filtered, is formed by the fenestrated capillary endothelium, the extracellular glomerular basement membrane (GBM), and the diaphragm bridged filtration slitsbetween podocyte foot processes. The endothelial pore size in the soft-shelled turtles is 80–185 nm and the thickness of the GBM was measured to 300–600 nm Similar data are not available for other reptiles. In humans, rats and platypus the GBM was measured to 314 ± 98.3 nm, 110–160 nm and 160–250 nm, respectively (Tsujii et al., 1992). In anuran and caecilian amphibians the basement membrane varies between 200 and 600 nm (Møbjerg et al., 1998, 2000, 2004). Endothelial pore size in rats (Fujita et al., 1976), and platypus is 30–150 nm and 40–100 nm; 100–300 nm and 50–200 nm in the caecilian amphibians and toad larvae. Glomerular basement membrane thickness in fish lungfish (Ojeda et al., 2006) is 1 m. The present data demonstrated that the pores of the glomerular endothelium are conspicuously irregular in dimension. Therefore the thickness of GBM and endothelial pore size in the soft-shelled turtle is nearly the same as that of toad larvae. We suggested the status of basement membrane thickness has to do with living environment of animal, or with the animal species. Capillary endothelial pore size is consistent with filtration molecular size, thus, 185 nm of the protein molecules is filtered by the soft-shelled turtle glomerular. The pores of the glomerular endothelium of soft-shelled turtle shows localize distribution characteristics, and its quantities were less which is one of important factors to affect the glomerular filtration rate. The endothermic vertebrate classes (birds and mammals) have significantly higher
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glomerular filtration rates than the ectothermic groups, as the reptiles tending to possess the lowest rates of glomerular filtration (Yaoita et al., 1999). Possibly because of the necessity of the softshelled turtle to conserve water, which is formed further argued by measurement data of glomerular morphological characteristics. The podocytes on the glomerular basement membrane (GBM) are the terminal element in the filtration barrier and display an elaborate morphology. Long cytoplasmic processes extend radially from the cell body and branch into several foot processes, which cover the outer aspect of the GBM. Podocytes in renal glomeruli contribute significantly to these filters’ important properties of hydraulic and macromolecular permeability (Yaoita et al., 1999). The secondary processes of C. mydas L. (Solomon, 1985) is developed, a podocyte only to encircle a single capillary of the glomerular capillaries, therefore, the capillary loop of curved shape can be observed in SEM, while, the podocytes in M. caspica (Meseguer et al., 1987) with short primary processes. The podocyte in P. dolloi (Ojeda et al., 2006) show numerous major cytoplasmic processes that extend over contiguous capillary loops, the podocyte primary processes of the P. dolloi is shorter in SEM, secondary processes and tertiary in the more developed, a podocytes were only attached to a single capillaries. The secondary processes and the tertiary processes in Mammals (Nizze and Csikós, 1980) and birds (Pak Poy and Robertson, 1957) are relatively well developed. A podocyte only encircle a single capillary, therefore, glomerular capillary bending is clearly visible. However, the podocyte proccesses of soft-shelled turtle is well developed, and the whole of glomerular capillary is covered by the podocyte processes. wider and bigger primary processes extend from the podocyte cell body, a little wider secondary processes extend from primary processes, and further extend to a number of little tertiary processes. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. We speculated that a podocyte processes encircles several capillaries. Thus, we can only observe the glomerular surface was covered by the podocyte processes, while a single capillary is seen difficultly. The podocytes of morphological features in soft-shelled turtle, have not previously been reported in other vertebrates kidney. It might be suggested that the podocyte morphological features above had a low filtration rate. Acknowledgments This study was supported by grants from the National Science Foundation of China (No. 31272521) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20110097110012), and Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors are grateful to Ph.D N. MØBJER (Department of Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark) for advice and for kindly supporting English language in the paper. References Anderson, E., 1960. The ultramicroscopic structure of a reptilian kidney. Journal of Morphology 106 (March), 205–241. Bankir, L., de Rouffignac, C., 1985. Urinary concentrating ability: insights from comparative anatomy. American Journal of Physiology 249 (6 Pt 2), R643–R666. Carvalho, E.T., Junqueira, L.C., 1999. Histology of the kidney and urinary bladder of Siphonops annulatus (Amphibia-Gymnophiona). Archives of Histology and Cytology 62 (March (1)), 39–45. Casotti, G., Braun, E.J., 2000. Renal anatomy in sparrows from different environments. Journal of Morphology 243 (March (3)), 283–291. Clothier, R.H., Worley, R.T., Balls, M., 1978 Dec. The structure and ultrastructure of the renal tubule of the Urodele amphibian, Amphiuma means. Journal of Anatomy 127 (Pt 3), 491–504.
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Dantzler, W.H., Holmes, W.N., 1974. Water and mineral metabolism in reptilia. In: Florkin, M., Scheer, B.T. (Eds.), Chemical Zoology. Academic Press, New York, London, pp. 277–377. Dantzler, Bradshaw, 2009. Osmotic and ionic regulation in reptiles. In: Evans, D.H. (Ed.), Osmotic and Ionic Regulation: Cells and Animals. CRC Press, Boca Raton. Dantzler, W.H., Schmidt-Nielsen, B., 1966. Excretion in freshwater turtle (Pseudemys scripta) and desert tortoise (Gopherus agassizii). American Journal of Physiology 210, 198–210. Davis, L.E., Schmidt-Nielsen, B., Stolte, H., 1976. Anatomy and ultrastructure of the excretory system of the lizard Sceloporus cyanogenys. Journal of Morphology 149 (July (3)), 279–326. Del Conte, E., Tamayo, J.G., 1973. Ultrastructure of the sexual segments of the kidney in male and female lizards, Cnemidophorus l. lemniscatus (L.). Zeitschrift für Zellforschung und mikroskopische Anatomie 144 (November (3)), 325–337. Fujita, T., Tokunaga, J., Edanaga, M., 1976. Scanning electron microscopy of the glomerular filtration membrane in the rat kidney. Cell and Tissue Research 166 (February (3)), 299–314. Gabri, M.S., Butler, R.D., 1984. The ultrastructure of the renal corpuscle of a lizard. Tissue Cell 16 (4), 627–634. Gabri, M.S., 1983a. Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) taurica. Journal of Morphology 175, 131–142. Gabri, M.S., 1983b. Seasonal changes in the ultrastructure of the kidney collecting tubule in the lizard Podarcis (= Lacerta) taurica. Journal of Morphology 175, 143–151. Hillyard, S.D., Møbjerg, N., Tanaka, S., Larsen, E.H., 2009. Osmotic and ion regulation in amphibians. In: Evans, D.H. (Ed.), Osmotic and Ionic Regulation: Cells and Animals. , 1st ed. CRC Press, Boca Raton, pp. 367–441. Meseguer, J., Ayala, A.G., Agulleiro, B., 1987. Ultrastructure of the nephron of freshwater turtles, Pseudemys scripta elegans and Mauremys caspica. Cell and Tissue Research 248, 381–391. Moore, B.C., Hyndman, K.A., Cox, A., Lawler, A., Mathavan, K., Guillette Jr., L.J., 2009. Morphology and histochemistry of juvenile American alligator (Alligator mississippiensis) nephrons. The Anatomical Record (Hoboken) 292 (October (10)), 1670–1676. Morild, I., Bohle, A., Christensen, J.A., 1985. Structure of the avian kidney. Anatomical Record 212 (May (1)), 33–40. Møbjerg, N., Jespersen, A., Wilkinson, M., 2004. Morphology of the kidney in the West African caecilian, Geotrypetes seraphini (Amphibia, Gymnophiona, Caeciliidae). Journal of Morphology 262 (November (2)), 583–607. Møbjerg, N., Larsen, E.H., Jespersen, A., 2000. Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae). Journal of Morphology 245 (September (3)), 177–195. Møbjerg, N., Larsen, E.H., Jespersen, Å., 1998. Morphology of the nephron in the mesonephros of Bufo bufo (Amphibia, Anura, Bufonidae). Acta Zoologica (Stockholm) 79, 31–51. Nizze, H., Csikós, A., 1980. Scanning electron microscopy of the rat renal glomerulus. International Urology and Nephrology 12 (1), 3–14. Ojeda, J.L., Icardo, J.M., Wong, W.P., Ip, Y.K., 2006. Microanatomy and ultrastructure of the kidney of the African lungfish Protopterus dolloi. The Anatomical Record. Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 288 (June (6)), 609–625. Pak Poy, R.K., Robertson, J.S., 1957. Electron microscopy of the avian renal glomerulus. Journal of Biophysical and Biochemical Cytology 3 (March (2)), 183–192. Peek, W.D., McMillan, D.B., 1979a. Ultrastructure of the renal corpuscle of the garter snake Thamnophis sirtalis. American Journal of Anatomy 155 (May (1)), 83–102. Peek, W.D., McMillan, D.B., 1979b. Ultrastructure of the tubular nephron of the garter snake Thamnophis sirtalis. American Journal of Anatomy 154 (January (1)), 103–127. Peek, W.D., Shivers, R.R., McMillan, D.B., 1977. Freeze-fracture analysis of junctional complexes in the nephron of the garter snake Thamnophis sirtalis. Cell and Tissue Research 179 (April (4)), 441–451. Sakai, T., Billo, R., Kritz, W., 1986. The structural organization of the kidney of Typhlonectes compressicaudus (Amphibia, Gymnophiona). Anatomy and Embryology 174, 243–252. Sakai, T., Kawahara, K., 1983. The structure of the kidney of Japanese newts, Triturus (Cynops) pyrrhogaster. Anatomy and Embryology (Berlin) 166 (1), 31–52. Sever, D.M., Hopkins, W.A., 2005a. Renal sexual segment of the ground skink, Scincella laterale (Reptilia, Squamata, Scincidae). Journal of Morphology 266, 46–59. Sever, D.M., Siegel, D.S., Bagwill, A., Eckstut, M.E., Alexander, L., Camus, A., Morgan, C., 2008. Renal sexual segment of the cottonmouth snake, Agkistrodon piscivorous (Reptilia, Squamata, Viperidae). Journal of Morphology 269 (June (6)), 640– 653. Sever, D.M., Hopkins, W.A., 2005b. Renal sexual segment of the ground skink, Scincella laterale (Reptilia, Squamata, Scincidae). Journal of Morphology 266 (October (1)), 46–59. Schmidt-Nielsen, B., Skadhauge, E., 1967. Function of the excretory system of the crocodile (Crocodylus acutus). American Journal of Physiology 212 (May (5)), 973–980. Schmidt-Nielsen, B., Davis, L.E., 1968. Fluid transport and tubular intercellular spaces in reptilian kidneys. Science 159 (March (819)), 1105–1108. Soares, A.M., Fava-de-Moraes, F., 1984. Morphological and morphometrical study of the kidney of the male tropical lizard Tropidurus torquatus. Anatomischer Anzeiger 157 (5), 365–373. Solomon, S.E., 1985. The morphology of the kidney of the green turtle (Chelonia mydas L). Journal of Anatomy 140, 355–369.
462
C.-S. Xu et al. / Micron 44 (2013) 451–462
Taugner, R., Schiller, A., Ntokalou-Knittel, S., 1982. Cells and intercellular contacts in glomeruli and tubules of the frog kidney. A freeze-fracture and thin-section study. Cell and Tissue Research 226 (3), 589–608. Tsujii, T., Inoue, S., Takamiya, H., Liszczynsky, H.R., Naora, H., Seno, S., 1992. Morphology of the kidney of the platypus (Ornithorhynchus anatinus: Monotremata). The Anatomical Records 234 (3), 348–358. Wake, M.H., 1970. Evolutionary morphology of the caecilian urogenital system. II. The kidneys and urogenital ducts. Acta Anatomica 75, 321–358.
Yaoita, E., Franke, W.W., Yamamoto, T., Kawasaki, K., Kihara, I., 1999. Identification of renal podocytes in multiple species: higher vertebrates are vimentin positive/lower vertebrates are desmin positive. Histochemistry and Cell Biology 111 (February (2)), 107–115. Zuasti, A., Ferrer, C., Ballesta, J., Pastor, L.M., 1987. Ultrastructure of the tubular nephron of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals. Histology and Histopathology 2 (October (4)), 391–400.
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Ultrastructure of the nephron in the soft-shelled turtle, Pelodiscus sinensis (Reptilia, Chelonia, Trionychidae) Chun-Sheng Xu a,b , Ping Yang a , Hui-Jun Bao a , Xun-Guang Bian a , Qiu-Sheng Chen a,∗ a b
Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People’s Republic of China College of Animal Science and Technology, Shihezi University, Shihezi, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 September 2012 Accepted 4 October 2012 Keywords: Kidney Soft-shelled turtle Podocyte Nephron Ultrastructure
a b s t r a c t The structure of the nephron in adult soft-shelled turtles (Pelodiscus sinensis) was studied by light microscopy, transmission and scanning electron microscopy. The kidney contained 5–6 renal lobes. Nephrons of P. sinensis are composed of a renal corpuscle (RC) and of a renal tubule that appears divided morphologically into five distinct segments: neck segment (NS) (This segment is only present in approximately 10% of the nephrons), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The RCs and most of the convoluted DTs lie in the central zone, while the PTs and the CDs lie in the peripheral zone of the renal lobe. The renal corpuscle is relatively large with especial processes in podocytes and a thick basement membrane. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. The neck segment when presentis short and has a relatively narrow lumen, consisting of cuboidal or squamous cells. There is a well-developed endocytic-lysosomal apparatus in the apical cytoplasm of the PT. The proximal tubule and intermediate segment cells show some differences between male and female. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. Not previously reported in vertebrate kidneys. The IS is the narrowest nephron segment, formed by multiciliated as well as nonciliated cells. In DT cells, basolateral interdigitations and infoldings are particularly well-developed. The CD contains clear cells with numerous secretory granules and dark cells with dense mitochondria and an elaborate Golgi complex. This study was undertaken in order to disclose specific kidney features in P. sinensis that could be related to function. In addition, the ultrastructure of the nephrons in P. sinensis are discussed in relation to other turtles and vertebrates. © 2012 Published by Elsevier Ltd.
1. Introduction As holds for all vertebrates, the main function of the reptilian kidney is to eliminate waste products and maintain salt and water balance by regulating the composition of the body fluids (Solomon, 1985). In all vertebrates, the kidney has the nephron as the basic structural and functional unit. The reptilian nephron lacks a loop of Henle characteristic of kidneys that produce concentrated urine and hence reptiles cannot produce urine hyperosmotic to plasma. The basic components of the reptilian nephron are: a renal corpuscle and a renal tubule consisting of a short ciliated neck
∗ Corresponding author. Tel.: +86 25 84398669; fax: +86 25 84398669. E-mail address: [email protected] (Q.-S. Chen). 0968-4328/$ – see front matter © 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.micron.2012.10.001
segment, a proximal tubule, a short ciliated thin intermediate segment and a distal tubule, the latter opening into the collecting duct (Dantzler and Bradshaw, 2009). Urinary excretion of ions and water involves regulation of glomerular filtration as well as tubular reabsorption and secretion (Dantzler and Holmes, 1974). Studies made on different species among the orders of living reptiles have demonstrated structural variations among their nephrons (Meseguer et al., 1987; Solomon, 1985; Gabri and Butler, 1984; Gabri, 1983a,b; Schmidt-Nielsen and Davis, 1968) In the present study we investigate nephron structure in the Chinese Soft Shelled Turtle, Pelodiscus sinensis, which lives in a wide range of habitats in China and its kidneys have some structural differences when compared to those of other reptiles and vertebrates. Kidney structure has only been scantily documented in this species and considering previous physiological reports (Dantzler and Schmidt-Nielsen,
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1966; Schmidt-Nielsen and Skadhauge, 1967; Dantzler and Holmes, 1974), data on renal ultrastructure of reptiles, mainly Chelonia, lizards, and crocodiles could be morphological and physiological significant. In the present paper, we highlight the ultrastructure of nephrons in P. sinensis and discuss the functional significance of the findings. The morphological features have been compared to those of other vertebrates, mainly in other orders of reptiles. The present study, employing both light microscopy based reconstructions and electron microscopy, considerably improves the structural understanding of the reptilian kidney. This work is a contribution to the knowledge of the kidney of P. sinensis, frequently used as an experimental model for physiological and biochemical studies. 2. Materials and methods Twenty male and female adult soft-shelled turtles, Pelodiseus sinensi, weighing 850–950 g/each, were purchased from a wild breeding base in the Jiangsu province of China at the beginning of May. The animals were anesthetized with ethyl ether or chloroform and their ventral caparaces removed. Tissue was subsequently removed for light microscopy, transmission electron microscopy and scanning electron microscopy. All protocols were approved by the Chinese Committee for Animal Use for Research and Education.
Fig. 1. P. sinensis. Transverse sections of the renal lobe. Paraffin sections, 5 m, stained with hematoxylin–eosin. When viewed in transverse sections the kidney can be divided into a central zone (cz) and two peripheral zones (pz). The central zone is characterized by the presence of renal corpuscles, distal tubules, and intermediate segments. The peripheral zones are dominated by the presence of proximal tubules and the collecting duct. cd, collecting duct; dt, distal tubules; iv, intralobular vein; ra, renal artery; g, glomerulus; pt, proximal tubule; fc, fibrous capsule.
2.1. Light microscopy 3. Results Tissue fixed in 10% cold formalin PBS solution was rinsed for 1 h in tap water and then dehydrated by placing the tissue in a graded series of ethyl alcohol (70, 85, 95, and 100% for an hour each). After dehydration, the tissue was embedded in paraffin blocks for sectioning (5 m) with a Leica RM2015 microtome. Alternate slides were stained with hematoxylin–eosin (for general cytology). 2.2. Transmission electron microscopy (TEM) Blocks (1 mm3 ) of tissue were fixed by immersion for 5 h at 4 ◦ C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, then postfixed in 1% buffered osmium tetroxide and embedded in araldite. Ultrathin sections were obtained using a LKB III ultramicrotome, stained with uranyl acetate and lead citrate and examined in a JEM-1200EX electron microscope. 2.3. Scanning electron microscopy (SEM) Slices of tissue 1 mm thick were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate, bufferwashed (pH 7.2) and dehydrated through a series of acetones. The material was critically point-dried in CO2 and sputter-coated with gold/palladium for 3 min in an Emscope sputter coater. Blocks were viewed using a Philips 501B scanning electron microscope. 2.4. Reconstruction of nephrons Serial transverse and longitudinal sections of the araldite- and the paraffin-embedded kidney tissue were used for graphic reconstructions of nephrons. The reconstructions were performed using a light microscope to trace the course of the nephron segments in successive sections. The diameter of the different segments of the nephron was determined from TEM pictures of the specimens by using the TEM m markers as calibration. The magnification of the drawing was determined by means of a calibrated microscope slide. The thickness of the different components of the glomerular basement membrane (GBM) was measured on TEM micrographs.
3.1. Kidney zonation and nephron segmentation The paired kidneys of the soft-shelled turtle are hemicycle in shape, lobed bodies closely applied to the posterior wall of the pleuroperitoneal cavity. They are metanephric in origin and collecting ducts empty into the ureter in the ventral surface center of each kidney. When viewed in transverse sections the renal lobe can be divided into a central zone and two peripheral zones (Fig. 1). The central zone is characterized by the presence of renal corpuscles, distal tubules, and intermediate segments. The perpheral zones are dominated by the presence of proximal tubules and the collecting duct system. The TEM analysis of the specimens allowed identification of the following divisions within the nephron: the renal corpuscle, the neck segment, the proximal tubule, the intermediate segment, the distal tubule and the collecting duct. The renal corpuscle is large and consisted of a glomerulus (capillary endothelium, glomerular basement membrane and mesangium) and Bowman’s capsule (visceral layer and parietal layer). The main features of the P. sinensi nephron are summarized in Fig. 2. Reconstructions of the nephron, were made from serial transverse and longitudinal sections. The spatial location of the different nephron components allowed recognition of a renal lobe with three main divisions (Fig. 1): a central zone and two peripheral zones. The transverse sections ratio of proximal and distal tubules was 2:1 by counting a single female or male lobe. 3.2. Renal corpuscle and neck segment (Figs. 3–5) The P. sinensis renal corpuscle is roughly ovoid in shape, with a diameter of 120 m (n = 7) and a smooth outer surface. The renal corpuscle is formed by two structures: the glomerulus formed by blood capillaries and the capsule of Bowman, which surrounds the glomerulus. The latter is composed of a visceral layer and a parietal layer with a urinary space in between (Fig. 4B). The urinary pole of the RC is continuous with either a small the neck segment or the
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Fig. 2. Schematic representation of the nephron in P. sinensis in which the tubules are spread apart. The figure moreover provides a scheme of cell types from proximal tubule (A–C); intermediate segment (D and E); distal tubule (F–H) and collecting duct (I and J). Male proximal tubule (A); female proximal tubule clear cell (B) and dark cell (C); male intermediate segment (D); female intermediate segment (E); distal tubule initial (F), middle (G) and terminal (H) regions; collecting duct dark cell (I) and clear cell (J). ba, basal body; bm, basement membrane; ci, cilium; rer, rough endoplasmic reticulum; mc, mitochondria; mv, microvilli; nu, nucleus; no, nucleolus; ly, lysosome; sg, secretory granules; in, infold; va, vacuole; g, glomerulus; ns, neck segment; pt, proximal tubule; is, intermediate segment; dt, distal tubule; cd, collecting duct.
PT (Fig. 3). None of the usual components of the juxtaglomerular apparatus (juxtaglomerular cells and macula densa) are observed in our material. The epithelium of the parietal layer is composed of fusiform shaped squamous cells. The cells have a height of 2–4 m and bulge slightly into the urinary space (Fig. 4A). A large and irregular
Fig. 3. Paraffin section 5 m, stained with HE. In some nephrons a small neck segment (ns) is present, connecting the urinary pole to the proximal tubule. Central zone of a renal lobe showing renal corpuscles with glomerulus (g), neck segment (ns) andproximal tubule (pt), distal tubule (dt), intralobular vein (iv).
nucleus is situated centrally in the cell. The cytoplasm moreover contains a number of scattered rough endoplasmic reticulum, free ribosomes and a few rounded mitochondria. Lateral and basal intercellular spaces contain microvillar extensions of the lateral membrane. The intercellular spaces are narrow beneath the apical junctional complex. The cells constituting the parietal layer epithelium gradually become cuboidal towards the urinary pole, where they merge either with a small NS or directly with the PT (Fig. 3). NS is present in approximately 10% of the nephrons in male and female kidney (Fig. 3A). When present, the NS is the shortest segment of the renal tubule. Podocytes form the epithelium of the capsule’s visceral layer. They contain an indented nucleus and send out cell processes. The podycyte cell body sends out wide and flat primary processes, the latter stretch out to form secondary processes that eventually extend into a few tertiary processes. The finest branches of the cell, are the pedicels or foot processes (Fig. 4C). The alternating foot processes rest on a 300–600 nm thick basement membrane which separates the podocytes from the juxtaposed fenestrated capillary endothelium (Fig. 4B). Between the foot processes are narrow filtration slits which are bridges by a diaphragm (Fig. 4B insert). Transmission electron microscopy demonstrated a large indentation nucleus within the podocyte cell body, often with peripherally clumped chromatin. The cytoplasm contain small mitochondria, a Golgi complex, lysosomes, numerous free ribosomes and granular endoplasmic reticulum. These organelles extended into the terminal foot processes see Fig. 5. The glomerular basement membrane consists of a lamina rara beneath the podocyte processes, a central lamina densa (basal lamina), and a lamina rara bordering the endothelium of the
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Fig. 4. (A) Parietal layer of renal capsule. Cells contain lateral intercellular spaces and basal labyrinth (B). The filtration barrier of the renal corpuscle in the soft-shelled turtle. The podocytes consist of cell bodies containing the indented nucleus that send out cell processes with foot processes, all of which rest on the glomerular basement membrane. Insert: filtration slits with diaphragm. (C) The epithelium of the glomerular capillary is composed of cell bodies that bulge into the capillary lumen and send out cell processes that line the blood side of the glomerular basement membrane. (D) Few mesangial cell. TEM. Pl, parietal layer; bm, basement membrane; en, endothelial cell; fp, foot processes; fe, fenestration; nu, nucleus; no, nucleolus; po, podocyte; rer, rough endoplasmic reticulum; e, erythrocyte; ca, capillary space; us, urinary space; go, golgi apparatus; mc, mitochondria; jc, junctional complexes; lis, lateral intercellular space; bis, basal intercellular space; ecp, endothelial cell process; me, mesangial cell; pp, primary processes; sp, secondary processes; tp, third processes.
glomerular capillaries. The interna lamina rara and lamina densa together have a constant thickness of 200–350 nm (Fig. 4A–D). The capillary endothelium is composed of fenestrated endothelial cells with a large nucleus (Fig. 4C). The cytoplasm of these cells contain numerous free ribosomes sparse rough endoplasmic reticulum and vacuoles. This epithelium is composed of cell bodies that bulge into the capillary lumen and send out cell processes (Fig. 4C). These processes line the basement membrane and form the fenestrations of the endothelium (80–185 nm in size) (Fig. 4B). In the
soft-shelled turtle, this endothelium usually presented a reticulated appearance; fenestrated regions are occasionally found (Fig. 4B). Few thin mesangial cells are distinguishable at the base of the capillary loops (Fig. 4D). 3.3. Proximal tubule (Figs. 6 and 7) In the soft-shelled turtle, the proximal tubule showed some differences between specimens. In female soft-shelled turtles (n = 10)
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Fig. 5. (A) SEM micrograph showing a renal corpuscle. Partial elimination of Bowman’s capsule allows observation of the glomerular capillary tufts. The afferent arteriole ( ); the efferent arteriole ( ); the efferent arterioles often have a smaller diameter than the afferent arteriole. The parietal epithelium of Bowman’s capsule ( ); Bowman’s capsule ( ); podocyte cell body ( ). (B) Detailed SEM view of a glomerulus showing podocyte morphology with developed primary processes. pcb, podocyte cell body; pp, primary processes; sp, secondary processes; tp, tertiary processes.
the tubules are heterocellular, composed of large, clear and dark, higher columnar cells (Fig. 7A). They have long microvilli forming the brush border characteristic of this segment, and a round nucleus located towards the center area (Fig. 7A). In the females, the dark cells appeared as single intercalated cells between the clear cells. In males (n = 10), the proximal tubules contain tree cell type: cells with microvilli, bald-headed cells (Fig. 6C) and basal cells (Fig. 6D). The cytoplasm of proximale tubule cells in both males and females is dominated by a large numbers of rounded mitochondria, numerous lysosomes and scattered endoplasmic reticulum. Between adjacent cells, junctional complexes are found just beneath the free surface (Figs. 6A, B and 7A). In the females investigated, the lateral cell membranes are straight between neighboring cells and no intercellular spaces are observed (Fig. 7A). While, in the males, lateral intercellular spaces are present and the basal cell membranes formed a labyrinth (Fig. 6A). In the males clear basal cells and bald-headed cells can occasionally be observed. The former is restricted to the basal region of the epithelium; nuclei are located towards the center area of the cells. The latter appeares as single intercalated cells between the microvilli bearing cells. The apical surfaces of the bald-headed cell carried few microvilli. Endoplasmic reticulum, mitochondria andvesicles of various sizes are scattered throughout the cytoplasm of these cells. The average cell height of the proximal tubule cells is 20 m. The average external diameter of the proximal tubule is 60 m. SEM showed that the PT contain, in transverse sections, the PT cells are tall pyramidal or columnar in shape. The cells exhibit a brush border of microvilli and lateral and basal intercellular spaces are evident in the SEM preparation (Fig. 7B). 3.4. Intermediate segment (Fig. 8) The IS is a short, straight, well-defined segment. The transition from the proximal tubule is defined by a considerable decrease in the cell height (Fig. 8A and B). The intermediate segment contain numerous multiciliated cells and a few non-ciliated. The apical surface of non-ciliated cells are covered with dense short microvilli. The epithelium of this multiciliated segment consisted of 8–10 m high cuboidal cells with basal located nuclei (Fig. 8A–C). The cells have an irregular or indented nucleus and few organelles (Fig. 8A). The apical cytoplasm in the multiciliated cells is characterized by the presence of basal bodies. Some mitochondria, lysosomes and vacuoles are found in the cytoplasm (Fig. 8A and B).
The IS in male and female showed some differences. In male, the lateral cell membranes form interdigitating microvillar projections, whereas the basal cell membranes lack infoldings (Fig. 8A). In females the intermediate segment, consisted of the cuboidal cells. The cell membrane is without basal and lateral infoldings. One round, or oval nuclei, containing one nucleolus, is located towards the basal area. The apical junctional complex consists of a zonula occludens, a zonula adherens, and a desmosome (Fig. 8C). The SEM showed that the IS consisted of shorter cuboidal multiciliated cells and non-ciliated cells. The apical cytoplasm of the latter display a few microvilli (Fig. 8D). 3.5. Distal tubule (Fig. 9) The distal tubules begin near the renal corpuscles and ran mainly within the central zone, but also partly in the peripheral zone. Cell height is measured to 8–16 m. The transition from the distal tubule to collecting ducts is gradual and cannot be discerned as a definite junction. The distal tubule can be subdivided into initial, middle and terminal regions according to differences in ultrastructure of the epithelial cells. The initial region (8–9 m cell high) is composed of low cuboidal cells with a centrally located nucleus with relatively large amounts of heterochromatin, and tubule diameter is 37 m. The apical surface of the cells in this part of the distal tubule has small, irregular microvilli and the lateral and basal cell membranes have extensively well-developed infoldings (Fig. 9A). Elongated mitochondria are associated with the basal plasma membrane infoldings (Fig. 9A and B). In all parts of the distal tubule, the interdigitations of the lateral cell membranes often reach the apical cell membrane and are linked by apical cell junction (Fig. 9A–C). The cytoplasm contains numerous mitochondria and endoplasmic reticulum (Fig. 9A and C). A few lysosomes are often found in the apical cytoplasm. In the initial region, a single central cilium is present on each cell (Fig. 9D). The 11–14 m high cells forming the middle region’s epithelium are characterized by the presence of rod-shaped nuclei with a distinct nucleolus (Fig. 9B). Nuclei is located towards the apical area. The apical surface of the cells has sparse, short microvilli, while lateral and basal cell membranes are extensively infolded, and the region has a very narrow lumen. Elongated mitochondria are associated with the basal plasma membrane infoldings (Fig. 9B). The interdigitations of the lateral cell membranes often reach the apical cell membrane and are linked by numerous apical cell junctions. Mitochondria are mainly found in the apical and
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Fig. 6. (A–D) Proximal tubule cells from the kidney of male soft-shelled turtle. TEM. (A) The lateral intercellular space is narrow beneath the junctional complex and becomes wider towards the base of the cell. (B) The apical brush border consists of microvillar evaginations of the cell. The apical cytoplasm possesses a well-developed endocytoticlysosomal apparatus. Vesicles, absorptive vacuoles, and tubular inclusions are seen within the apical cytoplasm. The cytoplasm of the cells contains a large number of rounded mitochondria. (C) Bald-headed cells. (D) Basal cells. av, apical invagination; bb, brush border; bh, bald-headed cells; bis, basal intercellular space; jc, apical junctional complex; lis, lateral intercellular space; mc, mitochondria; mf, microfilament; nu, nucleus; rer, rough endoplasmic reticulum; va, vacuole; ve, vesicle.
basal regions, relatively rare in the middle region. Apical cytoplasm contains numerous lysosomes (Fig. 9B), but the Golgi apparatus and rough endoplasmic reticulum are poorly developed. The terminal region is composed of higher cuboidal cells with well-developed lateral and basal cell membrane infoldings, but they reach below the apical cell membrane. The cells in this region, are 11–16 m cell high and have a centrally located nucleus (Fig. 9C), and a very narrow lumen is observed. The apical cytoplasm bulged into the tubular lumen. Occasionally, microvilli are found on the apical surface. The lateral interdigitations nearly reach the
apical cell junctions. The cytoplasm of these cells contains numerous mitochondria, ribosomes, rough endoplasmic reticulum, a few vacuoles and lysosomes (Fig. 9C). 3.6. Collecting duct system (Fig. 10) The collecting ducts are formed by columnar cells with a height of 10–18 m. The epithelium of the collecting tubules is composed of clear and dark columnar cells. In the collecting tubule both cell types have basally situated nuclei, which are slightly
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Fig. 7. (A) TEM showing clear cell (c) and dark cell (d) of the proximal tubule in female. bb, brush border; jc, apical junctional complex; mc, mitochondria; nu, nucleus; rer, rough endoplasmic reticulum; ve, vesicle; ly, lysosome; c, clear cell; d, dark cell. (B) SEM micrographs of transverse fracture of the proximal tubule in male. bb, brush border; lis, lateral intercellular space; bis, basal intercellular space.
Fig. 8. The intermediate segment. A ciliated intermediate segment containing numerous multiciliated cells and a few non-ciliated is present in the nephrons of the soft shelled turtle. The cilia have a basal body. (A) TEM. Cells in male have lateral intercellular space. (B and C) TEM. Cells in female. A few mitochondria, lysosome and rough endoplasmatic reticulum is seen within the cytoplasm of the cells. ba, basal body; ci, cilia; nu, nucleus; rer, rough endoplasmic reticulum; no, nucleoli; mv, microvilli; mc, mitochondria; jc, junctional complexes; ly, lysosome; lis, lateral intercellular space; va, vacuole; za, zonula adherens; zo, zonula occludens; de, desmosome. (D) SEM micrograph showing the IS in transverse sections containing multiciliate cells and microvilli cells.
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Fig. 9. P. sinensis (A–C). TEM micrographs of initial (A), middle (B) and terminal regions (C) of the distal tubule illustrating the distal tubule cells. in, infoldings; mc, mitochondria; nu, nucleus; no, nucleolus; rer, rough endoplasmic reticulum; va, vacuole; jc, junctional complexes; ci, cilia; mv, microvilli; ly, lysosome. (D) SEM viewed in transverse sections the initial region of distal tubule.
heterochromatic (Fig. 10A). The clear cells have a round, or oval, indented euchromatinic nucleus with a nucleolus. The cytoplasm comprises a elaborate circularly arranges Golgi complex near the nucleus and scatter rough endoplasmic reticulum (Fig. 10B). The clear cells of the collecting tubule possess regular, sparse microvilli, numerous secretory granules and microtubules in the apical cytoplasm, and a basally situated nucleus (Fig. 10A). The dark cells have a condensed and lobated nucleus and an electron-dense cytoplasm. The nucleus of the dark cell is surrounded by variable numbers of dense elongated or rounded mitochondria with parallel cristae. The cytoplasm of the dark cells contain dense mitochondria, endoplasmic reticulum, polyribosomes and vesicles.
4. Discussion The nephron of the soft-shelled turtle is composed of a renal corpuscle and of a renal tubule that can be divided into five distinct segments: neck segment, proximal tubule, intermediate segment, distal tubule, and collecting duct, This is similar to the nephron composition found in freshwater turtles (Meseguer et al., 1987) and other reptiles (Moore et al., 2009; Solomon, 1985; Gabri, 1983a,b; Davis et al., 1976; Anderson, 1960; Soares and Fava-de-Moraes, 1984; Peek and McMillan, 1979a; Del Conte and Tamayo, 1973) (see Table 1). However, differences are also apparent as seen for example in the presence of the ciliated neck segment. Noticeably,
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Fig. 10. (A) P. sinensis, Collecting duct cell showing the presence of two cell types: clear cells and dark (mitochondria rich) cell type. (B) lateral cell walls interdigitate. The picture shows a circularly arranged Golgi complex in clear cell. TEM. c, clear cell; d, dark cell; jc, apical junctional complex; mc, mitochondria; mv, microvilli; nu, nucleus; no, nucleoli; ly, lysosome; rer, rough endoplasmic reticulum; go, golgi apparatus; ve, vesicle; lis, lateral intercellular space; sg, secretory granules.
the neck segment is not present in all nephrons of the soft-shelled turtle as the parietal layer of Bowman’s capsules in some nephrons connects directly to the proximal tubule. When compared to the nephrons of amphibians (Møbjerg et al., 1998, 2000, 2004; Carvalho and Junqueira, 1999; Sakai and Kawahara, 1983; Taugner et al., 1982; Clothier et al., 1978), birds (Casotti and Braun, 2000; Morild et al., 1985) and mammals (Tsujii et al., 1992; Bankir and de Rouffignac, 1985), there are also obvious differences, it is related to structural features and may make clear functional differences between segments. As discussed below. The renal corpuscle of the soft-shelled turtle is relatively large compared to that of other reptiles with a diameter of approximately 120 m. In freshwater turtles (Meseguer et al., 1987) and in juvenile alligators (Moore et al., 2009) a diameter of 50 m was reported and approximately 60 m in the Garter Snake (Peek and McMillan, 1979a) and lizard (Davis et al., 1976). The mesonephric renal corpuscles of amphibians are relatively large as compared to that of the reptilian metanephros. In the toad Bufo bufo renal corpuscle diameter was 150–200 m and in the Japanese newt a renal corpuscle diameter of 110–140 m was measured (Møbjerg et al., 1998; Sakai and Kawahara, 1983). In the gymnophione Siphonops annulatus the average renal corpuscle diameter was 95 m and in Geotrypetes seraphini a maximal size of 180 × 100 m was measured (Carvalho and Junqueira, 1999; Møbjerg et al., 2004). Exceptionally large renal corpuscles with sizes of to 270 × 370 m have been found in the freshwater gymnophione Typhlonectes compressicauda (see Wake,
1970; Sakai et al., 1986). In the African Lungfish, Protopterus dolloi the size was much smaller, approximately 74–92 m (Ojeda et al., 2006). And in mammals, approximate corpuscle diameters for platypus, rabbit and rat were 112, 150 and 120 m, respectively (Tsujii et al., 1992). When comparing nephron structure among reptiles some differences are seen among species. The parietal cells of Bowman’s capsule of freshwater turtles (Meseguer et al., 1987) may have a cilium. Toward the urinary pole, the parietal cells of the Garter Snake (Peek and McMillan, 1979a,b) bear numerous cilia, while the apical surface of parietal cell in the soft-shelled turtle is smooth and has no cilia. In the Garter Snake the parietal layer cells of the renal corpuscle became cuboidal toward the urinary pole (Peek and McMillan, 1979a). These cells represent transitional stages between the parietal epithelium and neck segment of the tubular nephron. We observed that the parietal layer cells of the soft-shelled turtle have a gradual transition from squamous cells to cuboidal cells. This may be a specific structural features of reptiles. The parietal layer cells of the soft-shelled turtle interdigitate with each other via conspicuous infoldings of the lateral and basal cell membranes, and the fluid movement across the parietal layer cells of the renal corpuscle of reptiles is correlated with the parietal layer intercellular spaces (Schmidt-Nielsen and Davis, 1968). This movement may role of reabsorb. As holds for microvilli that expand the absorptive surface area of cells, the infoldings of the parietal layer cells might be involved in reabsorption of selected elements of the
Table 1 The nephron construction of eight reptiles. Reptiles species
P. sinensis M. caspica1 Testudo graeca2 C. mydas L.3 Alligator mississippiensis4 Phrynosoma5 S. cyanogenys6 Thamnophis sirtalis7
Nephrons construction RC
NS
PT
IS
DT
CS
CT
CD
+ + + + + + + +
+or– + – + + – – +
+ + + + + + + +
+ 3 regions + 2 regions + + 2 parts
3 regions 3 regions + + + + 2 regions +
+ +
+ + +
+ + +
+, present; –, absent; , indicates no mention or no inclusion in literature cited. *present study; 1, Meseguer et al. (1987); 2, Zuasti et al. (1987); 3, Solomon (1985); 4, Moore et al. (2009); 5, Anderson (1960); 6, Davis et al. (1976); 7, Peek et al. (1977); CS, connecting segment; CT, collecting tubule.
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primary urine. This feature of parietal cells, have not previously been suggested in other vertebrate kidneys. A clear difference is observed between ciliated intermediate segments of different reptiles. Meseguer et al. (1987) studying two species of freshwater turtles, Pseudemys scripta elegans and Mauremys caspica, divided this segment into three regions; a middle region and two transition regions representing the gradual transition to respectively the proximal and distal tubules. In these species dark cells lacking cilia were observed among both light and dark cilia bearing cells. In the present study on the soft-shelled turtle, we found an intermediate segment consisting of multiciliated as well as a few non-ciliated cells and a well defined transition between this segment and the proximal and distal tubules. Similar observation have been made in other reptiles. The intermediate segment epithelium in the horned lizard, Phyrnosoma (Anderson, 1960) nephron has multiple ciliated cells and non-ciliated cells, while that of Lizard (Sceloporus cyanogenys) (Gabri, 1983) contains ciliated cells and brush border cells; the middle segment of freshwater turtle contains bright and dark cells with cilia; the middle segment of the green turtle (Chelonia mydas L.) (Solomon, 1985) comprise a initial ciliated region and a terminal mucous region. But cell numbers between bright and dark cells is different from other reptiles. In the present study we divided the distal tubule of P. sinensis into three segments, based primarily on the form and extent of the lateral and basal cell membrane infoldings, the form and location of the nucleus and on cell height (see Figs. 2 and 9). The transition between the three regions of the P. sinensis distal tubule is gradual. The cells forming the initial region have well developed lateral and basal plasma membrane infoldings, which extend to the apical zone of the cytoplasm. The membrane folds of the middle region are similar to that of the initial region, but in the middle region the lateral cell membranes interdigitations are more well-developed. In the terminal region the interdigitations of the lateral cell membrane occupy the cytoplasm under the nucleus. The initial and middle segments of the distal tubule bears the ultrastructural characteristics of the amphibian early distal tubule/diluting segment corresponding to the distal straight tubule/thick ascending limb of mammals (see e.g. Møbjerg et al., 2000; Hillyard et al., 2009). A similar subdivision of the distal tubule was found in the freshwater turtles P. scripta elegans and M. caspica (see Meseguer et al., 1987). The initial region cells have lateral interdigitated processes occupying a very narrow intercellular space. The middle region cells have more developed lateral interdigitated processes than those of the initial region, the intercellular spaces are filled by short and scarce interdigitated processes. In the terminal region of the distal tubule, the intercellular spaces are filled by short and scarce interdigitated processes. The cells of the distal tubule have short, irregular membrane infoldings, which are occasionally seen. The distal tubule of the green turtles (Solomon, 1985) was not divided into regions, the lateral and basal cell plasma membrane infoldings of that are also observed. The plasma membrane of the distal tubule of lizards (Anderson, 1960) is thrown into elaborate folds which interdigitate with similar structures in adjacent cells. The distal tubule of lizards (Gabri, 1983a) has been divided into two portions: a wide and a narrow segments. In the wide segments, the lateral surfaces have numerous projections which interdigitate with those of the adjacent cells, and in some sections these complicated intercellular spaces are located at the base of the cells. However, in narrow part of the distal tubule, the intercellular space has not complex as those of the wider region, and there is no basal plasma membrane infoldings in the distal tubule. The collecting duct of the soft-shelled turtle contains clear and dark cells, and only clear cell cytoplasm contains a number of mucus granules. Lateral intercellular space of collecting tube cells in soft-shelled turtle contains a number of finger-like projections
as lizards (Davis et al., 1976). The collecting tubule of the green turtles (Solomon, 1985) only contained the lateral plasma membrane folds, however it has no mucous granules. Mucous goblets and lateral interdigitated processes could be seen in P. scripta elegans and in M. caspica (Meseguer et al., 1987), but the extension of lateral interdigitated processes is less than that of soft-shelled turtle, and undeveloped than that of soft-shelled turtle. The mucous secretion pobably produce a luminal layer that protects the epithelium from precipitated inorganic salts and thereby facilitating the excretion of insoluble urates (Gabri, 1983b), which related to finger-like projections and lateral plasma membrane folds in the soft-shelled turtle are possess. Sever et al. (2005, 2008) and Del Conte and Tamayo (1973) reported on the renal sexual segment (RSS) in the Cottonmouth Snake (Agkistrodon piscivorous), the Ground Skink, (Scincella laterale) and lizards (Cnemidophorus l. lemniscatus). Mature squamates possess hypertrophied regions of the distal nephrons of male snakes and lizards, the renal sexual segment (RSS). The RSS is believed to provide seminal fluid that mixes with sperm and is released into the female cloaca during coitus (Sever and Hopkins, 2005a,b). The presence of a RSS can be considered a synapomorphy for Squamata and perhaps for Lepidosauria, but it is lacking in turtles (Sever et al., 2008). A long these lines we did not find evidence for such a segment in the soft-shelled turtle. However, in the middle segment and proximal tubule cells of soft-shelled turtle, there is some structure differences between male and female, which had not previously been reported in reptiles kidneys. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. The structure of intermediate segment and proximal tubule epithelial cell basal surface in softshelled turtle had some differences, whether these differences go with the reproductive activity needs further research. Additionally, the size of the intercellular spaces may be important for the material transport, the male proximal tubular reabsorption capacity may be stronger than the female. The glomerular filtration barrier, across which blood is filtered, is formed by the fenestrated capillary endothelium, the extracellular glomerular basement membrane (GBM), and the diaphragm bridged filtration slitsbetween podocyte foot processes. The endothelial pore size in the soft-shelled turtles is 80–185 nm and the thickness of the GBM was measured to 300–600 nm Similar data are not available for other reptiles. In humans, rats and platypus the GBM was measured to 314 ± 98.3 nm, 110–160 nm and 160–250 nm, respectively (Tsujii et al., 1992). In anuran and caecilian amphibians the basement membrane varies between 200 and 600 nm (Møbjerg et al., 1998, 2000, 2004). Endothelial pore size in rats (Fujita et al., 1976), and platypus is 30–150 nm and 40–100 nm; 100–300 nm and 50–200 nm in the caecilian amphibians and toad larvae. Glomerular basement membrane thickness in fish lungfish (Ojeda et al., 2006) is 1 m. The present data demonstrated that the pores of the glomerular endothelium are conspicuously irregular in dimension. Therefore the thickness of GBM and endothelial pore size in the soft-shelled turtle is nearly the same as that of toad larvae. We suggested the status of basement membrane thickness has to do with living environment of animal, or with the animal species. Capillary endothelial pore size is consistent with filtration molecular size, thus, 185 nm of the protein molecules is filtered by the soft-shelled turtle glomerular. The pores of the glomerular endothelium of soft-shelled turtle shows localize distribution characteristics, and its quantities were less which is one of important factors to affect the glomerular filtration rate. The endothermic vertebrate classes (birds and mammals) have significantly higher
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glomerular filtration rates than the ectothermic groups, as the reptiles tending to possess the lowest rates of glomerular filtration (Yaoita et al., 1999). Possibly because of the necessity of the softshelled turtle to conserve water, which is formed further argued by measurement data of glomerular morphological characteristics. The podocytes on the glomerular basement membrane (GBM) are the terminal element in the filtration barrier and display an elaborate morphology. Long cytoplasmic processes extend radially from the cell body and branch into several foot processes, which cover the outer aspect of the GBM. Podocytes in renal glomeruli contribute significantly to these filters’ important properties of hydraulic and macromolecular permeability (Yaoita et al., 1999). The secondary processes of C. mydas L. (Solomon, 1985) is developed, a podocyte only to encircle a single capillary of the glomerular capillaries, therefore, the capillary loop of curved shape can be observed in SEM, while, the podocytes in M. caspica (Meseguer et al., 1987) with short primary processes. The podocyte in P. dolloi (Ojeda et al., 2006) show numerous major cytoplasmic processes that extend over contiguous capillary loops, the podocyte primary processes of the P. dolloi is shorter in SEM, secondary processes and tertiary in the more developed, a podocytes were only attached to a single capillaries. The secondary processes and the tertiary processes in Mammals (Nizze and Csikós, 1980) and birds (Pak Poy and Robertson, 1957) are relatively well developed. A podocyte only encircle a single capillary, therefore, glomerular capillary bending is clearly visible. However, the podocyte proccesses of soft-shelled turtle is well developed, and the whole of glomerular capillary is covered by the podocyte processes. wider and bigger primary processes extend from the podocyte cell body, a little wider secondary processes extend from primary processes, and further extend to a number of little tertiary processes. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. We speculated that a podocyte processes encircles several capillaries. Thus, we can only observe the glomerular surface was covered by the podocyte processes, while a single capillary is seen difficultly. The podocytes of morphological features in soft-shelled turtle, have not previously been reported in other vertebrates kidney. It might be suggested that the podocyte morphological features above had a low filtration rate. Acknowledgments This study was supported by grants from the National Science Foundation of China (No. 31272521) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20110097110012), and Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors are grateful to Ph.D N. MØBJER (Department of Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark) for advice and for kindly supporting English language in the paper. References Anderson, E., 1960. The ultramicroscopic structure of a reptilian kidney. Journal of Morphology 106 (March), 205–241. Bankir, L., de Rouffignac, C., 1985. Urinary concentrating ability: insights from comparative anatomy. American Journal of Physiology 249 (6 Pt 2), R643–R666. Carvalho, E.T., Junqueira, L.C., 1999. Histology of the kidney and urinary bladder of Siphonops annulatus (Amphibia-Gymnophiona). Archives of Histology and Cytology 62 (March (1)), 39–45. Casotti, G., Braun, E.J., 2000. Renal anatomy in sparrows from different environments. Journal of Morphology 243 (March (3)), 283–291. Clothier, R.H., Worley, R.T., Balls, M., 1978 Dec. The structure and ultrastructure of the renal tubule of the Urodele amphibian, Amphiuma means. Journal of Anatomy 127 (Pt 3), 491–504.
461
Dantzler, W.H., Holmes, W.N., 1974. Water and mineral metabolism in reptilia. In: Florkin, M., Scheer, B.T. (Eds.), Chemical Zoology. Academic Press, New York, London, pp. 277–377. Dantzler, Bradshaw, 2009. Osmotic and ionic regulation in reptiles. In: Evans, D.H. (Ed.), Osmotic and Ionic Regulation: Cells and Animals. CRC Press, Boca Raton. Dantzler, W.H., Schmidt-Nielsen, B., 1966. Excretion in freshwater turtle (Pseudemys scripta) and desert tortoise (Gopherus agassizii). American Journal of Physiology 210, 198–210. Davis, L.E., Schmidt-Nielsen, B., Stolte, H., 1976. Anatomy and ultrastructure of the excretory system of the lizard Sceloporus cyanogenys. Journal of Morphology 149 (July (3)), 279–326. Del Conte, E., Tamayo, J.G., 1973. Ultrastructure of the sexual segments of the kidney in male and female lizards, Cnemidophorus l. lemniscatus (L.). Zeitschrift für Zellforschung und mikroskopische Anatomie 144 (November (3)), 325–337. Fujita, T., Tokunaga, J., Edanaga, M., 1976. Scanning electron microscopy of the glomerular filtration membrane in the rat kidney. Cell and Tissue Research 166 (February (3)), 299–314. Gabri, M.S., Butler, R.D., 1984. The ultrastructure of the renal corpuscle of a lizard. Tissue Cell 16 (4), 627–634. Gabri, M.S., 1983a. Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) taurica. Journal of Morphology 175, 131–142. Gabri, M.S., 1983b. Seasonal changes in the ultrastructure of the kidney collecting tubule in the lizard Podarcis (= Lacerta) taurica. Journal of Morphology 175, 143–151. Hillyard, S.D., Møbjerg, N., Tanaka, S., Larsen, E.H., 2009. Osmotic and ion regulation in amphibians. In: Evans, D.H. (Ed.), Osmotic and Ionic Regulation: Cells and Animals. , 1st ed. CRC Press, Boca Raton, pp. 367–441. Meseguer, J., Ayala, A.G., Agulleiro, B., 1987. Ultrastructure of the nephron of freshwater turtles, Pseudemys scripta elegans and Mauremys caspica. Cell and Tissue Research 248, 381–391. Moore, B.C., Hyndman, K.A., Cox, A., Lawler, A., Mathavan, K., Guillette Jr., L.J., 2009. Morphology and histochemistry of juvenile American alligator (Alligator mississippiensis) nephrons. The Anatomical Record (Hoboken) 292 (October (10)), 1670–1676. Morild, I., Bohle, A., Christensen, J.A., 1985. Structure of the avian kidney. Anatomical Record 212 (May (1)), 33–40. Møbjerg, N., Jespersen, A., Wilkinson, M., 2004. Morphology of the kidney in the West African caecilian, Geotrypetes seraphini (Amphibia, Gymnophiona, Caeciliidae). Journal of Morphology 262 (November (2)), 583–607. Møbjerg, N., Larsen, E.H., Jespersen, A., 2000. Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae). Journal of Morphology 245 (September (3)), 177–195. Møbjerg, N., Larsen, E.H., Jespersen, Å., 1998. Morphology of the nephron in the mesonephros of Bufo bufo (Amphibia, Anura, Bufonidae). Acta Zoologica (Stockholm) 79, 31–51. Nizze, H., Csikós, A., 1980. Scanning electron microscopy of the rat renal glomerulus. International Urology and Nephrology 12 (1), 3–14. Ojeda, J.L., Icardo, J.M., Wong, W.P., Ip, Y.K., 2006. Microanatomy and ultrastructure of the kidney of the African lungfish Protopterus dolloi. The Anatomical Record. Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 288 (June (6)), 609–625. Pak Poy, R.K., Robertson, J.S., 1957. Electron microscopy of the avian renal glomerulus. Journal of Biophysical and Biochemical Cytology 3 (March (2)), 183–192. Peek, W.D., McMillan, D.B., 1979a. Ultrastructure of the renal corpuscle of the garter snake Thamnophis sirtalis. American Journal of Anatomy 155 (May (1)), 83–102. Peek, W.D., McMillan, D.B., 1979b. Ultrastructure of the tubular nephron of the garter snake Thamnophis sirtalis. American Journal of Anatomy 154 (January (1)), 103–127. Peek, W.D., Shivers, R.R., McMillan, D.B., 1977. Freeze-fracture analysis of junctional complexes in the nephron of the garter snake Thamnophis sirtalis. Cell and Tissue Research 179 (April (4)), 441–451. Sakai, T., Billo, R., Kritz, W., 1986. The structural organization of the kidney of Typhlonectes compressicaudus (Amphibia, Gymnophiona). Anatomy and Embryology 174, 243–252. Sakai, T., Kawahara, K., 1983. The structure of the kidney of Japanese newts, Triturus (Cynops) pyrrhogaster. Anatomy and Embryology (Berlin) 166 (1), 31–52. Sever, D.M., Hopkins, W.A., 2005a. Renal sexual segment of the ground skink, Scincella laterale (Reptilia, Squamata, Scincidae). Journal of Morphology 266, 46–59. Sever, D.M., Siegel, D.S., Bagwill, A., Eckstut, M.E., Alexander, L., Camus, A., Morgan, C., 2008. Renal sexual segment of the cottonmouth snake, Agkistrodon piscivorous (Reptilia, Squamata, Viperidae). Journal of Morphology 269 (June (6)), 640– 653. Sever, D.M., Hopkins, W.A., 2005b. Renal sexual segment of the ground skink, Scincella laterale (Reptilia, Squamata, Scincidae). Journal of Morphology 266 (October (1)), 46–59. Schmidt-Nielsen, B., Skadhauge, E., 1967. Function of the excretory system of the crocodile (Crocodylus acutus). American Journal of Physiology 212 (May (5)), 973–980. Schmidt-Nielsen, B., Davis, L.E., 1968. Fluid transport and tubular intercellular spaces in reptilian kidneys. Science 159 (March (819)), 1105–1108. Soares, A.M., Fava-de-Moraes, F., 1984. Morphological and morphometrical study of the kidney of the male tropical lizard Tropidurus torquatus. Anatomischer Anzeiger 157 (5), 365–373. Solomon, S.E., 1985. The morphology of the kidney of the green turtle (Chelonia mydas L). Journal of Anatomy 140, 355–369.
462
C.-S. Xu et al. / Micron 44 (2013) 451–462
Taugner, R., Schiller, A., Ntokalou-Knittel, S., 1982. Cells and intercellular contacts in glomeruli and tubules of the frog kidney. A freeze-fracture and thin-section study. Cell and Tissue Research 226 (3), 589–608. Tsujii, T., Inoue, S., Takamiya, H., Liszczynsky, H.R., Naora, H., Seno, S., 1992. Morphology of the kidney of the platypus (Ornithorhynchus anatinus: Monotremata). The Anatomical Records 234 (3), 348–358. Wake, M.H., 1970. Evolutionary morphology of the caecilian urogenital system. II. The kidneys and urogenital ducts. Acta Anatomica 75, 321–358.
Yaoita, E., Franke, W.W., Yamamoto, T., Kawasaki, K., Kihara, I., 1999. Identification of renal podocytes in multiple species: higher vertebrates are vimentin positive/lower vertebrates are desmin positive. Histochemistry and Cell Biology 111 (February (2)), 107–115. Zuasti, A., Ferrer, C., Ballesta, J., Pastor, L.M., 1987. Ultrastructure of the tubular nephron of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals. Histology and Histopathology 2 (October (4)), 391–400.