Renal corpuscle of the sturgeon kidneyAn ultrastructural chemical dissection and lectin-binding study.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 272A:563–573 (2003) Renal Corpuscle of the Sturgeon Kidney: An Ultrastructural, Chemical Dissection, and Lectin-Binding Study 1 JOSÉ L. OJEDA,1* JOSÉ M. ICARDO,1 AND ALBERTO DOMEZAIN2 Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, Santander, Spain 2 Department of Research and Development, Piscifactoria de Sierra Nevada, Riofrio, Spain ABSTRACT The sturgeon is an ancient species of ﬁsh that thrives in a wide range of ecological environments, from freshwater to seawater. Basic in this process of adaptation is the ability of the kidney to control ﬂuid ﬁltration and urine formation. However, the morphological basis of this process is mostly unknown. The aim of the present study was to use microdissection techniques (scanning electron microscopy (SEM), transmission electron microscopy (TEM), and lectin-binding histochemistry) to examine the structure of the renal corpuscle of the sturgeon Acipenser nacarii in order to reveal morphologic features that could be related to function, phylogeny, and habitat. The renal corpuscles are aligned along the intrarrenal arteries. The urinary pole shows a siphon-like neck segment (NS) in 92% of the nephrons, whose structural characteristics are different from those of other ﬁsh. The podocytes have cuboidal cellular bodies, intercellular contacts, and poorly developed cell processes. The podocyte glycocalyx contains N-acetylglucosamine and lacks sialic acid. The structural and lectin-binding patterns are similar to those found in the immature mammalian kidney. The glomerular basement membrane (GBM) is very thick and consists of three layers: a lamina rara externa, a lamina densa, and a thick subendothelial lamina. The latter contains tubular microﬁbrils, collagen ﬁbers, and long mesangial cell processes. Frequently, the podocyte bodies attach directly to the GBM, and the area occupied by the ﬁltration slits is very small. Furthermore, the GBM shows a glycosylation pattern different from that observed in most vertebrates. Contrary to what would be expected in sturgeons living in freshwater, the A. nacarii renal corpuscle morphology suggests a low glomerular ﬁltration rate. Anat Rec Part A 272A:563–573, 2003. © 2003 Wiley-Liss, Inc. Key words: podocytes; glomerular basement membrane; glycoconjugates; Chondrosteans; phylogeny The vertebrate kidney is the main organ involved in the maintenance of body ﬂuid homeostasis. The morphology and function of the kidney have been modiﬁed through evolution to fulﬁll different physiological requirements, and the widest range of kidney types is found in ﬁshes (Hentschel and Elger, 1989). The structure of the ﬁsh kidney has been the subject of detailed studies in many ﬁsh taxa (for review, see Elger et al., 2000). Within the group of chondrosteans, the sturgeons constitute a family (Acipenseridae) of relic ﬁsh that is phylogenetically very interesting (Dettlaff et al., 1993). Sturgeons show great ecological plasticity and are able to thrive in both freshwater and marine environments. While the kidney should play a key role in this process of adaptation, the structure of the sturgeon kidney is largely unknown. Sturgeons are an endangered species, and are also considered to be valuable commercially. Thus, a systematic study of the sturgeon kidney would © 2003 WILEY-LISS, INC. be of considerable importance from an anatomical, phylogenetic, or environmental standpoint. The renal corpuscle is an elaborate structure that specializes in the production of the glomerular ﬁltrate and in the retention of plasma proteins in the circulation. It Grant sponsor: Ministerio de Ciencia y Tecnologı́a; Grant numbers: PB98-1428-CO2-02; BMC2000-0118-CO2-01; FIT0600002000127; Grant sponsor: CEDETI; Grant number: 20000007. *Correspondence to: Prof. José L. Ojeda, Departamento de Anatomı́a y Biologı́a Celular, Facultad de Medicina, Cardenal Herrera Oria s/n, 39011-Santander, Spain. E-mail: firstname.lastname@example.org Received 17 October 2002; Accepted 14 February 2003 DOI 10.1002/ar.a.10068 564 OJEDA ET AL. consists of a globular tuft of capillaries surrounded by a chalice-shaped, double-walled cup (termed Bowman’s capsule). The parietal layer of Bowman’s capsule is formed by epithelial cells. The visceral layer of Bowman’s capsule is applied against the capillary surface in such a way that its cellular components have been transformed into highly specialized cells or podocytes. A thick basement membrane, the glomerular basement membrane (GBM), is interposed between the podocytes and the capillaries. These three elements—the capillary wall, GBM, and podocytes— constitute the ﬁltration barrier. In mammals, the functional characteristics of the ﬁltration barrier depend largely on two factors: the structural characteristics of the barrier, and the presence and distribution of negative charges in each of its three components (Latta, 1980). The negative charge of the ﬁltration barrier depends on the presence of glycoconjugates in both the podocyte coat (Kerjaschki et al., 1984; Dekan et al., 1991) and the GBM (Kanwar and Farquhar, 1979; Kanwar et al., 1980). Glycoconjugates also appear to be involved in the maintenance of the podocyte morphology (Andrews, 1979). Negatively charged sites have been found in the GBM of primitive ﬁshes (Tsujii et al., 1984a, b) and in the more advanced teleosts (Boyd and DeVries, 1983, 1986; Elger et al., 1984), indicating the existence of a functional ﬁltration barrier. Lectins are ubiquitous proteins of nonimmune origin that show a high speciﬁcity for carbohydrate moieties (Lis and Sharon, 1986; Damjanov, 1987). This characteristic has been found to be a useful tool for detecting the presence of cellular and extracellular carbohydrates. Thus, the podocytes and the GBM of the mammalian kidney exhibit lectin-binding patterns that are considered to be species speciﬁc (Holthöfer, 1983; Ojeda et al., 1993). As stated above, these patterns determine the functional characteristics of the ﬁltration barrier. However, few lectin-binding studies have involved the ﬁsh kidney, and, to our knowledge, none have been carried out in the sturgeon. The aim of the present study was to use light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and chemical microdissection techniques to examine the structure of the renal corpuscle of the amphibiotic sturgeon Acipenser naccarii. In addition, several lectin-binding patterns were analyzed to disclose speciﬁc morphologic features that could be related to function, phylogeny, and habitat. MATERIALS AND METHODS A total of eight specimens of the amphibiotic sturgeon Acipenser naccarii (A. naccarii Bonaparte 1836), whose vital cycles were entirely completed in freshwater, were collected at the Sierra Nevada Fishery (Riofrio, Spain). The sturgeons ranged in age from 17 months (0.3 kg, n ⫽ 2), 20 months (0.5 kg, n ⫽ 3), and 6 years (8 kg, n ⫽ 3). The ﬁsh were killed by a blow to the head, the ventral body wall was opened, and the kidneys were exposed. The long columnar kidneys were visually divided into four parts, and samples were taken from the two middle segments. Conventional Light Microscopy Kidney fragments were immersed in Bouin’s ﬁxative for 24 hr, dehydrated in graded ethanol, cleared in xylene, embedded in Paraplast (Sherwood Medical Co., St. Louis, MO), and serially sectioned at 10 m. Thick sections were stained with Harris hematoxylin and eosin. For semithin sections, kidney fragments were immersed in 3% glutaraldehyde in phosphate-buffered saline (PBS) for 2–3 hr, postﬁxed in 1% osmium tetroxide for 1 hr, dehydrated in graded acetone and propylene oxide, and embedded in Araldite (Fluka, Chemie GmbH, Buchs, Switzerland). Sections (1 m thick) were cut with a LKB III ultratome, stained with 1% Toluidine blue, and observed with a Zeiss III photomicroscope. SEM For SEM, kidney fragments were ﬁxed in 3% glutaraldehyde as above. After ﬁxation, some samples were dehydrated in graded acetone, dried by the critical point method, coated with gold, and viewed with a Philips SEM 501. Other samples were ﬁrst subjected to chemical and mechanical microdissection. For chemical microdissection, the specimens were digested either with HCl (Evan et al., 1976) or KOH (Ushiki and Murakumo, 1991). In both cases, collagenase digestion was omitted. 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. In addition, some microdissected specimens were mounted in a 1:1 mixture of glycerol/water, and viewed and photographed with a stereomicroscope. TEM For TEM, two types of ﬁxatives were used. Some kidney fragments were ﬁxed in 3% glutaraldehyde. Additional samples were immersed in 3% glutaraldehyde and 0.5% tannic acid, pH 7.3, for 2–3 hr. Tannic acid was used to increase the contrast of the basement membranes (Simionescu and Simionescu, 1976). After ﬁxation the specimens were embedded in Araldite, as described above. Ultrathin sections were cut with a Leica Ultracut UCT, stained with uranyl acetate and lead citrate, and examined with a Zeiss ME 10C. Lectin-Binding Studies Kidney slices were immersed in cold ethanol-glacial acetic acid (99:1) (Saint-Marie, 1962) for 1 hr, dehydrated in cold 100% ethanol, cleared in cold xylene, and embedded in diethylene glycol distearate (DGD) (Polysciences, Warrington, PA), as previously described (Ojeda et al., 1989a). Semithin sections were cut at 2 m and dried on a warm (40°C) plate. Dewaxed sections were stained for 30 min with ﬂuorescein isothiocyanate (FITC)-conjugated lectins, 50 gr/ml in PBS, of different nominal sugar speciﬁcities (Table 1), purchased from Vector Laboratories (Burlingame, CA) or Sigma Chemicals (St. Louis, MO). The slides were then washed three times (10 min each) in PBS and mounted with Vectashield (Vector). Selected tissue sections were treated with neuraminidase prior to lectin staining. Neuraminidase from Clostridium perfringens (type V; Sigma) was diluted (1 Ul/ml) in 0.05 M acetate buffer, pH 5.3, and applied to the sections for 4 hr at 37°C. This procedure exposes the penultimate carbohydrate residues blocked by sialic acid (Uehara et al., 1985). The sections were then washed in acetate buffer and stained. Lectin-binding speciﬁcity was tested by preincubation of the lectin conjugates with 0.2-M solutions of the nominal speciﬁc sugars (Sigma) (Sarkar et al., 1981; Goldstein and Poretz, 1986; Damjanov, 1987). All of the controls were routinely negative. Labeling was assessed by means of a 565 STURGEON RENAL CORPUSCLE TABLE 1. Lectin source Arachy hypogaea Canavalia ensiformis Glycine maxumun Maclura pomifera Lycopersicon esculentum Ricinus communis Triticum vulgaris Triticum vulgaris Ulex europaeus Abbreviation Nominal saccharide speciﬁcity PNA Con A SBA MPA LEA RCA-I WGA WGAs UEA-I D-galactose(␤1-3)-N-acetyl-D galactosamine ␣-D-mannose; ␣-D-glucose N-acetyl-D-galactosamine, ⬎D-galactose ␣-acetylgalactosamine, ⬎ ␣-galactosamine N-acetyl lactosamine ␤-D-galactose N-acetyl-D-glucosamine; N-acetyl neuraminic acid N-acetyl-D-glucosamine ␣-L-fucose Nominal lectin saccharide speciﬁcities (Damjanov, 1987; Goldstein and Poretz, 1986; Sarkar et al., 1981). PNA, peanut agglutinin. Con A, concanavalin A. SBA, soybean agglutinin. MPA, maclura pomifera agglutinin. LEA, lycopersicon esculentum agglutinin. RCA-I, ricinus communis (castor bean) agglutinin. WGA, wheat germ agglutinin. WGAs, succinylated wheat germ agglutinin. UEA-I, ulex europeus (gorse) agglutinin. Zeiss Axiovert 100 inverted microscope attached to a BioRad MRC-1024 confocal SE scanning laser microscope equipped with an argon ion laser (488 nm). The confocal images were processed with Photoshop (Adobe, Inc.) software. RESULTS Structure of the Renal Corpuscle The renal corpuscles appear aligned along the intrarrenal arteries, while the renal tubules occupy the spaces between the arteries (Fig. 1). Thus, an overall zonation can be observed in the sturgeon kidney. Abundant hemopoietic tissue normally surrounds the renal corpuscles (Fig. 2). The renal corpuscles are globular and small (Fig. 2), with a diameter slightly larger than that of the proximal tubules (Fig. 3). Size apparently does not vary with the location of the renal corpuscle. The afferent and efferent arterioles enter the renal corpuscle by a clearly delineated vascular pole. In most cases (92 of 100 nephrons examined for this purpose), the urinary pole shows a neck segment (NS) at the boundary between Bowman’s capsule and the proximal tubule (Fig. 3). The parietal layer of Bowman’s capsule is composed of three different cell types (Figs. 4 –7). We found squamous epithelial cells with a prominent basal surface and an elaborate pattern of grooves and ridges (Fig. 4). Other cells are columnar or rounded and protrude into the urinary space (Fig. 5). They also have a prominent basal pole, but lack ridges. These two parietal cell types show a smooth apical surface (Figs. 6 and 7) with a long central cilium (Fig. 8). In addition, the parietal layer exhibits sparsely distributed multiciliary cells (Fig. 9) similar to those observed in the NS (see below). The distribution of these cellular types is variable. Any parietal layer can contain a single cell type or two types, or be formed by clusters of the three cell types. The external surface of the parietal layer always exhibits a thick basement membrane consisting of a thin lamina lucida, a thin lamina densa, and a very thick lamina ﬁbroreticularis. The latter appears in continuity with numerous collagen ﬁbers, which form a ﬁbrous capsule around the renal corpuscle (Figs. 1 and 6). No apparent correlation was observed between the structure of the parietal layer and the size of the renal corpuscle or the weight or sex of the sturgeon. In microdissected nephrons, the NS appears as a siphon-like structure (Fig. 3). The NS connects the renal corpuscle with the proximal tubule and consists of two parts: proximal and distal. The proximal part appears as a long, funnel-shaped tube composed of squamous or slightly rounded cells (Fig. 5). The distal part is much shorter, is continuous with the proximal tubule, and is formed by multiciliary cells (Figs. 8 and 9). The cilia form a tuft that almost occludes the NS lumen (Fig. 8). The cytoplasm of these cells contains the striated rootlets of the kinocilia (Fig. 10). Curiously, the external diameter of the proximal segment appears to be smaller than that of the distal segment, although its luminal diameter is wider. This apparent contradiction is due to the different epithelial composition (squamous vs. ciliary) of the wall of the two NS segments. The vascular tuft of the renal corpuscle looks like a bunch of grapes, the external surface which is dominated by the presence of the podocyte bodies (Fig. 11). The body of the podocytes is cuboidal and is often in direct contact with the GBM (Fig. 12). The cell nucleus occupies most of the cell body and is surrounded by a thin rim of cytoplasm. The nucleus shows a spherical nuclear compartment free of chromatin (Fig. 13), which we have previously referred to as the cleared extrachromosomal domain (Icardo et al., 2002). The cytoplasm contains a prominent endoplasmic reticulum and Golgi apparatus, but cytoskeletal elements (such as microtubules and microﬁlaments) are scarce. The lateral bodies of the podocyte are frequently connected by macula adherens (Fig. 13), and most podocytes exhibit a short cilium (Fig. 11). The major processes of the podocyte are short and scarce, and most foot processes arise directly from the cell body (Fig. 11). However, the morphology and arrangement of these processes are not homogeneous. Within a single renal corpuscle, some areas present regular foot processes showing the characteristic interdigitating pattern, with ﬁltration slits and well-developed slit diaphragms (Fig. 14a and b). However, most areas show poorly developed foot processes and large podocyte processes attached directly to the GBM (Fig. 15). In these cases, the lateral cell membranes of the processes are joined by occluding junctions, and normal ﬁltration slits are not observed. The GBM consists of three layers: a lamina rara externa located beneath the foot processes, a lamina densa, and a subendothelial lamina (Fig. 14a and b). The lamina rara shows strands of ﬁbrillar material and is thicker than the lamina densa (53 nm vs. 43 nm of average diameter, as measured on 50 – 60 points on transmission electron mi- 566 OJEDA ET AL. Fig. 1. Transverse section through the kidney showing the renal corpuscle arrangement along an intrarrenal artery (arrowheads). Hematoxylin-eosin. ⫻25. Fig. 2. Semithin section through a renal corpuscle. The parietal layer of Bowman’s capsule contains squamous (long arrowheads) and columnar cells (arrows). Cleared nucleoplasmic areas appear in the podocyte cell nucleus (arrowheads). Asterisk, hemopoietic tissue; star, vascular pole. ⫻400. Fig. 3. Microdissection of a renal corpuscle and the initial part of the proximal tubule. HCl digestion. The NS (arrow) adopts a siphon-like shape. ⫻40. Fig. 4. SEM micrograph of the basal surface of the parietal layer of a renal corpuscle. The basement membrane has been removed by KOH digestion. The parietal layer consists only of squamous epithelial cells. Note the presence of tortuous ridges between neighboring cells (arrowheads). ⫻1,375. crographs). The lamina densa may be irregular, and show segments of low electron density and reduced thickness (Fig. 14b). Between the lamina densa and the capillary surface lies an extended subendothelial lamina. This lamina consists of a loose network of microﬁbrils and associated amorphous material, and occasional collagen ﬁbers (Fig. 14a and b). The microﬁbrils appear in cross sections as tubular structures with a hollow center (Fig. 14b). The thickness of the subendothelial lamina is signiﬁcantly greater in the areas where mesangial cells are present than in the areas where these cells are absent (366 nm vs. 190 nm, calculated as above). The subendothelial lamina is normally separated from the endothelial cell surface by an electron-lucent area (45–207 nm thick) which is devoid of amorphous and ﬁbrillar material (Figs. 14a and 15). This clear space cannot be considered a lamina rara. Thus, a capillary basement membrane appears to be absent. The glomerular mesangial cells cover most of the glomerular capillary surface. The mesangial cells possess elongated cell bodies and long cytoplasmic processes (Figs. 14 –17) embedded in the subendothelial lamina. These processes contain longitudinal bundles of actin ﬁlaments and dense bodies (Fig. 17). The mesangial cells establish contact with each other and with the endothelial cells by means of the cellular processes (Figs. 16 and 17). The endothelium shows numerous open pores 260 –720 nm in diameter (Figs. 15 and 18), but also exhibits large areas without fenestrae (Figs. 12 and 14a). In these areas the endothelial thickness ranges between 225–900 nm. Secretory granules of high electron density are frequently observed in the cytoplasm of the endothelial cells. Micropinocytotic vesicles are not observed. Lectin-Binding Patterns Figure 19 shows the distribution of lectin-binding sites in the different components of the renal corpuscle. These results are summarized in Table 2. The podocyte coat reacts moderately to Con A, MPA, and LEA. Afﬁnity for PNA is high, but it only appears after the neuraminidase treatment. This indicates the presence of cryptic PNAbinding sites masked by terminal sialylation. The similar staining intensity to WGA, WGAs, and WGA after neuraminidase treatment indicates that podocyte coat reactivity to WGA is exclusively due to N-acetyl-D-glucosamine. The GBM is positive for all the lectins tested except UEA-I. In fact, UEA-I only reacts with some interstitial cells. Furthermore, the GBM contains both free and cryptic PNA-binding sites. As with the podocyte coat, the afﬁnity of the GBM for WGA is exclusively due to N-acetylD-glucosamine. The parietal cell coat (PCC) and the STURGEON RENAL CORPUSCLE 567 Fig. 5. SEM view of two microdissected renal corpuscles. KOH digestion. The renal corpuscles are partly embedded in hemopoietic tissue (asterisk). The parietal layer of both corpuscles is composed of rounded epithelial cells. The NS’s (arrows) separate the glomeruli from the proximal tubules (stars). The vascular poles are indicated by arrowheads. Inset: Detailed view of the basal pole of the parietal cells. ⫻320; inset: ⫻1,250. Fig. 6. TEM micrograph showing the ﬂattened cytoplasm (arrowheads) of the squamous parietal cells. Arrow, podocyte; star, collagen ﬁbers. ⫻3,150. Fig. 7. TEM micrograph showing columnar cells in the parietal layer. Asterisk, urinary space. ⫻3,150. Fig. 8. SEM micrograph of a renal corpuscle after tissue fracture. The glomerulus has been eliminated, and the inner aspect of the parietal epithelium and the NS (arrow) are exposed. All parietal cells exhibit a cilium. The proximal part of the NS is formed by cells similar to the parietal cells. The distal part of the NS contains cells with numerous long cilia occluding the neck lumen (arrowhead). ⫻830. Fig. 9. SEM micrograph showing the presence of multiciliary cells (thin arrows) in the glomerular parietal layer. Large arrow indicates the NS. Star, glomerulus. Inset: Detail of a ciliary bundle. ⫻1,500; inset: ⫻6,000. parietal basement membrane (PBM) show the same reactivity to all the lectins tested. They are positive to ConA, MPA, NaseMPA, and SBA, and negative to PNA, NasePNA, WGA, WGAs, UEA-I, and RCA-I. Curiously, the areas of the parietal layer formed by columnar cells show a faint reactivity to LEA in both PCC and PBM, and an increase in the staining intensity for Con A in the PBM. The endothelial cell coat is only faintly positive to SBA. No cryptic MPA-binding sites are observed in any of the renal corpuscle components (Fig. 19). As with the structure of Fig. 10. TEM micrograph illustrating the structural characteristics of a cell located in the distal part of the NS. The striated rootlets of several kinocilia (arrowheads) are clearly observed. Asterisk, NS lumen. ⫻16,000. Fig. 11. SEM micrograph of a glomerulus showing the podocyte morphology. Only short main podocyte cell processes can be observed (arrows). The area occupied by the ﬁltration slits is small. Arrowheads, cilia. ⫻2,750. Fig. 12. Panoramic view of a glomerulus. The podocyte (P) cell body is attached directly (arrowheads) to the GBM (star). Endothelial cells (EC) are joined by tight junctions and lack pores in this area. ⫻5,000. Fig. 13. TEM micrograph showing cell contacts between two podocyte bodies. Asterisk indicates the cleared extrachromosomal domain. Inset: detailed view of a macula adherens between podocytes, ⫻10,000; Inset ⫻20,000. Fig. 14. TEM micrographs showing the structure of the ﬁltration barrier. Broad podocyte processes (BPP) and well-developed foot processes (large arrowheads) can be observed in a and b. a: Electron-lucent areas (stars) appear between the subendothelial lamina (SL) and the endothelial cells (EC). Note the absence of pores in this ﬁeld. Mesangial cell processes (MP) and microﬁbrils (small arrowheads) appear embedded in the subendothelial lamina. The tubular structure of the microﬁbrils is clearly visible in cross sections (inset). The lamina densa is continuous in part a, but shows zones with decreased electron density and thickness in b (small arrows). Large arrows, slit diaphragm; asterisk, lamina rara externa. a: ⫻20,000. b: ⫻31,500. Inset: ⫻50,000. STURGEON RENAL CORPUSCLE 569 Fig. 15. TEM micrograph of a section through the ﬁltration barrier. A long mesangial process (MP) appears interposed between the foot processes and the endothelial cells (asterisk). No well-developed ﬁltration slits can be observed. Arrowheads, endothelial pores; stars, electron-lucent spaces beneath the subendothelial lamina; E, erythrocyte. ⫻16,000. Fig. 16. Collagen ﬁbers (arrowhead) in the subendothelial lamina establish close relationships with the mesangial cells (MC) and their cytoplasmic processes (arrows). Tannic acid ﬁxation. ⫻10,000. Fig. 17. TEM micrograph showing two mesangial cell processes in longitudinal (asterisk) and transverse (star) sections. Note the presence of bundles of thin ﬁlaments (arrowheads). Arrow, dense body; EC, endothelial cell; P, podocyte. ⫻16,000. Fig. 18. SEM micrograph showing a luminal view of a renal capillary. Numerous open pores can be observed. ⫻10,000. the renal corpuscle, the lectin-binding patterns do not vary with the age group or the sex of the specimens. ported. The present observations show that the sturgeon NS is different from that of other ﬁsh. In lampreys, elasmobranchs, holosteans, several teleosts, and lungﬁshes (Elger et al., 2000), the NS was rather straight and consisted of only multiciliary cells. In contrast, the sturgeon NS is siphon-like and contains two distinct parts composed of different cell types. Curiously, the structure of the proximal part resembles the NS of the rabbit nephron (Ojeda and Icardo, 1991). In both sturgeons and rabbits, the proximal NS part appears externally as a constricted zone of the nephron. However, its lumen is wider than that of other nephron segments. This apparent paradox can be explained by the fact that it is composed of squamous, ﬂattened cells. The presence of multiciliary cells in the elasmobranch NS indicates that these cells play a functional role in propelling urine. However, it seems obvious that the proximal part of the sturgeon NS cannot perform a similar function. The siphon shape of the sturgeon NS suggests, as was hypothesized in a previous study of rabbits (Schonheyder and Maunsbach, 1975), that it may inﬂuence the nephron ﬁltration rate. The morphology of the sturgeon podocytes is clearly different from that of adult mammals and of other ﬁsh. However, it shares morphological characteristics with the DISCUSSION The structure of the sturgeon renal corpuscle shows important differences from that of mammals and other ﬁsh. In most ﬁsh species, the parietal layer of Bowman’s capsule is composed of squamous cells (Elger et al., 2000), although some multiciliary cells have been observed in the parietal layer of the elasmobranchs (Lacy et al., 1987). In mammals, including man, the parietal layer is also formed by a single cell type, although tubule-like cells have frequently been observed near the urinary pole (Mayer and Ottolenghi, 1947; Andrews, 1981; Haley and Bulger, 1983). The sturgeon, however, has three cell types: squamous, columnar, and multiciliary. The columnar cells are morphologically different from the squamous cells, and also show different LEA reactivity. The functional significance of the three different cell types in the parietal layer of the sturgeon renal corpuscle is at present unknown. The presence of an NS has been observed in the renal corpuscle of several ﬁsh species (Hentschel and Elger, 1989), including the sturgeon (Gambaryan, 1988). However, ultrastructural data have not previously been re- 570 OJEDA ET AL. Fig. 19. Fluorescence micrographs of renal corpuscles stained with the FITC-conjugated lectins Con A (a and b), MPA (c), PNA (d), PNA after neuramidase (N-PNA) treatment (e), WGA (f), WGAs (g), UEA-I (h), RCA-I (i), SBA (j), and LEA (k and l). IC, interstitial cells. RC, renal corpuscle. Small arrowheads, podocyte coat. Large arrowheads, GBM. Single arrow, endothelial cells. Double arrow, parietal basement membrane. See text for description. a, c, d, f, and l: ⫻1,450. b: ⫻735. e: ⫻1,100. STURGEON RENAL CORPUSCLE TABLE 2. Lectin Con A MPA Nase MPA PNA Nase PNA WGA WGAs* UEA-I RCA-I SBA LEA PC GBM ECC PCC PBM ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹(⫹⫹) ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺(⫹) ⫺ ⫹ ⫺(⫹) Nase, neuraminidase pretreated sections. *Succinylated. ⫺, ⫹, ⫹⫹ and ⫹⫹⫹ Denote no detectable, faint, moderate and intense reactivity, respectively. PC, podocyte coat; GBM, glomerular basement membrane; ECC, endothelial cell coat; PCC, parietal cell coat; PBM, parietal basement membrane. (), Intensity in columnar PCC or in PBM of columnar cells. immature mammalian kidney, such as a cuboidal shape, the presence of intercellular contacts, and poor development of foot processes. In mammals, the podocytes have an unusual, highly negatively charged glycocalyx, which contains large amounts of podocalyxin (Kerjaschki et al., 1984). This molecule is a sialoprotein with a high afﬁnity for WGA, and for PNA after neuramidase digestion (Dekan et al., 1991). In the sturgeon, however, the podocytes contain N-acetylglucosamine and lack sialic acid. A negatively charged glycocalyx appears to be essential to maintain the characteristic shape of mature podocytes in vertebrates. The podocyte body becomes cuboidal and the foot processes retract when the sialic acid is removed by neuraminidase digestion, or the negative charge is neutralized (Andrews, 1979). The lack of sialic (N-acetyl neuraminic) acid could therefore account for the particular shape of the podocytes in the sturgeon kidney. Despite the absence of sialic acid, the afﬁnity of PNA for the podocyte glycocalyx is similar in sturgeons and mammals (Holthöfer and Virtanen, 1987; Holthöfer et al., 1988). In both cases, the podocytes are negative for PNA but show cryptic PNA-binding sites. To our knowledge, the presence of N-acetylglucosamine in the podocyte glycocalyx is an exceptional characteristic in the vertebrate kidney. Although dogﬁsh podocytes exhibit pronounced binding of WGA (Hentschel and Walther, 1993), the speciﬁc sugar responsible for this binding has not been characterized. Thus, a direct comparison cannot be made between the two species. Each cell type within the renal corpuscle showed a characteristic lectin-binding pattern. The podocytes are positive for WGA, WGAs, and PNA after neuraminidase treatment, as well as for Con A, LEA, and MPA. The endothelial cells are only positive for SBA. Positivity for SBA was also described in the human renal endothelium (Holthöfer et al., 1981). In contrast, the parietal cells only showed a faint reactivity to Con A, MPA, and RCA I. The ﬁltration barrier of A. naccarii is characterized by the fact that the podocyte bodies are frequently in direct contact with the GBM, and may occlude the ﬁltration slits. This represents an obstacle to the free passage of ﬂuids. Furthermore, the renal corpuscle exhibits an extensive mesangium and a very thick GBM formed by three layers: 571 a lamina rara externa located beneath the foot process, a lamina densa, and a thick subendothelial lamina. Finally, the endothelium is relatively thick and has large zones without pores. These features are all characteristic of ﬁsh with a low glomerular ﬁltration rate (Bulger and Trump, 1968; Accini et al., 1976). The GBM in A. naccarii is about three times as thick as that of the freshwater and marine teleosts (Hickman and Trump, 1969; Zuasti et al., 1983), and about 15% thicker than that of the freshwater sturgeon A. ruthenus (Gambaryan, 1988). In mammals, the mature GBM forms by fusion of three initially independent BMs: the podocyte BM, the mesangial BM, and the capillary BM (Abrahamson, 1987). We suggest that this fusion process does not occur in A. naccarii. For instance, the lamina rara externa is thinner than the lamina densa. This has been observed in the mammalian kidney when fusion of the podocyte BM with the other BMs is prevented (Ojeda et al., 1989b). The structure and the close spatial relationship of the subendothelial lamina with the mesangium suggest that this lamina represents the mesangial component of the GBM. The tubular microﬁbrils embedded in the subendothelial lamina have been found in the GBM of several ﬁsh groups (Bargmann and Hehn, 1971; Tsujii et al., 1984a, b) and appear to be produced by the mesangial cells (Sakai and Kriz, 1987). The wide electron-lucent space that appears beneath the basal surface of the endothelial cells has previously been described in the sturgeon kidney (Gambaryan, 1988) and is unlikely to be an artifact. We suggest that it is due to the lack of production of the endothelial component of the GBM. The glycosylation pattern of the GBM in A. naccarii is very different from that in most vertebrates. However, the lack of systematic studies of lectin distribution in ﬁsh species precludes any signiﬁcant comparative analysis. This type of analysis is more easily done with higher vertebrates. The GBM of A. naccarii contains N-acetylglucosamine and lacks sialic acid, as revealed by the use of WGAs and neuramidase-WGA sequences. Sialic acid appears to be a universal marker for the GBM (Holthöfer, 1983), and appears very early during normal kidney development. It is also detected in experimental conditions (Ojeda et al., 1993), when the GBM is exclusively produced by the podocytes. The presence of free PNA-binding sites in the sturgeon GBM is another striking feature. In the mammalian kidney, free PNA-binding sites are only present in the embryonic GBM, and soon become crypted by sialylation (Laitinen et al., 1989). Also, the GBM of A. naccarii exhibits LEA-positive sites (N-acetylactosamine), in contrast with what occurs in the adult mammalian kidney (unpublished observations). The presence of numerous mesangial cells with long processes extending into the GBM has been described in other sturgeon species (Gambaryan, 1988). The mesangial cells represent an obstacle to the free passage of molecules through the ﬁltration barrier. The presence of large actin ﬁlament bundles suggests that these cells are able to contract. Contraction of mesangial cells in response to functional requirements may quickly modify the structural arrangement of the ﬁltration barrier and, consequently, the ﬁltration rate. Indeed, it has previously been suggested (Hickman, 1968) that mesangial cells are the structure responsible for quick adaptation from a freshwater to a saline environment. On the other hand, low blood serum osmolarity appears to be maintained in fresh- 572 OJEDA ET AL. water sturgeons by kidney intensive potassium excretion (Natochin et al., 1987). We are currently studying the structural characteristics of the renal tubuli in order to gain insights into their possible role in controlling the ionic balance. In conclusion, A. naccarii presents a renal corpuscle with poorly developed podocytes with a cell coat devoid of sialic acid. The GBM conserves a primitive structure and exhibits an extensive mesangium. Furthermore, the pattern of glycosylation is more similar to that found in the developing compared to the adult renal corpuscle of higher vertebrates. The renal corpuscle of the sturgeon has many structural features in common with those of immature mammalians. 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