The Dipnoi (lungfishes) constitute a monophyletic group of Sarcopterygii fishes that are able to breathe through primitive lungs. There are about 50 extinct genera and 6 living species grouped into the genera Neoceratodus, Lepidosiren, and Protopterus (Greenwood,1986). The lungfishes are air-breathing freshwater fish that live in the tropical regions of Australia, South America, and Africa (Graham,1997). The lungfishes occupy a critical phylogenetic position: the appearance of aerial respiration in ancestral fishes was a pivotal development in the evolution of terrestrial vertebrates. Thus, they constitute a model for the study of the adaptative changes involved in the evolutionary transition from aquatic to terrestrial life. Lungfishes share many characteristics with both freshwater fish and amphibians. For instance, aerial respiration and the presence of endocrine glands involved in osmoregulation bring the lungfish closer to the amphibians than to freshwater fish (Sawyer et al.,1982). Other anatomic and functional features, such as the presence of gills, the absence of limbs, the kidney response to arginine vasotocin, and the production of an osmotically diluted urine, make the lungfish closer to other freshwater fish (Sawyer,1970; Sawyer et al.,1976,1982; Babiker and Rankin,1979).
The lungfish Protopterus dolloi has the ability to estivate through the dry seasons that characterize part of tropical life (Fishman et al.,1986). In this condition, P. dolloi survives for months without ingesting food or water and breathing only by the lungs. Estivation also implies a dramatic change in renal function. In freshwater fish, the main physiological function of the kidney is to excrete water. In contrast, in lungfish, the kidney must retain water during estivation. Therefore, the lungfish kidney must be designed to respond to these radical functional changes. After the pioneering work of Cordier (1929,1937), there have been few studies devoted to the microanatomy and structure of the lungfish kidney. In P. dolloi, a single study has reported on the ultrastructure of the distal tubule (Hentschel and Elger,1987). These studies, however, did not report whether the lungfishes were estivating. Estivation may induce changes in kidney morphology and the habitat is therefore of great importance for structural reports.
The aim of the present study is to examine the three-dimensional microanatomy and the structure of the kidney of the African lungfish Protopterus dolloi when living in freshwater conditions. We have used light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and chemical microdissection techniques. This study was undertaken to disclose specific features that could be related to function, phylogeny, and habitat. The data on the structure of the P. dolloi kidney in freshwater conditions will be used as a baseline for future studies that will determine the morphological changes that occur during estivation.
MATERIALS AND METHODS
Maintenance of Specimens
The study was performed on six specimens of the lungfish Protopterus dolloi with a body mass weight of 100–150 g. Sex identification within this range of body mass is not possible due to the lack of distinct external features. The specimens were collected from Central Africa and imported through a local fish farm in Singapore. Species identification was performed according to Poll (1961). The animals were maintained in plastic aquaria filled with dechlorinated tap water (pH 7.1–7.2) containing 0.71 mM Na+, 0.32 m MK+, 0.72 mM Ca2+, 0.06 mM Mg2+, 2.2 mM Cl−, and 0.2 mM HCO3− at 25°C (Wood et al.,2005). The water also contained small amounts of phosphates (0.10 mM) and sulfates (0.04 mM). Water was changed daily. The specimens were acclimatized to laboratory conditions for at least 1 month. During the adaptation period, the animals were fed frozen blood worms. The fish were killed by a blow to the head and the ventral body wall was opened. The long columnar kidneys were exposed and the central portion was divided into four parts about 0.5 cm in length.
For conventional light microscopy, small kidney fragments were fixed in 3% glutaraldehyde in phosphate-buffered saline (PBS), postfixed in 1% osmium tetroxide for 1 hr, dehydrated in graded acetone and propylene oxide, and embedded in Araldite (Fluka, Buchs, Switzerland). Semithin (1 μm) sections were cut with a Leica ultracut UCT, stained with 1% toluidine blue, and observed with a Zeiss III photomicroscope.
Transmission Electron Microscopy
For TEM, two types of fixatives were used. Selected kidney fragments were fixed in 3% glutaraldehyde in PBS. Other fragments were fixed in 3% glutaraldehyde containing 0.5% tannic acid. In both cases, the specimens were postfixed in 1% osmium tetroxide for 1 hr and embedded in Araldite as above. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 208.
Scanning Electron Microscopy
For SEM, selected kidney fragments were fixed in 3% glutaraldehyde. After fixation, a first group of samples was dehydrated in graded acetone, dried by the critical point method, coated with gold, and observed with a Philips SEM 501. A second group of samples was subjected to chemical and mechanical microdissection prior to desiccation. For chemical microdissection, the specimens were digested either with NaOH (Takashashi-Iwanaga and Fugita,1986) or KOH (Ushiki and Murakumo,1991). After chemical digestion, the specimens were carefully microdissected under a stereomicroscope with sharpened tungsten needles. The specimens were then processed for SEM as described above.
The diameter of the different segments of the nephron was determined in SEM pictures of the microdissected specimens by using the SEM μm markers as calibration. To this end, we have assumed that any size changes that the samples undergo during chemical digestion and the dehydration are similar in all the segments studied. This method allowed very precise determination of the level at which measurements were made. Three different animals were sampled. Thirty measurements were made per segment.
The height of the cells in each nephron segment was measured on semithin transverse sections using a camera lucida coupled to the microscope. The magnification of the drawing was determined by means of a calibrated microscope slide. Fifty measurements were made per cell studied, in at least three animals. The thickness of the different components of the glomerular basement membrane (GBM) was measured on TEM micrographs as previously described (Ojeda et al.,1989).
The kidney of Protopterus dolloi appeared as a long, narrow, symmetrical organ located along the ventral surface of the vertebral column, adjacent to the body cavity. It presented a convex laterodorsal surface covered by a fibrous capsule (Fig. 1) and a more flattened medioventral surface that contained the renal vessels. Abundant hematopoietic tissue and dark pigmented cells were present under the fibrous capsule. Often, this area contained encapsulated parasites (not shown). The renal corpuscles were located in the middle area of the kidney (Fig. 1). The kidney tissue between this area and the laterodorsal surface was mostly occupied by the proximal tubules. The distal tubules occupied the area between the renal corpuscles and the medioventral kidney surface. The spatial location of the different nephron components allowed recognition of a kidney zonation with three main divisions (Fig. 1): a middle zone, a laterodorsal zone, and a medioventral zone.
The SEM analysis of the microdissected 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, the collecting tubule, the collecting duct, and the archinephric duct. The main features of the microanatomy of the P. dolloi nephron are summarized in Figure 2.
The renal corpuscles (RCs) were seen to form small clusters (3–4 units per cluster; Fig. 1). Each RC was roughly ovoid in shape (Figs. 2 and 3a), with a diameter of 74–92 μm. Its outer surface was smooth. The basal pole of the parietal cells of Bowman's capsule exhibited elaborate grooves and microplicae and showed well-defined cell boundaries (Fig. 3b). The urinary pole of the RC was continuous with a well-delimited neck segment (NS). The NS appeared as a straight, narrow (21–27 μm in outer diameter), and short (45–56 μm in length) tubular segment that runs parallel to the medial surface of the kidney (Figs. 2 and 3a). The basal pole of the NS cells was similar to that of the cells of Bowman's capsule, but lacked microplicae (Fig. 3c). The transition from the NS to the proximal tubule (PT) was sharply defined. This transition was defined by a considerable increase in the outer tubular diameter (from 21–27 to 36–47 μm; Fig. 3a) and by a change in the configuration of the basal cell pole (Fig. 3d). The initial PT portion suddenly changed direction and formed a small loop that entered the laterodorsal kidney zone. This short loop was followed by a straight segment (first straight portion) that ran toward the kidney surface (Fig. 3a). Near the subcapsular hematopoietic tissue, the PT turned back to form an arch (arcuate portion; Fig. 4) that was continuous with a second short straight portion (Fig. 5). This last segment ran toward the renal corpuscle area. Overall, the PT looked like an inverted fish hook located in the laterodorsal zone of the kidney (Fig. 2). The arcuate portion of the PT was the widest (54–71 μm in outer diameter) of all the tubular portions of the nephron. The total PT length was 600–766 μm. The basal pole of the PT cells exhibited a scaly appearance with ill-defined cell boundaries (Fig. 3d). In addition, the second, straight portion of the PT contained cells exhibiting smooth basal poles and well-delineated cell boundaries (Fig. 5). Accordingly, the PT was divided into two portions: PT segment I (PTI), extending from the end of the neck segment to the start of the second straight portion, and PT segment II (PTII), which corresponded to this latter straight portion. This PT division was justified ultrastructurally.
The PT was continuous with the intermediate segment (IS; Figs. 2 and 4). The IS was a short, straight, well-defined portion. The IS appeared as the narrowest portion of the nephron (17–21 μm in outer diameter; Fig. 5), with a length of 200–380 μm. The IS ran into the laterodorsal kidney zone in the opposite direction but parallel to the first straight portion of the PT (Fig. 5). The final portion of the IS reached the level of the renal corpuscle, i.e., it entered the mid-zone of the kidney. The basal pole of the IS cells appeared similar to that of the second cell type of the PT (Fig. 6).
The transition from the IS to the distal tubule (DT) was defined by an abrupt increase in the external tubular diameter (from 17–21 to 30–38 μm; Figs. 2 and 6) and by a change in the basal cell pole configuration. The DT ran through the medioventral kidney zone, forming 3–4 large loops (Fig. 7). The total DT length was 851–986 μm. The basal pole of the DT cells had a scaly appearance, similar to that of the PTI cells (Fig. 6, inset). However, the distal part of the DT showed, in addition to these cells, a second cell type. The latter cells were characterized by a round prominent basal cell pole (Fig. 8). Accordingly, the DT could also be subdivided into two portions, DTI and DTII, based on the presence of these prominent cells (Fig. 2). TEM showed these cells to be flask cells.
The transition between the DT and the collecting tubule (CT) was not defined by changes in tubular diameter. However, the transition could clearly be recognized by a marked increase in the number of flask cells (Fig. 8). The CT coursed through the mid-zone of the kidney and entered the laterodorsal zone. Several CTs joined each other before opening into a collecting duct (CD; Fig. 8a). The CT had an outer diameter of 31–40 μm and were 246–291 μm in length. The CT was formed by principal cells and flask cells (Figs. 2 and 8) in a 5:1 ratio. The abundance of flask cells gave to the outer surface of the CT a cobblestone appearance. The collecting duct (CD) followed a characteristic course: it was the only tubular portion arranged in a dorsoventral direction (Fig. 2). The CD opened into the archinephric duct (AD). The AD ran craniocaudally along the ventral side of the kidney.
Ultrastructure of Nephron Segments
As stated above, the RCs appeared in clusters of three or, more frequently, four units (Fig. 9). The RCs within each cluster were tightly apposed. Only the parietal basement membrane and some collagen fibers were interposed between contiguous RCs. Each RC cluster was supplied by a single long unbranched arteriole that appeared enveloped by pericytes (Fig. 10). Two types of pericytes were present. Some pericytes showed a polygonal cell body and short irregular cytoplasmic processes (Fig. 10a). Other pericytes showed a prominent cell body and ribbon-like cytoplasmic processes that completely bounded the afferent arteriole (Fig. 10b).
The parietal layer of the Bowman's capsule was formed by squamous epithelial cells that presented a smooth apical surface, a long central cilium, and rows of short microvilli located near the cellular junction (Fig. 11a). TEM showed the presence of numerous cytoplasmic vesicles, preferentially aligned along the basal cell surface, and of a large number of intermediate filaments (Fig. 11b). The basement membrane of Bowman's capsule was composed of a thin lamina lucida, a thin lamina densa, and a thick lamina fibroreticularis that contained abundant amorphous material and some collagen fibers (Fig. 11b).
The glomerular capillary tuft was formed by 3–4 capillary loops (Fig. 12a). Capillary branching or interlooping anastomoses were not observed. Most podocyte bodies occupied a strategic position, being located on the inner side of the areas of capillary bending (Fig. 12a). Each podocyte showed numerous major cytoplasmic processes that extended over contiguous capillary loops, thus covering a large capillary area. Furthermore, the major podocyte processes formed a very specific interdigitating pattern. Many of them, instead of branching directly, expanded to form a flattened network over the capillary surface (Fig. 12b). Side branches occurred at the outer network boundaries. Other major cytoplasmic processes ran under the network, ending at the level of the network holes. Pedicels, and the corresponding filtration slits, occurred both at the level of the network holes and between the final branches (Fig. 12b). Tight and gap junctions occurred between the major podocyte processes in the crossing areas (Fig. 13a and b). However, the junctions between the pedicels were scarce. On the whole, the foot processes appeared regularly arranged along the outer surface of the GBM, with filtration slits and well-developed slit diaphragms (Fig. 13a). The podocytes exhibited a prominent cytoskeleton formed by intermediate filaments (Fig. 13) arranged along the long axis of the major cell processes and some microtubules (Fig. 13c). Neither the podocyte bodies nor the major podocyte processes were directly attached to the GBM. Thus, a subcellular space could clearly be delineated (Fig. 13a).
The GBM was very thick, with a maximum thickness of about 1 μm. It was formed by three layers: a lamina rara, a lamina densa, and a subendothelial lamina (Fig. 13a). The lamina rara, which underlay the foot processes, was the thinnest GBM layer (31–37 nm thick). The lamina densa showed a uniform electron density (92–112 nm thick). The subendothelial lamina consisted of two distinct zones. The first, apposed to the lamina densa, showed a variable thickness (between 266 and 433 nm). It consisted of a loose network of microfibrils, amorphous material, and a few collagen fibers (Fig. 13a). The microfibrils appeared in cross-sections as tubular structures with a hollow center (Fig. 13a). The collagen fibers were arranged parallel to the lamina densa. The second, the subendothelial zone, was located under the endothelial basal surface. It consisted of an electron-lucent space (110–408 nm thick) devoid of extracellular material. Thus, it was clear that the endothelium lacked a basement membrane. Mesangial cell processes were frequently observed under the endothelium.
Chemical digestion with NaOH completely removed the GBM. Following digestion, two types of mesangial cells could be discerned. The most numerous mesangial cells showed elongated cell bodies and long, thick cytoplasmic processes (Fig. 14a) that bounded most of the capillary circumference. The long processes gave rise to numerous short processes that contacted the short processes of neighboring mesangial cells. Thus, the mesangial cell processes formed a loose network embedded in the subendothelial lamina of the GBM. The second mesangial cell type consisted of small stellate cells that showed short cell processes (Fig. 14b). These processes also established contacts with the processes of neighboring mesangial cells of the same type. TEM showed that the cytoplasmic processes of the mesangial cells contained slender mitochondria and intermediate filaments (Fig. 14c).
The endothelium of the glomerular capillaries showed numerous open fenestrations grouped into small oval areas (Fig. 14a). The endothelial basal cell pole was irregular and possessed numerous short cytoplasmic processes (Fig. 14b) that occasionally established contact with mesangial cell processes. Secretory granules and micropinocytotic vesicles were not observed in the cytoplasm of the endothelial cells.
The epithelium of the NS consisted of cuboidal (7–8 μm tall) multiciliated cells that showed a large nucleus (Fig. 15a). There were 6–7 cells in cross-section. The apical cell cytoplasm showed the presence of basal bodies, striated rootlets, and microvesicular membranous profiles (Fig. 15b). The cilia had a regular 9+2 pattern and formed a tuft that almost occluded the NS lumen (Fig. 16c). The basal and lateral cell membranes were mostly smooth (Fig. 15c). The lateral cell membranes were joined through desmosomes near the apical cell pole (Fig. 15b), but were separated by an intercellular space in the rest of its extension. The basement membrane of the NS showed the conventional three-layer structure, being formed by a lamina rara, a lamina densa, and a well-developed lamina fibroreticularis. This structure was maintained through the full length of all the tubular segments of the nephron.
The PT contained, in transverse sections, 8–9 cells at the beginning of the first portion (PTI), and 12–13 cells in the zone of the largest diameter (arcuate portion). As stated above, the PTI contained a single cell type. These cells showed a truncated pyramidal shape (17–22 μm tall) and exhibited a regular brush border of microvilli (4–5 μm tall) and a central solitary cilium (Fig. 16a and b). Thus, these cells were brush border (BB) cells. Owing to the narrowness of the tubular lumen at the PTI level, the microvilli occupied most of the lumen. The apical cell cytoplasm contained a prominent endocytic-lysosomal apparatus consisting of apical cytoplasmic invaginations, small vesicles, large vacuoles with fine granular contents, and numerous lysosomes (Fig. 16b). A large and smooth-contoured nucleus, with abundant heterochromatin distributed in a “blots-of-ink” pattern, was basally located (Fig. 16b; most tubular cells along the nephron showed the same heterochromatin pattern). Numerous rounded mitochondria were located in the perinuclear zone. The lateral and basal cell membranes exhibited prominent folds, clearly visible by SEM as numerous microplicae (Fig. 16a). The scaly appearance of the basal pole of these cells (Fig. 3d) was due to the presence of the basal microplicae. The PTI cells were joined through apical junctional complexes containing desmosomes. However, below these junctional complexes, the cells were separated by lateral intercellular spaces that became wider toward the cell base. These spaces were occupied by the interdigitating lateral microplicae. The basal microplicae delimited a basal extracellular space continuous with the lateral intercellular spaces (Fig. 16b). Thus, a continuous basolateral labyrinth was formed. Postfixation with tannic acid revealed the presence of densely stained, basement membrane-like material between the basal microplicae (Fig. 16c).
The second portion of the proximal tubule (PTII) was composed of BB cells and of a small number of multiciliated cells (Fig. 17a). The BB cells of the PTII portion were morphologically different from those of PTI (compare Figs. 16a and b and 17b and c). They were taller (20–29 μm tall) columnar cells with shorter (2–3 μm tall) microvilli (Fig. 17). The supranuclear zone exhibited a characteristic appearance containing numerous vacuoles filled with granular content and many elongated mitochondria (Fig. 17c). The nucleus was very large, occupying about two-thirds of the cell volume. The lateral cell membranes presented desmosomes at the apical junctional complex and toward the cell base (Fig. 17c), and the lateral and basal microplicae were less prominent and numerous. Consequently, the lateral extracellular spaces between the BB cells of PTII were narrower than those located between the BB cells of PTI. Also, the basal extracellular space was not well delineated, and a basolateral labyrinth could not be discerned. Multiciliated cells were seen intercalated between the BB cells. They had a dark appearance under the light microscope (Fig. 17a) and on TEM (Fig. 17c). Their structure was similar to that of the NS cells, but the cilia were longer and extended down the lumen for a distance of about 2–3 cells.
The IS consisted of cuboidal (13–16 μm tall) multiciliated cells (Fig. 18). There were 5–6 cells in each cross-section. These cells showed some differences with regard to the multiciliated cells of the NS and PTII. The lateral cell membranes exhibited numerous and prominent folds (Fig. 18a; compare with Fig. 15c), and the nucleus appeared more centrally located (Fig. 18b). Also, the IS cells were wider than the multiciliated PTII cells and did not have a dark appearance. The supranuclear cytoplasm contained many small vesicles, and a few minute microvilli were present at the apical cell surface (Fig. 18c).
The DT contained 7–8 cells in cross-section. The DT could be subdivided into proximal (DTI) and distal (DTII) portions according to differences in the cell composition. The DTI consisted exclusively of columnar (17–21 μm tall) epithelial cells (Fig. 19a) that displayed a highly complex lateral cell membrane surface due to the presence of cell processes (Fig. 19). Under SEM, two types of lateral cell processes could be distinguished. The first type consisted of prominent ridges extending from the apical cell surface to the cell base. These ridges bifurcated as they approached the cell base (Fig. 19a). The second type consisted of numerous microplicae (Fig. 19a). The interdigitating lateral cell projections gave the lateral spaces a labyrinthic appearance (Fig. 19b). The presence of basal microplicae also formed a basal labyrinth. However, continuity with the lateral labyrinth was not apparent (Fig. 19b). The apical cell surface showed a few short and thick irregular microvilli (Fig. 19a) and occasional short solitary cilia. The apical cytoplasm contained numerous small vesicles. Mitochondria were found in the perinuclear zone and near the basolateral cell membrane (Fig. 19b). The cytoplasm contained large pale areas, presumably occupied by extracted glycogen. The nuclei were large with abundant heterochromatin.
The distal portion of the distal tubule (DTII) contained two cell types. The first showed similar features to those of DTI cells (Fig. 20a). Small differences between the two cell types were that the principal cells of DTII were more prismatic and had less prominent lateral and basal labyrinths and narrower intercellular spaces (Fig. 20b; compare Figs. 19 and 20). The second DTII cell type consisted of a small number of so-called flask cells. The structural characteristics of these cells will be described with the collecting tubule segment.
The CT was formed by 7–8 cells in cross-section. The CT contained two cell types, principal and flask cells. The first represented almost 80% of all CT cells. The principal cells were prismatic (20–23 μm tall), with a smooth apical surface and occasional short solitary cilia (Fig. 21a). The apical cytoplasm displayed a characteristic spongy appearance due to the presence of numerous vesicles containing a flocculent material (Fig. 21b). The cytoplasm also contained mitochondria, free ribosomes, microfilaments, and glycogen deposits. The nucleus was large and ovoid, was basally located, and contained abundant heterochromatin (Fig. 21b). The lateral cell membrane displayed abundant microplicae (Fig. 21) that protruded into the lateral intercellular spaces. The basal microplicae were poorly developed.
The flask cells showed a characteristic pear shape with a large and prominent rounded basal pole (Figs. 22 and 23). They were more voluminous and taller (25–29 μm) than the principal cells. The cytoplasm of these cells showed a clear appearance under the light microscope (Fig. 22) and on TEM (Fig. 23). The cell nucleus was rounded, was located basolaterally, and showed less heterochromatin than the principal cell nucleus (Fig. 22). Nonetheless, the most prominent feature of the flask cells was the presence of a central cavity communicating with the tubular lumen. This cavity adopted two different configurations. Most frequently, the cavity appeared as a narrow tortuous canaliculus (Fig. 23a and b), but in some cases, a large cavity opened into the tubular lumen through a funnel-shaped communication (Fig. 23c). The flask cell central cavity was often occupied by amorphous material that stained intensely with toluidine blue (Fig. 22) and showed a uniform moderate electron density under TEM (not shown). This material could also be observed in the tubular lumen (Fig. 22). The cell membrane bounding the cavity displayed microplicae, which were especially numerous and thick in the basal zone of the cavity (Fig. 23c). In both types of flask cells, the cytoplasm located around the cavity showed abundant mitochondria (Fig. 23b). Curiously, the basal cell cytoplasm was very poor in organelles. Only a few mitochondria, small lipid droplets, and free ribosomes appeared in this area (Fig. 23a and b).
The present observations provide new data on the microanatomy and structure of the P. dolloi kidney. These data may be useful to understand the special physiological requirements and the phylogenetic position of this species. In addition, this study provides a framework to study the structural changes that should occur in the kidney of P. dolloi during estivation. A characteristic feature of the P. dolloi kidney is the spatial distribution of the different nephron components: proximal and distal tubules lie apart, separated by the renal corpuscles. This agrees with previous observations (Hentschel and Elger,1987) and makes it possible to divide the kidney into three different zones. While kidney zonation is typical of the tetrapods, it can also be observed in other fish classes (Polypterus, Protopterus, and marine Elasmobranchii) (Hentschel and Elger,1987). However, there is not any indication that may suggest the existence in Protopterus of a countercurrent system involved in osmoregulation.
The renal corpuscles aggregate into small clusters in the mid-zone of the kidney. This also agrees with previous observations made in Protopterus (Cordier,1929; Hentschel and Elger,1987). In P. dolloi, the renal corpuscles within a cluster are supplied by a single afferent arteriole surrounded by pericytes. This feature has not previously been reported. However, it could be highly significant from the functional point of view. Pericytes are contractile cells that respond to vasoactive hormones (Kelly et al.,1988; Helbig et al.,1992) and are able to control the flow of blood through the small vessels (Sims,1986; Díaz-Flores et al.,1991). Thus, each RC cluster appears to represent a functional unit with a common hemodynamic regulatory mechanism. Furthermore, a relatively small number of arterioles can control the filtration activity of a large number of renal corpuscles. This may represent a functional advantage in response to changes in the environment.
The podocyte processes interdigitate in a characteristic way, different from that observed in other vertebrates (for review, see Takahashi-Iwanaga,2002). Many major podocyte processes form a flattened network that is undercrossed by other major processes. In the crossing areas, the podocyte processes are joined by gaps and tight junctions. While the presence of junctions between the pedicels has often been reported (Reeves et al.,1978; Lacy et al.,1987; Ojeda et al.,2003), junctions between the major cell processes are exceptional. To our knowledge, they have only been reported in the hagfish (Kühn et al.,1975,1980) and in the Rana esculenta (Taugner et al.,1982). Gap junctions are important for cell-to-cell communication (Alexander and Goldberg,2003; Nicholson,2003) and make it possible to establish integrated cell networks (Watsky,1995; Ojeda et al., 2001) with a strong capacity for cell communication and cohesion. The numerous gap junctions found in our study suggest that the podocytes may also be forming a synchronized system. Thus, within a renal corpuscle, the podocytes could respond quickly to environmental changes, modifying both the spatial arrangement of the foot processes and synthesis of basement membrane components.
The GBM is formed by three layers: a lamina rara underlying the foot processes, a lamina densa, and a subendothelial lamina. In P. dolloi, the GBM is extremely thick: two to five times the thickness recorded in some freshwater and marine teleosts (Hickman and Trump1969; Zuasti et al., 1983) and in the freshwater sturgeons A. ruthenus and A. naccarii (Gambaryan,1988; Ojeda et al.,2003). The thickness of the GBM and the existence of a prominent mesangium are characteristic of species with a low glomerular filtration rate (Bulger and Trump,1968; Accini et al.,1976). Noticeably, the Protopterus excrete an osmotically diluted urine (Sawyer,1966, 1970), and the filtration barrier has been reported to be unusually permeable (Sawyer et al.,1976,1982). Similar discrepancies between morphological and functional data have also been reported in the frog kidney (Taugner et al.,1982). Taugner et al. (1982) suggested the existence of substantial phylogenetic differences in the physicochemical properties of the filtration barrier. In addition, the effects of several peptide hormones, which could compensate functionally for the presence of a thick physical barrier, should be considered. Arginine vasotocin increases the glomerular filtration rate even in the absence of hypertension (Sawyer et al.,1982), and proximal tubule cells have been suggested to control the transport of water across the tubular epithelium through the production of somatostatin (Masini et al.,1998) and of components of the renin-angiotensin system (Masini et al.,1996).
The characteristics of the mesangial cells have not previously been studied in lungfish. Indeed, SEM studies of the mesangial cells are scarce in any species (Takahashi-Iwanaga,1991, 1992). Thus, it is difficult to make any significant comparative analysis. We have observed two types of mesangial cells. The mesangial cells with large cell processes could be implicated in the regulation of glomerular filtration (Michael et al.,1980). Mesangial cells have been implicated in the rapid adaptation to changes in the hydrosaline environment (Hickman,1968). The network formed by the mesangial cell processes are an obstacle to the free passage of macromolecules through the filtration barrier. Furthermore, the arrangement of this sieve network may be rapidly modified by contraction or expansion of the mesangial processes (Kreisberg,1983). The small stellate mesangial cells may perform a different functional role. Their morphology is similar to that of dendritic cells (Hoefsmit,1982), suggesting that they may be involved in the uptake and processing of small particles and macromolecules. Indeed, this function has been assigned to the mesangium in the mammalian kidney (Seiler et al.,1986). The presence in the kidney of P. dolloi of two mesangial cell types with different functions (contractile and phagocytic) is in line with observations in the mammalian kidney (Schreiner and Cotran,1982).
The neck segment is present in the kidney of several fish species (Hentschel and Elger,1989) and of other vertebrates (Sakai et al.,1986; Uchiyama et al.,1990; Møbjerg et al.,2004), including mammals (Ojeda and Icardo,1991). The NS has been shown to present a valvular (Schonheyder and Maunsbach,1975) or a siphon (Ojeda et al.,2003) shape, and to consist of squamous cells lacking cilia (Ojeda and Icardo,1991), of cuboidal multiciliated cells (Elger et al.,2000), or of both cell types (Ojeda et al.,2003). The existence of multiple cytoarchitectural designs makes it difficult to attribute a precise functional role to the NS. It has been suggested that the cilia of NS cells propel the urine toward the proximal tubule, reducing the pressure in the urinary space and, consequently, increasing the glomerular filtration rate (Sawyer et al.,1982). If so, this function would be of great important in the lungfish since they have an unusually low arterial pressure (Sawyer,1970).
Our findings on the microanatomy of the proximal tubule differ from previous reports based on plastic (Cordier,1937) and on optic (Hentschel and Elger,1987) reconstructions. Thus, the previous division of the Protopterus PT into a pars convoluta and a pars recta cannot be maintained. The PT only exhibits a short loop at the start of its course. For the rest of the course, the PT looks like a fish hook, with two straight parts joined by an arcuate portion. Also, we have not found the diverticula described in other Protopterus species (Cordier,1937), or the numerous loops described in L. paradoxa (Guyton,1935). Some of these discrepancies may be species-specific. Others could be due to the different techniques used. It should be stressed that the microdissection studies allow a very precise definition of the course followed by the different tubular segments of the nephron. Finally, some of the discrepancies could be due to the study of animals under different physiologic conditions.
The PT of elasmobranches and actinopterygii fish has classically been divided into proximal and distal portions based on the cell morphology and on tracer studies with peroxidase (Hentschel and Elger,1989; Elger et al.,2000). Our SEM and TEM observations demonstrate that the lungfish PT can also be divided into proximal (PTI) and distal (PTII) portions. The PTI is exclusively formed by BB cells. Our observations on the structure of these cells agree with those made in the lungfish (Hentschel and Elger,1989) and in other vertebrates (Maunsbach,1973; Maunsbach and Boulpaep,1984; Elger et al.,2000; Møbjerg et al.,2004). The present and previous observations provide structural evidence that the PTI is the principal site of reabsorption of the renal corpuscle filtrate in vertebrates. One remarkable finding is that the lateral intercellular spaces in PTI widen toward the base and are continuous with the basal extracellular space. This spatial continuity forms a basolateral labyrinth (see also Elger et al.,2000). While this arrangement is atypical in fish, it has been observed in several amphibian (Taugner et al.,1982; Maunsbach and Boulpaep,1984) and reptilian (Davis et al.,1976; Peek and McMillan,1979) species (see also Elger et al.,2000). It may represent an evolutionary advantage during adaptation to terrestrial life. It is also interesting that, in elasmobranchs and teleosts, the intercellular spaces are very narrow and the basal and lateral cell membranes are characterized by the presence of membrane infoldings, instead of membrane projections (Hentschel and Elger,1989; Elger et al.,2000).
The PTII corresponds to the “segment rectiligne” or “segment sexual” described by Cordier (1937), indicating the existence of sexual dimorphism in the P. dolloi nephron. However, the use of small-size animals does not allow us to confirm the existence of structural differences related to the sexual cycle of males. The PTII is formed by multiciliated and BB cells. The morphologic characteristics of the BB cells in the PTII portion of P. dolloi are different from those of the PTI and have not been reported previously. However, columnar BB cells possessing numerous vesicles and lacking lysosomes have been described in the PTII portion of several marine elasmobranchii and actinopterygii (Hentschel and Elger,1989). The ultrastructural differences with regard to PTI cells suggest that the BB cells in the PTII portion may have additional or different functional activities that must be determined in future studies.
The intermediate segment is characterized both by its specific microanatomy and by the fact that it is formed only by multiciliated cells. The IS is well defined in archaic freshwater fish (Henstchel and Elger,1989), in Amphibia (Clothier et al.,1978; Taugner et al.,1982; Sakai et al.,1986; Møbjerg et al.,2004), and in Reptilia (Davis et al.,1976; Peek and McMillan,1979). In the lungfish, the presence of the IS has been reported in L. paradoxa (Guyton,1935) and in P. annectens (Sawyer et al.,1982), but appears to be absent in N. forsteri (Jespersen,1969). In P. dolloi, the IS was previously termed the “segment grêle” (Cordier,1929), but it was not recognized in a later study (Hentschel and Elger,1987). Little is known of the functional role of the IS. It is generally accepted that the ciliated segments of the nephron are involved in fluid propulsion. If true, this function may be very important in P. dolloi since the narrow lumen of the distal and collecting segments must offer a high resistance to urinary flow.
The DT forms several loops in the medioventral kidney zone, being the only portion of the P. dolloi nephron that is clearly convoluted. Many of these loops establish close contacts with the glomerular areas. However, we could not discern the presence of tubuloglomerular junctions that might suggest the existence of tubular control of the glomerular filtrate. On the basis of its microanatomy and ultrastructure, we have divided the DT into portions I and II (the early and late distal tubule of Hentschel and Elger,1987). The DTI has a regular outside diameter and consists of a single cell type. The DTII also contains flask (“intercalated”) cells. The presence of intercalated cells in the distal segment of the nephron is common to Polypterus, lungfish, amphibian, and mammals (Hentschel and Elger,1987). However, previous studies in the lungfish have only reported the presence of flask cells with a central cavity. We show here that the flask cells in P. dolloi adopt two different configurations: with a large cavity, or with a narrow canaliculus. Curiously, flask cells with a canaliculus, “canaliculi cells” (Bargmann and Welsch,1972), have been described in the kidney of several amphibians (Brown,1980; Jonas et al.,1988; Møbjerg et al.,2000) and are considered to be equivalent to the dark cells of the mammalian kidney. These cells are important in ion transport and may also have, as it has been suggested in anurans (Jonas et al.,1991), a secretory role.
The outer diameter (OD) of the tubular portions of the nephron varies according to the segment considered. The PT (specially its arcuate portion) is the thickest of all segments, while the transition between this tubule and the IS is the narrowest. The two ciliated segments, NS and IS, are slender, and the distal and the collecting tubules have a similar, intermediate OD. However, owing to the prominent flask cell basal pole, the OD of the collecting tubule is not uniform. Instead, it shows a characteristic cobblestone appearance that has not previously been described in any fish or amphibian species. The OD of the tubular segments of the P. dolloi nephron is in the diameter range reported in several fish (Youson and McMillan,1970; Lacy and Reale,1985) and amphibian (Sakai and Kawahara,1983; Møbjerg et al.,2004) species. In Dipnoi, differences in the OD of the tubular segments of the nephron have been reported in L. paradoxa (Guyton,1935) and in P. dolloi (Hentschel and Elger,1987). However, some previous observations do not agree with the present results. Although the arcuate portion of the PT is the widest tubular segment of the nephron, we have not observed the exceptionally wide lumen reported by Hentschel and Elger (1987). Indeed, very large dilatations of the PT lumen occur in P. dolloi during the estivation period only (data not shown). Also, the DT of P. dolloi shows a very narrow lumen under freshwater conditions. Again, widening of the DT only occurs during the estivation period (data not shown). Thus, it seems plausible that some of the previous observations were made in animals collected during estivation.
Finally, the tubular cells of the P. dolloi nephron, except those in the NS and IS, are very large (see also Hentschel and Elger,1987). They are about 2–3 times larger than in other fish classes (lamprey, Youson and McMillan,1970; European eel and Striped mullet, data not shown), and in amphibians (Møbjerg et al.,2004). However, the most striking difference between the tubular cells in the P. dolloi and those in other fish classes is the size of the nucleus, which is about three times larger than in the European eel or in the Striped mullet (data not shown). Furthermore, the nucleus shows large heterochromatin masses with a characteristic “blots-of-ink” distribution. The high nucleus/cytoplasm ratio may be due to polyploidy. This is very frequent in fish (Schultz,1979; Van de Peer et al.,2003) and seems to have played an important role in animal evolution (McLysaght et al.,2002). On the other hand, cell size in fish has been related to the amount of ribosome genes (Pedersen,1971), although this correlation has not been observed in the tetraploid Cyprinids (Schmidtke et al.,1975). Cell size has also been related to metabolic activity, with larger cells having a relatively lower metabolic rate than smaller cells (Szarski,1970). This may be of functional importance during the adaptation to climate changes.
The authors thank R. Garcia-Ceballos and M. Mier for expert technical assistance.