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The site of ultrafiltration in the kidney sac of the pulmonate gastropod Biomphalaria glabrata
TISSUE AND CELL, 1990 22 (6) 911-923 @ 1990 Longman Group UK Ltd.
0040-8166/90/0@22-0911/$10.00
M. MATRICON-GONDRAN
THE SITE OF ULTRAFILTRATION IN THE KIDNEY SAC OF THE PULMONATE GASTROPOD BIOMPHALARIA Keywords:
GLABRATA Eiomphalaria
excretion, internal defence system, kidney, podocyte-like barrier
glabrata,
cells, ultrafiltration
ABSTRACT. Ultrastructural observation of the renopericardial region in B. glabrata revealed the presence of a site for ultrafiltration located in the proximal region of the kidney sac, in the vicinity of the pericardium. This region comprises podocyte-like cells covering large hemal spaces supplied by a renal artery. Extracellular material observed in spaces between podocyte foot processes resembled small septate junctions rather than diaphragms such as observed in various kidneys. The effective ultrafiltration barrier may be the thick basal lamina of the podocytes. The hemal spaces have a connective tissue frame rich in fixed phagocytes and migrating hemocytes which may play a role in the infernal defence system of the snail.
Introduction
urine and the presence of inulin in primary urine after its injection into the circulatory system. In bivalves and in prosobranch gastropods, hemolymph ultrafiltration occurs through the walls of the cardiac cavities and primary urine passes from the pericardial cavity to the kidney(s) through the renopericardial duct(s) (Harrison, 1962; Florey and Cahill, 1977). Podocyte-like cells have been observed on the pericardial surface of heart cavities: auricles and the pericardial glands in Myth eduh (Pirie and George, 1979) and the auricle of prosobranchs (Boer et al., 1973; Andrews, 1976; Boer and Sminia, 1976). Such is not the case in Pulmonates in which ultrafiltration does not occur in the pericardium but must take place in the kidney sac itsefl (Vorwohl, 1961; Martin et al., 1965; Khan and Saleuddin, 1979). In Lymnaea stagnaiis, Wendelaar Bonga and Boer (1969) suggest that it may occur at the base of renal folds covered by flattened cells. While studying the internal defence system of the snail Biomphalaria glabrata, we examined the connective tissue and the hemal spaces of the pericardial region. The peculiar organization found in the proximal part of the kidney which has the features of an ultrafiltration site is described below. It comprises
The kidney
of gastropods is a blind sac located along the dorsal region of the mantle cavity. The proximal part of the renal sac is connected to the pericardial cavity by a renopericardial duct. In pulmonates, the renal sac ends as a ureter which opens near the pneumostome. The wall of the kidney has a uniform structure with epithelial infoldings, the cells of which, called nephrocytes, are characterized by apical microvilli forming a brush border, a large apical vacuole containing a spherocristal and a basal labyrinth consisting of intricated cytoplasmic processes associated with mitochondria (Bouillon, 1960; Skelding, 1973; Wendelaar Bonga and Boer, 1969; Khan and Saleuddin, 1979). These cells are well suited to reabsorption and secretion. The actual site of formation of primary urine in pulmonates is still a matter for speculation. According to Potts (1967), there is physiological evidence for the occurrence of ultrafiltration such as identical concentrations of minerals in hemolymph and primary Laboratoire d’Histolopie et Cytologie des Invert&b& Marins, Universitt Pierre et Marie Curie, Paris, France. Received 23 January 1990. Revised 18 July 1990. 911
912
MATRICON-GONDRAN
large hemal spaces projecting into the kidney lumen and covered by podocyte-like cells the basal lamina of which forms an ultrafiltration barrier. Hemolymph of suitable hydrostatic pressure is supplied by a renal artery (Basch, 1969; Pan, 1971). Hemal spaces, supported by connective tissue, harbour circulating hemocytes and may play a role in the snail’s internal defence. Material and Methods Albino B. glubrutu snails of the Porto Rico strain, reared in the laboratory were used in this study. They were kept in aerated water and fed with lettuce ad libitum. Electron microscope observations
The shell was cut along the suture of the terminal spire, the columellar muscle detached from the shell and the whole snail immersed in a fixative of 1% glutaraldehyde
in 0.05 M cacodylate buffer pH 7.2 at room temperature for 30 min. The renopericardial region was then dissected and left in fixative for an hour. The specimens were then rinsed overnight in 0.05 M cacodylate buffer pH 7.2 at 4°C osmicated in 2% 0~0, in the same buffer at 4°C for 1 or 2 hr, rinsed in cold buffer, dehydrated in graded ethylic alcohols, treated with propylene oxide and embedded in Epon. Semi-thin sections stained by Azur II and methylene blue were used to select regions containing the ultrafiltration site. Thin sections were contrasted by uranyl acetate and lead citrate and observed with a Philips EM 300 Electron microscope at 80 KV; magnification calibrations were controlled with a cross ruled grating (Agar Scientific). Some snails were fixed with 2% tannic acid in glutaraldehyde fixative to demonstrate the structures associated with the plasma membrane of the podocytes and. when present. the slit diaphragms between their pedicels.
Fig. la. Organization of the pcricardial region and the kidney in B. glabrata. The proximal region of the kidney (Rl) lies along the left side of the pericardial cavity (PC) and its main part (R2) follows the roof of the palleal cavity (p). The renopericardial duct (arrow head) opens into the main part of the kidney. The hemolymph collected from the renal sinus (rs) and from the vena reno-pulmonalis (double arrow) enters the auricle (A); it is pumped by the ventricle (V) into the common aorta (ca) which divides into posterior and anterior aortae (pa and aa). The anterior aorta twists around the intestine (I) and runs ventrally towards the head region. Fig. lb. Blood supply of the proximal region of the kidney (the pericardium and heart have been removed). The renal artery (ra) leaves the anterior aorta (aa), runs along the pericardial cavity and enters the proximal kidney (Rl). AG: lobes of the albumen gland; D: digestive gland; hpo: site of the hemocyte producing organ; m: columellar muscle.
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Results Organization of the proximal region of the kidney
The blind proximal end of the kidney is closely associated with the posterior region of the pericardial cavity. The sac continues forwards as a cylindrical tube located on the left side of the heart and then passes along the roof of the palleal chamber (Fig. la). In contrast with the main region of the kidney characterized by epithelial folds covered by nephrocytes, the very proximal part has a reticular aspect due to the presence of large hemal spaces covered by podocyte-like cells (Figs 2, 3). There is a sharp transition between the proximal and the main regions, podocyte like cells merging with typical nephrocytes (Figs 3,6), but the hemal spaces seem continuous with the axial space of nephrocyte epithelial folds. The strongly ciliated renopericardial duct opens into the kidney lumen at the beginning of the nephrocyte region (Figs la, 2). The proximal kidney is well supplied with hemolymph by the renal artery previously described by Basch (1969) and Pan (1971). Hemal spaces, supported by connective tissue strands, radiate from the renal artery (Figs. 2, 3). After fixation, the hemal spaces appear swollen with hemolymph, thus the podocytes seem stretched and their foot processes disconnected. Sectioning of the main aorta prior to fixation prevented the swelling of the hemal spaces but caused the proximal region structures to collapse.
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The podocytes
Most organelles of podocytes are gathered in the perinuclear region which sometimes projects into the lumen and sometimes lies on the basal lamina (Figs 4, 5). Podocyte thick basal extensions reach the basal lamina on the surface of which they branch and interdigitate. Thus they form almost typical foot processes which are irregular in size and unevenly spaced, although this may be a consequence of the swelling of the underlying hemal spaces. Apical cytoplasmic extensions run parallel to the surface, leaving a large baso-lateral space between apical and basal regions. They attach to similar extensions of neighbouring cells by a short zonula adherens followed by a short septate junction (Fig. 5; inset). We searched the pericardial surface of the heart cavities for the presence of podocyte like cells. In B. glabrata, the auricle is covered by a flat epithelium and the ventricle by thicker cells; in several places, this covering is differentiated into interdigitating processes attached to a thin basal lamina (not illustrated). Thus the wall of the heart may be the site of a limited hemolymph filtration. The ultrafiltration barrier
The presence of an ultrafiltration barrier in the proximal region of the kidney is strongly suggested by the striking difference between the hemal spaces retaining the collo’idal fraction of hemolymph which appears as grey coagulated material and the empty-looking urinary lumen (Figs 4, 5, 6).
Fig 2. On semi-thin sections, the proximal kidney (Rl) has a spongy structure due to the hemal spaces radiating from the renal artery (ra). The urinary lumen (L) in Rl is lined by podocytes. The main part of the kidney (R2) comprises epithelial infoldings covered with nephrocytes. Arrow: renopericardial duct. X 100. Fig. 3. Hemal spaces (H) of the proximal kidney are supported by a connective tissue frame (c), There is a sharp transition (arrows) between the proximal region lined by podocytes (P) and the main region lined by nephrocytes (N.). He: hemocyte; L: urinary lumen. X 650. Fig. 4. Podocytes (P) and their foot processes (p) cover the basal lamina (B) which limits the hemal spaces (H); their apical processes connected by short junctional complexes (arrows) delimit a wide baso-lateral space (S). He: hemocyte; L: urinary lumen. X 5600. Fig. 5. Basal extensions of podocytes form unevenly spaced foot processes (p). B: basal lamina; H: hemal space; J: junctional complex: L: urinary lumen; S: baso-lateral space. X 18,000. Inset: apical junctional complex comprising a desmosome (D) and a short septate junction (sj). X 40,000. Fig. 6. Sharp transition between a podocyte (P) and a nephrocyte (N) characterized apical brush border (mv) and pinocytotic vesicles. X 13,ooO.
by its
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There is no continuous endothelial layer on the hemal side of the filtration barrier which comprises only the thick basal lamina of podocytes and their pedicels (Fig. 7). Coagulated proteins were observed behind the inner side of the basal lamina. The main component of the barrier appears to be the basal lamina itself which exhibits a fibrillar or lamellar structure. Its thickness is about 50nm but is uneven; it increases and contains more layers in older snails (6 or 12 months old) (Fig. 10). Compared with the podocyte layer, the basal lamina of true nephrocytes is thin and even (Fig. 9). The foot processes of the podocytes attach to the basal lamina by thin filaments; hemidesmosomes are present (Fig. 10). There were no typical slit diaphragms between the foot processes, but thin bridges persisted in places where contacts had not been disrupted by the swelling of hemal spaces (Fig. 8) and in samples in which this swelling had been prevented. In tannic acid-treated specimens, the cell coat is well preserved and roughly equidistant bridges are seen between foot processes the membranes of which are about 15 nm apart (14.6 2 3.5 nm) (Fig. 10).
As controls, we examined other structures in B. glabrata held to have a filtering function i.e. basal cytoplasmic processes of nephrocytes and pedicels of pore cells from the connective tissue, observed in tannic acidtreated specimens. In nephrocytes near the basal lamina, the interdigitating cytoplasmic processes are connected by a few bridges; adjacent membranes are about 15 nm apart and the intervals between bridges about 1.5nm (Fig. 11). Pore cells possess filtration devices consisting of pedicels bridged by true slit diaphragms as described in Lymnaea stagnalis by Boer and Sminia (1976). In B. glabrata these structures are very similar and characterized by a 20 nm-wide space between membranes and equidistant double bridges 20nm apart (Figs 12, 13, 14). The hemal spaces
These contained coagulated hemolymph and circulating hemocytes. They are supported by a thin frame of connective tissue comprising fibroblasts, smooth muscle cells and fixed phagocytes; all these cells attach to the podocyte basal lamina by small hemidesmosomal junctions.
Fig. 7. The ultrafiltration barrier consists of podocyte lamina (B) (aged specimen). H: coagulated hemolymph 40,oGQ. Fig. 8. Adjacent membranes this two months old specimen,
foot processes (p) and the thick basal in hemal space; L: urinary lumen. X
of foot processes are connected by thin bridges the basal lamina (B) is thin. X 84,000.
Fig. 9. In the basal labyrinth of nephrocytes, interdigitated lamina consisting of a simple lamina dema (d). F: fibroblast.
cell processes X 40,OOU.
(arrows).
In
lie on a thin basal
Fig. 10. Tannic acid treatment enhances fibrillar material and bridges (arrows) connecting podocyte foot processes (p). In this six months old snail, the basal lamina shows a lamina densa (d) doubled by a thick tibrillar or lamellar layer (f) and an inner granular layer (g). X 84,000. Fig. 11. In the basal labyrinth of nephrocytes, tannic acid treatment intervals connecting cytoplasmic processes (arrows). X 84,ooO.
reveals bridges at regular
Fig. 12-14. Pedicels of pore cells (tannic acid treated specimen). 12. Tangential section of the surface of a pore cell: pedicels (circles) connected by slit diaphragms delimitate a superficial labyrinth (s) resulting from plasma membrane invagination. x 52,ooo. 13. Section perpendicular to the surface of a pore cell: pedicels (circles) are connected by double bridges (arrows). B: basal lamina. X 84,CW. 14. Tangential section: bridges between plasma membranes of cell and pedicel (circle) are evenly spaced. The cytoplasmic leaflet of both membranes is lined by dense librillar material. x. 84,ooo.
1 :
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The most typical fibroblasts are fusiform (2 or 3 pm in D.) with a small elongated nucleus and dense cytoplasm loaded with glycogen ((Yand /3 particles) (Fig. 15). Thin cytoplasmic extensions (0.2pm in D.), together with a few collagen fibers attach on the podocyte basal lamina. Muscle cells have thick extensions (2pm in D.) which contain (Yglycogen particles and dilated granular endoplasmic reticulum cisternae (Fig. 16). Myofilament bundles stretch between opposite sides of the epithelial folds and consist of thick and thin filaments attached to dense plates facing the podocyte basal lamina. Collagen fibers are also present on the surface of muscle cell extensions. Sections of nervous fibers and synapses were observed. Fixed phagocytes have long extensions (Fig. 17), their cytoplasm is abundant, rather clear and rich in organelles: llexuous mitochondria, dense granules taken to be peroxisomes, extensive granular and smooth endoplasmic reticulum and several dictyosomes comprising a few small concave saccules surrounded by small vesicles. They also contain organelles involved in cellular digestion: multivesicular bodies, secondary lysosomes and large residual bodies containing whole cells partly digested. All these features suggest intense metabolic activity. Hemocytes were rather scarce in the hemal spaces of two-months-old non-infested snails and they appeared to be mature: they had an elongated shape and thick pseudopods (Fig. 15). The most superficial part of the cytoplasm forms a dense ectoplasm with large superficial vacuoles and contains free ribosomes and /3 particles of glycogen, microfilaments and smooth endoplasmic reticulum cisternae. Most organelles are
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restricted to the endoplasm and consist of numerous small mitochondria, areas filled with (Yparticles of glycogen, granular endoplasmic reticulum, dictyosomes, and multivesicular bodies.
Discussion Our study of the anatomy and fine structure of the kidney in B. glabrata brings to light facts that confirm or explain certain known physiological processes. The production of primary urine by a typical ultrafiltration process is suggested by the following: - The proximal part of the kidney has a specialized structure in which large hemal spaces are separated from the urinary lumen by podocyte-like cells. This provides a surface area large enough to ensure efficient blood filtration. - The ultrafiltration barrier consists of a thick basal lamina and interdigitating podocyte foot processes. This ultrafiltration site of a pulmonate kidney can be compared with the renal corpuscle of vertebrates or with the filtration sites found in excretory organs of various invertebrates: the nephridia of annelids (Koechlin, 1966), the antenna1 gland of crustaceans (Kiimmel, 1967), etc. . .In pulmonates, most authors, van Aardt (1968) excepted, think that primary urine formation takes place in the kidney sac itself (Vorwohl, 1961; Martin et al., 1965; Potts, 1967, 1975; Khan and Saleuddin, 1979). While podocyte-like cells have been observed at sites of ultrafiltration in bivalves (Pirie and George, 1979), cephalopods (Witmer and Martin, 1973) and prosobranchs (Andrews and Little, 1972; Boer et
Fig. 15. Fibroblast in the hemal space (H): cytoplasmic processes (arrows) and collagen bundles (co) attach to the inner side of the podocyte basal lamina. The type 4, hemocyte (He) has a dense cytoplasm, pseudopods and superficial vacuoles. X 10,000. Fig. 16. Muscle cells contain glycogen particles and dilated rough endoplasmic reticulum; thin filaments attach to dense plates (arrows) connected to the podocyte basal lamina. N: nerve fiber. X 16,500. Fig. 17. The fixed phagocyte is rich in organelles. High phagocytic activity is suggested by the presence of pseudopodia and of numerous lysosomal structures and residual bodies (Ly). g: dictyosome; H: hemal space; L: urinary lumen; mb: multivesicular body; Ny: part of the nucleus. X 17,500.
MATRICON-GONDRAN
al., 1973; Boer and Sminia, 1976; Andrews,
1976, 1979), ultrastructural studies revealed typical nephrocytes only in the kidney of various pulmonates (Bouillon, 1960; Wendelaar Bonga and Boer, 1973; Skelding, 1973; Khan and Saleuddin, 1979). It was therefore proposed that ultrafiltration may occur through nephrocytes and their basal lamina (Vorwohl, 1961; Skelding, 1973; Khan and Saleuddin, 1979; Saleuddin et al., 1983). Compared to other transporting epithelia, the structural polarity of nephrocytes with apical microvilli and basal labyrinth, suggests a re-absorptive function rather than filtration. Podocytes of B. glubratu resemble those covering the auricle of Viuiparus (Andrews, 1979) the ‘filtration pouches’ of which seem equivalent to the wide baso-lateral spaces observed here. In B. glubrata, primary urine may accumulate in baso-lateral spaces and diffuse easily through the short apical junctional complexes of podocytes. In our species, the presence of a conspicuous filtration system in the proximal region of the kidney is an indication that, in pulmonates, primary urine is formed by the same process as in most animals. Similar filtration structures could probably be identified in other pulmonates. The ultrafiltration barrier
The ultrafiltration barrier has two main components: the thick basal lamina and the slit diaphragms of the podocytes. The basal lamina, which consists of several fibrillar layers, would appear to be very effective in molecular filtration; coagulated hemolymph is always seen on its inner side. It may also serve a mechanical function in offering resistance to blood pressure. The actual structure of the slit diaphragms was difficult to discern. Foot processes were often distorted by the swelling of hemal spaces and the material between adjacent membranes was not preserved. Previously, Andrews and Little (1972) failed to find any diaphragms in podocyte-like cells on the ventricle of Poteria. In our species, tannic acid treatment revealed atypic diaphragms either as a fuzzy material resembling the cell coat or as roughly equidistant septa spaced at about 15 nm; the mean distance between adjacent membranes was only about 15 nm.
Other known filtering structures have different features: in the mammalian renal corpuscle, spaces 39 nm wide are closed by bridges attached to a central filament (Rodewald and Karnowsky, 1974). In the coelomic sac of the crayfish kidney, two parallel helical structures occupy the 37 nm wide space and form a sieve structure with pores 5-6 nm wide (Schaffner and Rodewald, 1978). Narrower slits, 22-30nm wide, are reported in podocytes of Viuiparus auricle (Boer and Sminia, 1976), in pericardial cells of Calliphora (Crossley, 1972), in pore-cells of Lymnaea stagnalis (Boer and Sminia. 1976) and now. in pore-cells of B. glabruta (Table 1). In B. glubrutu podocytes, the material between foot processes forms 3 or 4 septa instead of a single diaphragm. Similar structures observed in the basal labyrinth of nephrocytes resemble septate junctions with respect to the distance between membranes and the intervals between bridges. but their location is basal instead of apical. We consider that podocyte foot processes and their ‘diaphragms’ do not play a major role in the ultrafiltration barrier. The thick basal lamina may be sufficient to retain a variety of hemolymph molecules and to resist fluid pressure. Granath et al. (1987) have shown that B. glubratu hemolymph proteins consist mainly of hemoglobin subunits ranging from 160-10 KD. The actual role of the ultrafiltration barrier should now be investigated by tracer studies to determine the size of the molecular filter. Blood supply of the ultrajiltration site
The renal blood supply of gastropods is essentially venous (Fretter and Graham, 1962; Bekius, 1972) and this is the case for the main portion of the kidney in B. glubrutu. In contrast, renal arteries were observed in Helix pomatia (Schmidt, 1916) and in B. glubratu (Basch, 1969; Pan, 1971) in which the renal artery, arising from the anterior aorta, reaches the proximal part of the kidney and opens abruptly into the large hemal spaces. According to Pan (1971), intricated muscle fibers form a valve regulating the blood flow to the kidney. This artery must provide the hydrostatic pressure necessary to counterbalance the colloid osmotic pressure of hemolymph and allow the formation of primary urine.
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Table 1. Characteristicsof various sieve structures Structure of slit diaphragm
Distance between adjacent membranes
Intervals of bridges
Mammalian glomerulus Rodewald and Karnovsky (1974)
39 nm
Axial filament and lateral bridges
Crayfish coelomic sac Schaffner and Rodewald (1978)
37 nm
2 parallel helical structures
Podocytes on Viviparus auricles Boer and Sminia (1976)
22 nm
Alternating half bridges (11 nm) in a double row
Podocytes in B. glabrata present study
15 nm
bridges
El5 nm
Nephrocytes in B. glabrata present study
15 nm
bridges
=15 nm
Pore cells in B. glabrata present study
20 nm
bridges double row
20 nm
Pore cells in Lymnaea stagnalis Boer and Sminia (1976)
22 nm
Half bridges (10 nm) facing each other double row
20 nm
Pericardial cells in (Calliphora Crossley (1972)
30 nm
2 rows of 12 nm bridges at a 15 nm distance
Relationship between the pericardial cavity and the kidney lumen What is the role of a functional renopericardial connection if large amounts of primary urine can be produced in the proxi-
mal kidney? Its presence may be related to the fact that the refilling of molluscan hearts is brought about by the constant volume mechanisms proposed by Krijgsman and Divaris (1955) and Ramsay (1962) and further demonstrated by Jones (1971), Dale (1973) and Florey and Cahill (1977). It implies the presence of pericardial fluid at a suitable low pressure. The pericardial surface of the heart seems permeable enough to allow the formation of sufficient pericardial fluid though it does not possess podocytes similar to those observed in prosobranchs by Boer et al. (1973) or Andrews and Little (1972). The renopericardial duct not only drains the excess of pericardial fluid as suggested by Jones (1971,1983), but also prevents the flow of primary urine into the pericardial cavity.
11 nm
15 nm
are supported by a thin frame of connective tissue comprising a few fibroblasts and muscle cells. Other types of cell which are common in B. glabrata connective tissue such as glycogen vesicular cells, pore cells (Sminia, 1972) and calcium spherule cells (Vovelle and Grasset, 1979) do not occur in this region. Numerous fixed phagocytes reminiscent of those in L. stagnalis (Sminia, 1981) were however seen, and several hemocytes were trapped in hemal spaces: most of these were fully differentiated type 4 hemocytes (Joky et al., 1983). The occurrence of fixed phagocytes and of hemocytes suggests that the proximal kidney participates in the snail defence system since these cells can ingest and retain foreign particles (Tripp, 1961; Brown and Brown, 1965; Sminia, 1981). Studies on snails injected with foreign substances or particles or harbouring parasitic organisms are under progress.
Acknowledgements Connective tissue and hemocyte components of the site of ultrafiltration The hemal spaces of the ultrafiltration site
I wish to thank Monique Letocart and Mich-
elle Roumane assistance.
for their valuable
technical
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MATRICON-GONDRAN References
Andrews, E. B. 1976. The ultrastructure of the heart and kidney of the pilid Gastropod Mollusc Marlsu cornuarietis. with special reference to filtration throughout the Architaeniglossa. J. Zool., Land., 179, 85-106. Andrews, E. B. 1979. Fine structure in relation to function in the excretory system of two species of Viuiparur. J. Mollu.xan studies. 45, 186206. Andrews, E. B. and Little, C. 1972. Structure and function in the excretory systems of some terrestrial prosobranch snails (Cyclophoridae). J. Zool., London., 168, 39>422. Basch, P. F. 1%9. The arterial system of Biomphalaria glabrata (Say). Malacologia, 7, 160-181. Bekius, R. 1972. The circulatory system of Lymnaea stagnaliF (L.). Neth. J. Zool., 22, l-58. Boer, H. H., Algera, N. H. and Lommerse A. W. 1973. Ultrastructure of possible sites of ultratiltration in some Gasteropoda, with particular reference to the auricle of the fresh-water Prosobranch Viuiparw uiuiparus L. Z. Zellforsch. Microsk. Anat., 143, 32%341.
Boer, H. H. and Sminia, T. 1976. Sieve structure of slit diaphragms of podocytes and pore cells of Gasteropod Molluscs. Cell T&s. Res., 170, 221-229. Bouillon, J. 1960. Ultrastructure des cellules r&tales des Mollusques I. Gasteropodcs pulmonca terrestrea (Helix pomatia). Ann. Sci. Nat. (Zool.), 2, 719-749. Brown, A. C. and Brown, R. J. 1965. The fate of thorium dioxide injected into the pedal sinus of &&a (Gastropoda: Prosobranchiata). J. Exp. Biol., 42, 509-519. Crossley, A. C. 1972. The ultrastructure and function of pericardial cells and other nephrocytes in an insect: C‘ulliphoru erythrocephala.
Tissue & Cell. 4, 529-560.
Dale, B. 1973. Blood pressure and its hydraulic functions in Helix pomatia L. J. Exp. Biol., 59, 477-490. Florey, E. and Cahill, M. A. 1977. Hemodynamics in Lamellibranch Molluscs: confirmation of constant volume mechanism of auricular and ventricular filling. Remarks on the heart as site of ultrafiltration. Camp. B&hem. Physiol., 57A, 47-52.
Fretter, V. and Graham, A. 1962. British Prosobranch Molluscs. Ray Society, London. Granath, 0. W. Jr., Spray, F. J. and Judd, R. C. 1987. Analysis of Biomphalaria glubrata (Gastropoda) hemolymph by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high performance liquid chromatography and immunoblotting. J. Invert. Pathol., 49, 198-208. Harrison, F. M. 1962. Some excretory processes in ti-c abalone Hal&is rufescens. J. Exp. Blol., 39, 17’)_lY2. Joky, A., Matricon-Gondran, M. and Benex, J. 1983. Fine structural differences in the amoebocytes of Biomphalariu glabrata. Deu. Camp. Immunol., 7, 669-672. Jones, H. D., 1971. Circulatory pressures in Helix pomatia L. Camp. Biochem. Phys~ol., 39A, 28%295. Jones, H. D. 1983. Circulatory systems of Gastropods and Bivalves. In The Mollurca (Eds A. S. M. Saleuddin and K. M. Wilbur), A. P., vol. 5, Physiology, Part 2, pp. 189-238. Khan, H. R. and Saleuddin, A. S. M. 1979. Effects of osmotic changes and nemosecretory extract\ on kidney ultrastructure in the fresh-water pulmonate Helisoma Can. J. Zoo/. . 57, 12561270. Koechlin, N. 1966. Ultrastructure du plexus sanguin perioesophagien; ses relations avec la ntphridie de Sabella paaonina Savigny. C. R. Acad. Sci. (Paris). 262, Ser. D, 12661269. Krijgsman, B. J. and Divaris, G. A. 1955. Contractile and pacemaker mechanism of the heart of molluscs. Bzol. Reu. Cambridge Philos. Sot., 30, l-39. Kiimmel, G. 1967. Die Podocyten. Zool. Beitr., N.F. 13, 245-262. Martin, A. W., Stewart, D. M. and Harrison. F. M. 1965. Urine formatton in a pulmonate land snail. Achatina fulicu. J. exp. Biol., 42, 99-123.
Pan, C. T. 1971. The arterial system of the Planorbid snail Biomphaluria glabrutu. Trans. Amer. microsc. Sot..
90,
434-440.
Pirie, B. J. S. and George, S. G. 1979. Ultrastructure
of the heart and excretory system of Myti[u_sedulis (L.). J. Mar.
Biol. Ass. U.K., 59, 819-829.
Potts, W. T. W. 1967. Excretion in the Molluscs. Biol. Reu.. 42, l-41. Potts, W. T. W. 1975. Excretion in Gastropods. In Exkretion (ed. A. Wening), Gustav Fischer Verlag, Stuttgart,
pp.
76-88.
Ramsay, J. A. 1952. ‘A physiological approach to the lower animals’. Cambridge University Press. London. Rodewald, R. and Karnovsky, M. J. 1974. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J. Cell Biol.,
60,423-433.
Saleuddin, A. S. M., Farrel, C. L., Gomot, L. and Khan, H. R. 1983. Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix asperse Miiller. J. Morph., 178, 313-322.
Schaffner, A. and Rodewald, R. 1978. Filtration barriers in the coelomic sac of the Crayfish. Procumbarus clarkii. J. Ultrastruct. Res., 65, 3tG47.
Schmidt. G. 1916. Blutgefaszsystem und Mantelhole der Weinbergschnecke 261.
(Helixpomatia).
Z. Wiss. Zool., 115,201-
SITE OF ULTRAFILTRATION
IN A PULMONATE
KIDNEY
923
Skelding, J. M. 1973. The fme structure of the kidney of Achatina achatina (L.). Z. ZeNforsch. Mikrosk. Anat., 147, l-29.
Sminia, T. 1972. Structure and function of blood and connective tissue cells of the freshwater pulmonate Lymnaea sragnalk studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. Mikrosk. Anat., 130,497-526. Sminia, T. 1981. Phagocytic cells in Molluscs. In Aspects of Developmental and Comparatioe Immunology (cd. J. B. Salomon), Pergamon Press. Oxford, New York, pp. 125-132. Tripp, M. R. 1961. The fate of foreign materials experimentally introduced into the snail Australorbis glabratus. J. Parasit., 47, 745-751.
Van Aardt, W. .I. 1968. Quantitative aspects of the water balance in Lymnaea stagnalis (L.). Neth. J. Zool., 18, 253312. Vorwhol, G. 1961. Zur Funktion der Exkretions-organe van Helix pomaria L. und Archachatina uentricosa Gould. Z. Vergl. Physiol., 45, 12-49.
Vovelle, J. and Grassct, M. 1979. Approche histophysiologique et cytologique du role des cellules g sphtrules calciques du repli operculaire chez Pomatias elegans (Mtiller), Gasteropode Prosobranche. Proc. Sixth. Europ. M&c. Congr., Malacologia, 18, 557-560. Wendelaar Bonga, S. E. and Boer, H. H. 1969. Ultrastructure of the rcnopericardial system in the pond snail Lymnaea stagnalis (L.). Z. Zellforsch. Mikrosk. Anat. 94, 51%529. Witmer, A. and Martin, A. W. 1973. The fine structure of the branchial heart appendage of the cephalopod Octopus dofleini martini. Z. Zellforsch. Mikrosk. Anat.. 136, 54>568.
0040-8166/90/0@22-0911/$10.00
M. MATRICON-GONDRAN
THE SITE OF ULTRAFILTRATION IN THE KIDNEY SAC OF THE PULMONATE GASTROPOD BIOMPHALARIA Keywords:
GLABRATA Eiomphalaria
excretion, internal defence system, kidney, podocyte-like barrier
glabrata,
cells, ultrafiltration
ABSTRACT. Ultrastructural observation of the renopericardial region in B. glabrata revealed the presence of a site for ultrafiltration located in the proximal region of the kidney sac, in the vicinity of the pericardium. This region comprises podocyte-like cells covering large hemal spaces supplied by a renal artery. Extracellular material observed in spaces between podocyte foot processes resembled small septate junctions rather than diaphragms such as observed in various kidneys. The effective ultrafiltration barrier may be the thick basal lamina of the podocytes. The hemal spaces have a connective tissue frame rich in fixed phagocytes and migrating hemocytes which may play a role in the infernal defence system of the snail.
Introduction
urine and the presence of inulin in primary urine after its injection into the circulatory system. In bivalves and in prosobranch gastropods, hemolymph ultrafiltration occurs through the walls of the cardiac cavities and primary urine passes from the pericardial cavity to the kidney(s) through the renopericardial duct(s) (Harrison, 1962; Florey and Cahill, 1977). Podocyte-like cells have been observed on the pericardial surface of heart cavities: auricles and the pericardial glands in Myth eduh (Pirie and George, 1979) and the auricle of prosobranchs (Boer et al., 1973; Andrews, 1976; Boer and Sminia, 1976). Such is not the case in Pulmonates in which ultrafiltration does not occur in the pericardium but must take place in the kidney sac itsefl (Vorwohl, 1961; Martin et al., 1965; Khan and Saleuddin, 1979). In Lymnaea stagnaiis, Wendelaar Bonga and Boer (1969) suggest that it may occur at the base of renal folds covered by flattened cells. While studying the internal defence system of the snail Biomphalaria glabrata, we examined the connective tissue and the hemal spaces of the pericardial region. The peculiar organization found in the proximal part of the kidney which has the features of an ultrafiltration site is described below. It comprises
The kidney
of gastropods is a blind sac located along the dorsal region of the mantle cavity. The proximal part of the renal sac is connected to the pericardial cavity by a renopericardial duct. In pulmonates, the renal sac ends as a ureter which opens near the pneumostome. The wall of the kidney has a uniform structure with epithelial infoldings, the cells of which, called nephrocytes, are characterized by apical microvilli forming a brush border, a large apical vacuole containing a spherocristal and a basal labyrinth consisting of intricated cytoplasmic processes associated with mitochondria (Bouillon, 1960; Skelding, 1973; Wendelaar Bonga and Boer, 1969; Khan and Saleuddin, 1979). These cells are well suited to reabsorption and secretion. The actual site of formation of primary urine in pulmonates is still a matter for speculation. According to Potts (1967), there is physiological evidence for the occurrence of ultrafiltration such as identical concentrations of minerals in hemolymph and primary Laboratoire d’Histolopie et Cytologie des Invert&b& Marins, Universitt Pierre et Marie Curie, Paris, France. Received 23 January 1990. Revised 18 July 1990. 911
912
MATRICON-GONDRAN
large hemal spaces projecting into the kidney lumen and covered by podocyte-like cells the basal lamina of which forms an ultrafiltration barrier. Hemolymph of suitable hydrostatic pressure is supplied by a renal artery (Basch, 1969; Pan, 1971). Hemal spaces, supported by connective tissue, harbour circulating hemocytes and may play a role in the snail’s internal defence. Material and Methods Albino B. glubrutu snails of the Porto Rico strain, reared in the laboratory were used in this study. They were kept in aerated water and fed with lettuce ad libitum. Electron microscope observations
The shell was cut along the suture of the terminal spire, the columellar muscle detached from the shell and the whole snail immersed in a fixative of 1% glutaraldehyde
in 0.05 M cacodylate buffer pH 7.2 at room temperature for 30 min. The renopericardial region was then dissected and left in fixative for an hour. The specimens were then rinsed overnight in 0.05 M cacodylate buffer pH 7.2 at 4°C osmicated in 2% 0~0, in the same buffer at 4°C for 1 or 2 hr, rinsed in cold buffer, dehydrated in graded ethylic alcohols, treated with propylene oxide and embedded in Epon. Semi-thin sections stained by Azur II and methylene blue were used to select regions containing the ultrafiltration site. Thin sections were contrasted by uranyl acetate and lead citrate and observed with a Philips EM 300 Electron microscope at 80 KV; magnification calibrations were controlled with a cross ruled grating (Agar Scientific). Some snails were fixed with 2% tannic acid in glutaraldehyde fixative to demonstrate the structures associated with the plasma membrane of the podocytes and. when present. the slit diaphragms between their pedicels.
Fig. la. Organization of the pcricardial region and the kidney in B. glabrata. The proximal region of the kidney (Rl) lies along the left side of the pericardial cavity (PC) and its main part (R2) follows the roof of the palleal cavity (p). The renopericardial duct (arrow head) opens into the main part of the kidney. The hemolymph collected from the renal sinus (rs) and from the vena reno-pulmonalis (double arrow) enters the auricle (A); it is pumped by the ventricle (V) into the common aorta (ca) which divides into posterior and anterior aortae (pa and aa). The anterior aorta twists around the intestine (I) and runs ventrally towards the head region. Fig. lb. Blood supply of the proximal region of the kidney (the pericardium and heart have been removed). The renal artery (ra) leaves the anterior aorta (aa), runs along the pericardial cavity and enters the proximal kidney (Rl). AG: lobes of the albumen gland; D: digestive gland; hpo: site of the hemocyte producing organ; m: columellar muscle.
SITE OF ULTRAFILTRATION
IN A PULMONATE
Results Organization of the proximal region of the kidney
The blind proximal end of the kidney is closely associated with the posterior region of the pericardial cavity. The sac continues forwards as a cylindrical tube located on the left side of the heart and then passes along the roof of the palleal chamber (Fig. la). In contrast with the main region of the kidney characterized by epithelial folds covered by nephrocytes, the very proximal part has a reticular aspect due to the presence of large hemal spaces covered by podocyte-like cells (Figs 2, 3). There is a sharp transition between the proximal and the main regions, podocyte like cells merging with typical nephrocytes (Figs 3,6), but the hemal spaces seem continuous with the axial space of nephrocyte epithelial folds. The strongly ciliated renopericardial duct opens into the kidney lumen at the beginning of the nephrocyte region (Figs la, 2). The proximal kidney is well supplied with hemolymph by the renal artery previously described by Basch (1969) and Pan (1971). Hemal spaces, supported by connective tissue strands, radiate from the renal artery (Figs. 2, 3). After fixation, the hemal spaces appear swollen with hemolymph, thus the podocytes seem stretched and their foot processes disconnected. Sectioning of the main aorta prior to fixation prevented the swelling of the hemal spaces but caused the proximal region structures to collapse.
KIDNEY
913
The podocytes
Most organelles of podocytes are gathered in the perinuclear region which sometimes projects into the lumen and sometimes lies on the basal lamina (Figs 4, 5). Podocyte thick basal extensions reach the basal lamina on the surface of which they branch and interdigitate. Thus they form almost typical foot processes which are irregular in size and unevenly spaced, although this may be a consequence of the swelling of the underlying hemal spaces. Apical cytoplasmic extensions run parallel to the surface, leaving a large baso-lateral space between apical and basal regions. They attach to similar extensions of neighbouring cells by a short zonula adherens followed by a short septate junction (Fig. 5; inset). We searched the pericardial surface of the heart cavities for the presence of podocyte like cells. In B. glabrata, the auricle is covered by a flat epithelium and the ventricle by thicker cells; in several places, this covering is differentiated into interdigitating processes attached to a thin basal lamina (not illustrated). Thus the wall of the heart may be the site of a limited hemolymph filtration. The ultrafiltration barrier
The presence of an ultrafiltration barrier in the proximal region of the kidney is strongly suggested by the striking difference between the hemal spaces retaining the collo’idal fraction of hemolymph which appears as grey coagulated material and the empty-looking urinary lumen (Figs 4, 5, 6).
Fig 2. On semi-thin sections, the proximal kidney (Rl) has a spongy structure due to the hemal spaces radiating from the renal artery (ra). The urinary lumen (L) in Rl is lined by podocytes. The main part of the kidney (R2) comprises epithelial infoldings covered with nephrocytes. Arrow: renopericardial duct. X 100. Fig. 3. Hemal spaces (H) of the proximal kidney are supported by a connective tissue frame (c), There is a sharp transition (arrows) between the proximal region lined by podocytes (P) and the main region lined by nephrocytes (N.). He: hemocyte; L: urinary lumen. X 650. Fig. 4. Podocytes (P) and their foot processes (p) cover the basal lamina (B) which limits the hemal spaces (H); their apical processes connected by short junctional complexes (arrows) delimit a wide baso-lateral space (S). He: hemocyte; L: urinary lumen. X 5600. Fig. 5. Basal extensions of podocytes form unevenly spaced foot processes (p). B: basal lamina; H: hemal space; J: junctional complex: L: urinary lumen; S: baso-lateral space. X 18,000. Inset: apical junctional complex comprising a desmosome (D) and a short septate junction (sj). X 40,000. Fig. 6. Sharp transition between a podocyte (P) and a nephrocyte (N) characterized apical brush border (mv) and pinocytotic vesicles. X 13,ooO.
by its
02
MATRICON-GONDRAN
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There is no continuous endothelial layer on the hemal side of the filtration barrier which comprises only the thick basal lamina of podocytes and their pedicels (Fig. 7). Coagulated proteins were observed behind the inner side of the basal lamina. The main component of the barrier appears to be the basal lamina itself which exhibits a fibrillar or lamellar structure. Its thickness is about 50nm but is uneven; it increases and contains more layers in older snails (6 or 12 months old) (Fig. 10). Compared with the podocyte layer, the basal lamina of true nephrocytes is thin and even (Fig. 9). The foot processes of the podocytes attach to the basal lamina by thin filaments; hemidesmosomes are present (Fig. 10). There were no typical slit diaphragms between the foot processes, but thin bridges persisted in places where contacts had not been disrupted by the swelling of hemal spaces (Fig. 8) and in samples in which this swelling had been prevented. In tannic acid-treated specimens, the cell coat is well preserved and roughly equidistant bridges are seen between foot processes the membranes of which are about 15 nm apart (14.6 2 3.5 nm) (Fig. 10).
As controls, we examined other structures in B. glabrata held to have a filtering function i.e. basal cytoplasmic processes of nephrocytes and pedicels of pore cells from the connective tissue, observed in tannic acidtreated specimens. In nephrocytes near the basal lamina, the interdigitating cytoplasmic processes are connected by a few bridges; adjacent membranes are about 15 nm apart and the intervals between bridges about 1.5nm (Fig. 11). Pore cells possess filtration devices consisting of pedicels bridged by true slit diaphragms as described in Lymnaea stagnalis by Boer and Sminia (1976). In B. glabrata these structures are very similar and characterized by a 20 nm-wide space between membranes and equidistant double bridges 20nm apart (Figs 12, 13, 14). The hemal spaces
These contained coagulated hemolymph and circulating hemocytes. They are supported by a thin frame of connective tissue comprising fibroblasts, smooth muscle cells and fixed phagocytes; all these cells attach to the podocyte basal lamina by small hemidesmosomal junctions.
Fig. 7. The ultrafiltration barrier consists of podocyte lamina (B) (aged specimen). H: coagulated hemolymph 40,oGQ. Fig. 8. Adjacent membranes this two months old specimen,
foot processes (p) and the thick basal in hemal space; L: urinary lumen. X
of foot processes are connected by thin bridges the basal lamina (B) is thin. X 84,000.
Fig. 9. In the basal labyrinth of nephrocytes, interdigitated lamina consisting of a simple lamina dema (d). F: fibroblast.
cell processes X 40,OOU.
(arrows).
In
lie on a thin basal
Fig. 10. Tannic acid treatment enhances fibrillar material and bridges (arrows) connecting podocyte foot processes (p). In this six months old snail, the basal lamina shows a lamina densa (d) doubled by a thick tibrillar or lamellar layer (f) and an inner granular layer (g). X 84,000. Fig. 11. In the basal labyrinth of nephrocytes, tannic acid treatment intervals connecting cytoplasmic processes (arrows). X 84,ooO.
reveals bridges at regular
Fig. 12-14. Pedicels of pore cells (tannic acid treated specimen). 12. Tangential section of the surface of a pore cell: pedicels (circles) connected by slit diaphragms delimitate a superficial labyrinth (s) resulting from plasma membrane invagination. x 52,ooo. 13. Section perpendicular to the surface of a pore cell: pedicels (circles) are connected by double bridges (arrows). B: basal lamina. X 84,CW. 14. Tangential section: bridges between plasma membranes of cell and pedicel (circle) are evenly spaced. The cytoplasmic leaflet of both membranes is lined by dense librillar material. x. 84,ooo.
1 :
?
SITE OF ULTRAFILTRATION
IN A PULMONATE
The most typical fibroblasts are fusiform (2 or 3 pm in D.) with a small elongated nucleus and dense cytoplasm loaded with glycogen ((Yand /3 particles) (Fig. 15). Thin cytoplasmic extensions (0.2pm in D.), together with a few collagen fibers attach on the podocyte basal lamina. Muscle cells have thick extensions (2pm in D.) which contain (Yglycogen particles and dilated granular endoplasmic reticulum cisternae (Fig. 16). Myofilament bundles stretch between opposite sides of the epithelial folds and consist of thick and thin filaments attached to dense plates facing the podocyte basal lamina. Collagen fibers are also present on the surface of muscle cell extensions. Sections of nervous fibers and synapses were observed. Fixed phagocytes have long extensions (Fig. 17), their cytoplasm is abundant, rather clear and rich in organelles: llexuous mitochondria, dense granules taken to be peroxisomes, extensive granular and smooth endoplasmic reticulum and several dictyosomes comprising a few small concave saccules surrounded by small vesicles. They also contain organelles involved in cellular digestion: multivesicular bodies, secondary lysosomes and large residual bodies containing whole cells partly digested. All these features suggest intense metabolic activity. Hemocytes were rather scarce in the hemal spaces of two-months-old non-infested snails and they appeared to be mature: they had an elongated shape and thick pseudopods (Fig. 15). The most superficial part of the cytoplasm forms a dense ectoplasm with large superficial vacuoles and contains free ribosomes and /3 particles of glycogen, microfilaments and smooth endoplasmic reticulum cisternae. Most organelles are
KIDNEY
919
restricted to the endoplasm and consist of numerous small mitochondria, areas filled with (Yparticles of glycogen, granular endoplasmic reticulum, dictyosomes, and multivesicular bodies.
Discussion Our study of the anatomy and fine structure of the kidney in B. glabrata brings to light facts that confirm or explain certain known physiological processes. The production of primary urine by a typical ultrafiltration process is suggested by the following: - The proximal part of the kidney has a specialized structure in which large hemal spaces are separated from the urinary lumen by podocyte-like cells. This provides a surface area large enough to ensure efficient blood filtration. - The ultrafiltration barrier consists of a thick basal lamina and interdigitating podocyte foot processes. This ultrafiltration site of a pulmonate kidney can be compared with the renal corpuscle of vertebrates or with the filtration sites found in excretory organs of various invertebrates: the nephridia of annelids (Koechlin, 1966), the antenna1 gland of crustaceans (Kiimmel, 1967), etc. . .In pulmonates, most authors, van Aardt (1968) excepted, think that primary urine formation takes place in the kidney sac itself (Vorwohl, 1961; Martin et al., 1965; Potts, 1967, 1975; Khan and Saleuddin, 1979). While podocyte-like cells have been observed at sites of ultrafiltration in bivalves (Pirie and George, 1979), cephalopods (Witmer and Martin, 1973) and prosobranchs (Andrews and Little, 1972; Boer et
Fig. 15. Fibroblast in the hemal space (H): cytoplasmic processes (arrows) and collagen bundles (co) attach to the inner side of the podocyte basal lamina. The type 4, hemocyte (He) has a dense cytoplasm, pseudopods and superficial vacuoles. X 10,000. Fig. 16. Muscle cells contain glycogen particles and dilated rough endoplasmic reticulum; thin filaments attach to dense plates (arrows) connected to the podocyte basal lamina. N: nerve fiber. X 16,500. Fig. 17. The fixed phagocyte is rich in organelles. High phagocytic activity is suggested by the presence of pseudopodia and of numerous lysosomal structures and residual bodies (Ly). g: dictyosome; H: hemal space; L: urinary lumen; mb: multivesicular body; Ny: part of the nucleus. X 17,500.
MATRICON-GONDRAN
al., 1973; Boer and Sminia, 1976; Andrews,
1976, 1979), ultrastructural studies revealed typical nephrocytes only in the kidney of various pulmonates (Bouillon, 1960; Wendelaar Bonga and Boer, 1973; Skelding, 1973; Khan and Saleuddin, 1979). It was therefore proposed that ultrafiltration may occur through nephrocytes and their basal lamina (Vorwohl, 1961; Skelding, 1973; Khan and Saleuddin, 1979; Saleuddin et al., 1983). Compared to other transporting epithelia, the structural polarity of nephrocytes with apical microvilli and basal labyrinth, suggests a re-absorptive function rather than filtration. Podocytes of B. glubratu resemble those covering the auricle of Viuiparus (Andrews, 1979) the ‘filtration pouches’ of which seem equivalent to the wide baso-lateral spaces observed here. In B. glubrata, primary urine may accumulate in baso-lateral spaces and diffuse easily through the short apical junctional complexes of podocytes. In our species, the presence of a conspicuous filtration system in the proximal region of the kidney is an indication that, in pulmonates, primary urine is formed by the same process as in most animals. Similar filtration structures could probably be identified in other pulmonates. The ultrafiltration barrier
The ultrafiltration barrier has two main components: the thick basal lamina and the slit diaphragms of the podocytes. The basal lamina, which consists of several fibrillar layers, would appear to be very effective in molecular filtration; coagulated hemolymph is always seen on its inner side. It may also serve a mechanical function in offering resistance to blood pressure. The actual structure of the slit diaphragms was difficult to discern. Foot processes were often distorted by the swelling of hemal spaces and the material between adjacent membranes was not preserved. Previously, Andrews and Little (1972) failed to find any diaphragms in podocyte-like cells on the ventricle of Poteria. In our species, tannic acid treatment revealed atypic diaphragms either as a fuzzy material resembling the cell coat or as roughly equidistant septa spaced at about 15 nm; the mean distance between adjacent membranes was only about 15 nm.
Other known filtering structures have different features: in the mammalian renal corpuscle, spaces 39 nm wide are closed by bridges attached to a central filament (Rodewald and Karnowsky, 1974). In the coelomic sac of the crayfish kidney, two parallel helical structures occupy the 37 nm wide space and form a sieve structure with pores 5-6 nm wide (Schaffner and Rodewald, 1978). Narrower slits, 22-30nm wide, are reported in podocytes of Viuiparus auricle (Boer and Sminia, 1976), in pericardial cells of Calliphora (Crossley, 1972), in pore-cells of Lymnaea stagnalis (Boer and Sminia. 1976) and now. in pore-cells of B. glabruta (Table 1). In B. glubrutu podocytes, the material between foot processes forms 3 or 4 septa instead of a single diaphragm. Similar structures observed in the basal labyrinth of nephrocytes resemble septate junctions with respect to the distance between membranes and the intervals between bridges. but their location is basal instead of apical. We consider that podocyte foot processes and their ‘diaphragms’ do not play a major role in the ultrafiltration barrier. The thick basal lamina may be sufficient to retain a variety of hemolymph molecules and to resist fluid pressure. Granath et al. (1987) have shown that B. glubratu hemolymph proteins consist mainly of hemoglobin subunits ranging from 160-10 KD. The actual role of the ultrafiltration barrier should now be investigated by tracer studies to determine the size of the molecular filter. Blood supply of the ultrajiltration site
The renal blood supply of gastropods is essentially venous (Fretter and Graham, 1962; Bekius, 1972) and this is the case for the main portion of the kidney in B. glubrutu. In contrast, renal arteries were observed in Helix pomatia (Schmidt, 1916) and in B. glubratu (Basch, 1969; Pan, 1971) in which the renal artery, arising from the anterior aorta, reaches the proximal part of the kidney and opens abruptly into the large hemal spaces. According to Pan (1971), intricated muscle fibers form a valve regulating the blood flow to the kidney. This artery must provide the hydrostatic pressure necessary to counterbalance the colloid osmotic pressure of hemolymph and allow the formation of primary urine.
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OF ULTRAFILTRATION
IN A PULMONATE
KIDNEY
921
Table 1. Characteristicsof various sieve structures Structure of slit diaphragm
Distance between adjacent membranes
Intervals of bridges
Mammalian glomerulus Rodewald and Karnovsky (1974)
39 nm
Axial filament and lateral bridges
Crayfish coelomic sac Schaffner and Rodewald (1978)
37 nm
2 parallel helical structures
Podocytes on Viviparus auricles Boer and Sminia (1976)
22 nm
Alternating half bridges (11 nm) in a double row
Podocytes in B. glabrata present study
15 nm
bridges
El5 nm
Nephrocytes in B. glabrata present study
15 nm
bridges
=15 nm
Pore cells in B. glabrata present study
20 nm
bridges double row
20 nm
Pore cells in Lymnaea stagnalis Boer and Sminia (1976)
22 nm
Half bridges (10 nm) facing each other double row
20 nm
Pericardial cells in (Calliphora Crossley (1972)
30 nm
2 rows of 12 nm bridges at a 15 nm distance
Relationship between the pericardial cavity and the kidney lumen What is the role of a functional renopericardial connection if large amounts of primary urine can be produced in the proxi-
mal kidney? Its presence may be related to the fact that the refilling of molluscan hearts is brought about by the constant volume mechanisms proposed by Krijgsman and Divaris (1955) and Ramsay (1962) and further demonstrated by Jones (1971), Dale (1973) and Florey and Cahill (1977). It implies the presence of pericardial fluid at a suitable low pressure. The pericardial surface of the heart seems permeable enough to allow the formation of sufficient pericardial fluid though it does not possess podocytes similar to those observed in prosobranchs by Boer et al. (1973) or Andrews and Little (1972). The renopericardial duct not only drains the excess of pericardial fluid as suggested by Jones (1971,1983), but also prevents the flow of primary urine into the pericardial cavity.
11 nm
15 nm
are supported by a thin frame of connective tissue comprising a few fibroblasts and muscle cells. Other types of cell which are common in B. glabrata connective tissue such as glycogen vesicular cells, pore cells (Sminia, 1972) and calcium spherule cells (Vovelle and Grasset, 1979) do not occur in this region. Numerous fixed phagocytes reminiscent of those in L. stagnalis (Sminia, 1981) were however seen, and several hemocytes were trapped in hemal spaces: most of these were fully differentiated type 4 hemocytes (Joky et al., 1983). The occurrence of fixed phagocytes and of hemocytes suggests that the proximal kidney participates in the snail defence system since these cells can ingest and retain foreign particles (Tripp, 1961; Brown and Brown, 1965; Sminia, 1981). Studies on snails injected with foreign substances or particles or harbouring parasitic organisms are under progress.
Acknowledgements Connective tissue and hemocyte components of the site of ultrafiltration The hemal spaces of the ultrafiltration site
I wish to thank Monique Letocart and Mich-
elle Roumane assistance.
for their valuable
technical
922
MATRICON-GONDRAN References
Andrews, E. B. 1976. The ultrastructure of the heart and kidney of the pilid Gastropod Mollusc Marlsu cornuarietis. with special reference to filtration throughout the Architaeniglossa. J. Zool., Land., 179, 85-106. Andrews, E. B. 1979. Fine structure in relation to function in the excretory system of two species of Viuiparur. J. Mollu.xan studies. 45, 186206. Andrews, E. B. and Little, C. 1972. Structure and function in the excretory systems of some terrestrial prosobranch snails (Cyclophoridae). J. Zool., London., 168, 39>422. Basch, P. F. 1%9. The arterial system of Biomphalaria glabrata (Say). Malacologia, 7, 160-181. Bekius, R. 1972. The circulatory system of Lymnaea stagnaliF (L.). Neth. J. Zool., 22, l-58. Boer, H. H., Algera, N. H. and Lommerse A. W. 1973. Ultrastructure of possible sites of ultratiltration in some Gasteropoda, with particular reference to the auricle of the fresh-water Prosobranch Viuiparw uiuiparus L. Z. Zellforsch. Microsk. Anat., 143, 32%341.
Boer, H. H. and Sminia, T. 1976. Sieve structure of slit diaphragms of podocytes and pore cells of Gasteropod Molluscs. Cell T&s. Res., 170, 221-229. Bouillon, J. 1960. Ultrastructure des cellules r&tales des Mollusques I. Gasteropodcs pulmonca terrestrea (Helix pomatia). Ann. Sci. Nat. (Zool.), 2, 719-749. Brown, A. C. and Brown, R. J. 1965. The fate of thorium dioxide injected into the pedal sinus of &&a (Gastropoda: Prosobranchiata). J. Exp. Biol., 42, 509-519. Crossley, A. C. 1972. The ultrastructure and function of pericardial cells and other nephrocytes in an insect: C‘ulliphoru erythrocephala.
Tissue & Cell. 4, 529-560.
Dale, B. 1973. Blood pressure and its hydraulic functions in Helix pomatia L. J. Exp. Biol., 59, 477-490. Florey, E. and Cahill, M. A. 1977. Hemodynamics in Lamellibranch Molluscs: confirmation of constant volume mechanism of auricular and ventricular filling. Remarks on the heart as site of ultrafiltration. Camp. B&hem. Physiol., 57A, 47-52.
Fretter, V. and Graham, A. 1962. British Prosobranch Molluscs. Ray Society, London. Granath, 0. W. Jr., Spray, F. J. and Judd, R. C. 1987. Analysis of Biomphalaria glubrata (Gastropoda) hemolymph by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high performance liquid chromatography and immunoblotting. J. Invert. Pathol., 49, 198-208. Harrison, F. M. 1962. Some excretory processes in ti-c abalone Hal&is rufescens. J. Exp. Blol., 39, 17’)_lY2. Joky, A., Matricon-Gondran, M. and Benex, J. 1983. Fine structural differences in the amoebocytes of Biomphalariu glabrata. Deu. Camp. Immunol., 7, 669-672. Jones, H. D., 1971. Circulatory pressures in Helix pomatia L. Camp. Biochem. Phys~ol., 39A, 28%295. Jones, H. D. 1983. Circulatory systems of Gastropods and Bivalves. In The Mollurca (Eds A. S. M. Saleuddin and K. M. Wilbur), A. P., vol. 5, Physiology, Part 2, pp. 189-238. Khan, H. R. and Saleuddin, A. S. M. 1979. Effects of osmotic changes and nemosecretory extract\ on kidney ultrastructure in the fresh-water pulmonate Helisoma Can. J. Zoo/. . 57, 12561270. Koechlin, N. 1966. Ultrastructure du plexus sanguin perioesophagien; ses relations avec la ntphridie de Sabella paaonina Savigny. C. R. Acad. Sci. (Paris). 262, Ser. D, 12661269. Krijgsman, B. J. and Divaris, G. A. 1955. Contractile and pacemaker mechanism of the heart of molluscs. Bzol. Reu. Cambridge Philos. Sot., 30, l-39. Kiimmel, G. 1967. Die Podocyten. Zool. Beitr., N.F. 13, 245-262. Martin, A. W., Stewart, D. M. and Harrison. F. M. 1965. Urine formatton in a pulmonate land snail. Achatina fulicu. J. exp. Biol., 42, 99-123.
Pan, C. T. 1971. The arterial system of the Planorbid snail Biomphaluria glabrutu. Trans. Amer. microsc. Sot..
90,
434-440.
Pirie, B. J. S. and George, S. G. 1979. Ultrastructure
of the heart and excretory system of Myti[u_sedulis (L.). J. Mar.
Biol. Ass. U.K., 59, 819-829.
Potts, W. T. W. 1967. Excretion in the Molluscs. Biol. Reu.. 42, l-41. Potts, W. T. W. 1975. Excretion in Gastropods. In Exkretion (ed. A. Wening), Gustav Fischer Verlag, Stuttgart,
pp.
76-88.
Ramsay, J. A. 1952. ‘A physiological approach to the lower animals’. Cambridge University Press. London. Rodewald, R. and Karnovsky, M. J. 1974. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J. Cell Biol.,
60,423-433.
Saleuddin, A. S. M., Farrel, C. L., Gomot, L. and Khan, H. R. 1983. Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix asperse Miiller. J. Morph., 178, 313-322.
Schaffner, A. and Rodewald, R. 1978. Filtration barriers in the coelomic sac of the Crayfish. Procumbarus clarkii. J. Ultrastruct. Res., 65, 3tG47.
Schmidt. G. 1916. Blutgefaszsystem und Mantelhole der Weinbergschnecke 261.
(Helixpomatia).
Z. Wiss. Zool., 115,201-
SITE OF ULTRAFILTRATION
IN A PULMONATE
KIDNEY
923
Skelding, J. M. 1973. The fme structure of the kidney of Achatina achatina (L.). Z. ZeNforsch. Mikrosk. Anat., 147, l-29.
Sminia, T. 1972. Structure and function of blood and connective tissue cells of the freshwater pulmonate Lymnaea sragnalk studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. Mikrosk. Anat., 130,497-526. Sminia, T. 1981. Phagocytic cells in Molluscs. In Aspects of Developmental and Comparatioe Immunology (cd. J. B. Salomon), Pergamon Press. Oxford, New York, pp. 125-132. Tripp, M. R. 1961. The fate of foreign materials experimentally introduced into the snail Australorbis glabratus. J. Parasit., 47, 745-751.
Van Aardt, W. .I. 1968. Quantitative aspects of the water balance in Lymnaea stagnalis (L.). Neth. J. Zool., 18, 253312. Vorwhol, G. 1961. Zur Funktion der Exkretions-organe van Helix pomaria L. und Archachatina uentricosa Gould. Z. Vergl. Physiol., 45, 12-49.
Vovelle, J. and Grassct, M. 1979. Approche histophysiologique et cytologique du role des cellules g sphtrules calciques du repli operculaire chez Pomatias elegans (Mtiller), Gasteropode Prosobranche. Proc. Sixth. Europ. M&c. Congr., Malacologia, 18, 557-560. Wendelaar Bonga, S. E. and Boer, H. H. 1969. Ultrastructure of the rcnopericardial system in the pond snail Lymnaea stagnalis (L.). Z. Zellforsch. Mikrosk. Anat. 94, 51%529. Witmer, A. and Martin, A. W. 1973. The fine structure of the branchial heart appendage of the cephalopod Octopus dofleini martini. Z. Zellforsch. Mikrosk. Anat.. 136, 54>568.