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Renal corpuscle of the sturgeon kidneyAn ultrastructural chemical dissection and lectin-binding study.

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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: ojedajl@unican.es
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. Similar structural features have been reported in hagfish (Kühn et al., 1975, 1980), lampreys
(Youson and McMillan, 1970), elasmobranchs (Lacy et al.,
1987), and English sole (Bulger and Trump, 1968). This
suggests the existence of a common evolutionary pattern
that has followed divergent adaptive changes to meet specific functional requirements. This fits well with the phyletic position of sturgeons, teleosts, and the so-called
higher vertebrates.
ACKNOWLEDGMENTS
The authors thank R. Garcia-Ceballos and M. Mier for
expert technical assistance.
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