THE AMERICAN JOURNAL OF ANATOMY 190:118-132 (1991)

Fine Structure of the Elasmobranch Renal Tubule: Neck and Proximal Segments of the Little Skate E.R. LACY AND E. REALE Department of Anatomy and Cell Biology, Medical University of' South Carolina, Charleston, South Carolina 29425 (E.R.L.);Laboratory of Cell Biology and Electron Microscopy, Hannover Medical School, 3000 Hannover 61, Germany ( E.R .)

ABSTRACT This is the first in a series of studies that examines the renal tubular ultrastructure of elasmobranch fish. Each subdivision of the neck segment and proximal segment of the renal tubule of the little skate (Rqja erinaceu) has been investigated using electron microscopy of thin sections and freeze-fracture replicas. Flagellar cells, characterized by long, wavy, flagellar ribbons, were observed in both nephron segments. They were found predominantly in the first subdivision of the neck segment, which suggests that propulsion of the glomerular filtrate is a primary function of this part of the renal tubule. In the non-flagellar cells of the neck segment (subdivisions I and 111, there were bundles of microfilaments, a few apical cell projections, and, in subdivision 11, numerous autophagosomes. In the proximal segment, the non-flagellar cells varied in size, being low in subdivision I, cuboidal in 11, tall columnar in 111, and again low in IV. Apical cell projections were low and scattered in subdivisions I and IV and were highest in I11 where the basolateral plasma membrane was extremely amplified by cytoplasmic projections. Furthermore, in these cells the mitochondria were numerous with an extensive matrix and short cristae. A network of tubules of the endoplasmic reticulum characterized the apical region of the non-flagellar cells in subdivisions I, 11, and IV. In the late part of subdivision I1 and the early part of 111, the cells were characterized by numerous coated pits and vesicles, large subluminal vacuoles, and basally located dense bodies, all of which are structures involved in receptor-rnediated endocytosis. Freeze-fracture replicas revealed gap junctions restricted to the cells of the first three subdivisions of the proximal segment. The zonulae occludentes were not different in the neck and proximal segments, being composed of several strands, suggesting a moderately leaky paracellular pathway.

itated by a renal countercurrent multiplier system which produces a urine that is hypertonic to the plasma. The presence of a renal countercurrent system in vertebrates has been regarded until recently as being associated with the production of a solute-rich, water-poor urine, a situation believed to be restricted to homeotherms. In the 1980's, computer-assisted reconstruction was used to confirm the presence of a renal countercurrent system in elasmobranch fish (Lacy et al., 1985). Elasmobranchs comprise a large, nearly entirely marine phylogenetic group including sharks, skates, and rays, all of which do not produce a hypertonic urine (Marshall, 1934; Smith, 1936; Kempton, 1953).These cartilagenous fish maintain a relatively constant internal solute environment by a number of mechanisms, including an elevated tissue and plasma concentration of urea,l so that the osmolality of the body fluids is approximately the same as that of the surrounding sea water (Smith, 1936). The capacity of these fishes' kidney to reabsorb the filtered urea has long been known from physiological studies which have shown that only a small fraction of plasma urea is excreted (Marshall, 1934; Smith, 1936; Kempton, 1953). The sequence of tubular segments and the epithelial morphology of the elasmobranch nephron have been elucidated only recently by light-microscopic methods (Lacy and Reale, 1985a,b), thus allowing physiological studies to determine the anatomical site of transepithelial solute and water transport (Friedman and Hebert, 1990; Hebert and Friedman, 1990). The marine elasmobranch nephron is extremely long and complex (Kempton, 1943, 1962; Borghese, 1966; Lacy et al., 1985; Lacy and Reale, 1985a,b, 1986), which no doubt has retarded the identification and extent of the tubular epithelium. Nevertheless, there is a definite sequence to the various morphologically distinct segments of the nephron as well as their juxtaposition to each other within the countercurrent system. The renal tubule consists of four loops (Fig. 1): two in a large 'Trimethylamine oxide constitutes a lesser but still significant proportion of the elevated plasma solutes (Smith, 1936).

INTRODUCTION

Land dwelling vertebrates have evolved mechanisms which minimize water loss to the environment. Osmotic homeostasis in both birds and mammals is facil1991 WILEY-LISS, INC.

Received January 18, 1990; Accepted August 30, 1990. Address reprint requests to Dr. Eric R. Lacy, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.

SKATE RENAL TUBULE: NECK AND PROXIMAL SEGMENT

119

Fig. 1 . Schematic drawing of the course of elasmobranch nephron into four loops, distal tubule, and the collecting duct. Loops I and 111 and the early distal tubule as well as the late distal tubule leaving the bundle (countercurrent system) are wrapped by the peritubular sheath. The subdivisions of each tubule segment are indicated by symbols and abbreviations. RC, Renal corpuscle. (Modified from Lacy and Reale, 1985b.3

blood sinus region of the kidney, the sinus zone, and two in a tightly compact region, the bundle zone. The countercurrent system is formed by the two nephron loops in the bundle zone and an exiting distal tubule, all of which are wrapped together by an investing peritubular sheath formed of squamous cells. This sheath separates each bundle of nephrons (the countercurrent system) from adjacent bundles as well as forming a barrier to the solutes and water surrounding the tubules in the bundle. At the proximal end, the sheath is pierced not only by the four limbs of the two tubule loops and by the distal segment but by entering and exiting capillaries which run the length of the two loops, giving this countercurrent system the likelihood of having a multiplier effect as found in the mammalian renal papilla (Lacy et al., 1985). Knowledge of the ultrastructure of the various epithelia along the renal tubule is necessary to correlate the anatomical specializations of the epithelial cells with the mechanisms of solute and water transport. The elasmobranchs remain the only major group of vertebrates in which the ultrastructural morphology of the nephron is not elucidated. In the present series of studies, we have used transmission electron microscopy of thin sections and freeze-fracture replicas t o document the fine structure of each segment and subdivision of the renaI tubule in the little skate, Raja

erinacea. This study concerns the neck segment and the proximal segment. MATERIALS AND METHODS

The capture and maintenance of the fish, the vascular-perfusion fixation method, the protocol for embedding the pieces of kidney in epoxy resin, and the techniques used for freeze-fracture have been detailed in previous publications (Lacy and Reale, 1985a,b, 1986; Lacy et al., 1987). Briefly, the kidneys of 12 female little skates (Raja erinacea) were fixed by vascular perfusion with solutions of either glutaraldehyde or glutaraldehyde and paraformaldehyde (Karnovsky, 1965). For thin sections, part of the specimens prefixed in either of the above solutions were postfixed in 1%OsO, solution in sodium cacodylate-HCl buffer or in ferrocyanide-reduced Os04 (Karnovsky, 1971). In addition, kidneys of the Atlantic sharpnose shark IRhizoprionodon terraenouae) were fixed by dripping a solution containing both aldehydes and osmium tetroxide (Hatae et al., 1986) on the renal surface in situ. All tissue specimens were dehydrated in ethanols and embedded in Epon-Araldite. Semithin sections were stained with alkalinized toluidine blue. Each segment or segment subdivision considered in the present investigation has been observed in a t least five tissue blocks of all 13 kidneys. The thin sections prepared from these

120

E.R. LACY AND E. REALE

blocks were stained with uranyl acetate and lead citrate. After aldehyde fixation, part of the little skate samples were cryoprotected (30%glycerol for 60 min), frozen with liquid Freon 22, fractured in a Balzers 300 BA device, and replicated by shadowing with platinum-carbon. Thin sections and replicas were examined in a Siemens Elmiskop 101 electron microscope. RESULTS

Neck Segment (Nk) Light microscopy showed that this segment has two subdivisions and forms part of the first loop of the renal countercurrent system. Beginning at the urinary pole of the renal corpuscle (Fig. 11, Nk runs for a very short distance in the interbundle connective tissue before piercing the peritubular sheath and forming part of the tubular countercurrent system. Once inside this sheath, Nk travels first straight and then convoluted, folding back on itself numerous times until it reaches the distal end of the sheath. At this position, it changes gradually into the first part of the proximal segment (Lacy and Reale, 1985b). The early neck segment (NkI) is readily identifiable by light microscopy since its wall contains a large number of flagellar cells whose numerous flagella nearly occlude the small tubular lumen. Non-flagellar cells are also present and become proportionately more numerous in the last portion of the neck segment (Nk-11). The flagellar cells (Fig. 2a) have the ultrastructural characteristics of the cells lining the urinary pole region of Bowman’s capsule (Lacy et al., 1987). The flagella are extremely long and are arranged on the cell surface in parallel rows, forming large ribbons with a n undulating pattern. The orientation of the ribbons toward the collecting duct suggests that they function to propel urine along the tubule. Other morphological specializations of these unique ribbons have been described elsewhere (Lacy e t al., 1989a,b). Among the flagella, there are some microvilli andlor microplicae that are flanked by a few coated and non-coated pits and vesicles at their bases. Each of these cells is connected by a distinct junctional complex (zonula occludens, zonula adhaerens, and small maculae adhaerentes). The lateral and basal cell limits are mostly straight, only occasionally being interrupted by a few folds. The cell body contains a n ovoid nucleus, numerous mitochondria (more than the non-flagellar cells, Fig. 2a), glycogen granules, some cisterns of the rough surfaced endoplasmic reticulum, the Golgi complex, a n abundant population of membrane-bound vesicles, and some dense bodies (presumably lysosomes). Among these components, there are distinct microtubules and microfilaments. Thin bundles of microfilaments are intermingled with the organelles in the apical part of the cell and form connections with the desmosomes. In addition, microfilament bundles are prominent in the basal part of the cell (Fig. 2). As seen in Figure 2b,c, most of the filaments measure 10-12 nm in diameter; smaller bundles, usually close and parallel to the basal plasma membrane, have a diameter of 5-7 nm. Based on these diameters, the filaments probably belong to the intermediate (10-12 nm) or to the actin (5-7 nrn) family of cytoskeletal proteins. The non-flagellar cells (Fig. 2a) are smaller and, in Nk-I, less numerous than the flagellar cells. The free

surface of non-flagellar cells is generally smooth with very few short and scattered microvilli andlor microplicae. The baso-lateral membrane is not generally interdigitated, but the basal plasma membrane often protrudes with the adjacent basement membrane into the surrounding connective tissue. The nucleus is irregularly shaped as compared to that of the flagellar cells. The cytoplasm displays a paucity of mitochondria, few tubuli of the endoplasmic reticulum, a small Golgi complex, and fewer, but still plentiful, bundles of microfilarnents than that of the flagellar cells. In the convoluted portion of the tubular bundle (Fig. 11, the second part of the neck segment (Nk-11) has low cuboidal cells, many of which possess flagella (Fig. 3a), although the number of these organelles is less than in Nk-I (Fig. 2aj. Nk-I1 non-flagellar cells also have numerous autophagosomes (Fig. 3b); otherwise, cytological details of both the flagellar and non-flagellar cells remained unchanged. Flagellar and non-flagellar cells of Nk-I and Nk-I1 lie on a continuous, uniformly thick basement membrane (Figs. 2,3). In summary, the epithelium of the neck segment is characterized by: 1) flagellar cells with extremely long flagellar ribbons throughout this segment but more prominent in the early (Nk-I) than the late (Nk-11)subdivision; 2) bundles of microfilaments in two sizes of classes (about 10-12 nm and 5-7 nm thick) in flagellar and non-flagellar cells; 3) a paucity of cell surface amplifications; and 4)numerous autophagosomes in Nk-11. Proximal Segment (Px)

Light microscopy showed that near the terminus of the tubular bundle (at the end distal to the renal corpuscle), the low cuboidal cells of the neck segment have microvilli; distally the cuboidal cells are replaced by columnar cells with a distinct brush border. These latter cells characterize the first two subdivisions of the proximal segment (Px-I and Px-TI), both of which are part of the tubular bundle (Fig. 1). The epithelial cells then become high columnar in the third segment (PxIII), which is found exclusively in the sinus zone. Finally the epithelium becomes low cuboidal (Px-IVj before merging into the granular cells of the first part of the intermediate segment (In-I). The transition from Px-IV to In-I always occurs near the renal corpuscle (Lacy and Reale, 1985b). Electron microscopy shows that flagellar and nonflagellar cells comprise the epithelium of the proximal segment. The flagellar cells have the same ultrastructural features observed previously in the neck segment and do not change their morphology along the various subdivisions of the proximal tubule. The surface of the

Fig. 2. Early neck segment (Nk-I).a: The non-flagellar cells have a characteristic luminal plasma membrane nearly devoid of apical projections. The flagellar cells are large, with numerous mitochondria and bundles of filaments. Filaments of two sizes belonging t o flagellar cells can be recognized in b (longitudinal section) and in c (cross section): thick filaments (about 10-12 nm), indicated by long arrows and insets on the right sides of the micrographs; and thin filaments (about 5-7 nm), indicated by arrowheads and insets on the left sides. a, X 8,000; b, x 60,000 (insets, x 100,000); c, X 36,000 (insets, x 60,000).

SKATE RENAL TIJBIJLE: NECK AND PROXIMAL SEGMENT

Fig. 2.

121

122

E.R. 1,ACY AND E. REALE

Fig. 3. Convoluted part of the tubular bundle. a Low euboidal cells of the late neck segment (Nk-IIf and the early proximal segment (Px-I).b: Autophagosomes (A) inside a non-flagellar cell of Nk-11. a, x 10,000;b, x 36,000.

low cuboidal non-flagellar cells bears tiny, irregularly shaped microvilli (Figs. 3a, 4a), which are probably sensitive to osmotic variations of the tubule content andlor of the fixative solution since they were very frequently swollen a t their tips (Fig. 3a), in contrast to the microvilli in other parts of the tubule. In the apical cell region, there is a conspicuous, tightly packed network of tubules of the smooth surfaced endoplasmic reticulum, which are intermingled with filaments of the terminal web, microtubules, and glycogen particles. The lateral cell borders, especially in the early part of Px-I (Fig. 3a), are not extensively interdigitated; and the basal plasma membrane is also straight. This cell type contains a few vacuoles as well as autophagosomes and dense bodies with heterogenous contents. The mitochondria have distinct, long cristae. Occluding junctions between non-f lagellar cells occasionally have characteristic bow-like shapes when viewed in thin sec-

tions, a peculiar aspect which may be due to the presence of some gap junctions inside the network of the tight junctional strands, as seen in freeze-fracture replicas. In the second subdivision of the proximal tubule (PxII), the non-flagellar cells (Fig. 5) are cuboidal or columnar; and the apical projections were higher (up to 350 nm in length) with a n irregular diameter and often separated from each other by pits, most frequently of the coated type. Although the lateral cell borders were straight, the basal plasma membrane has a n undulating pattern. The terminal web is conspicuous and has numerous small vesicles and tubules of the smooth endoplasmic reticulum intermingled within it (Fig. 5). These tubuli, like those of the previous Px-subdivision, form a tight network in the apical part of the cell, which occasionally is in continuity with the lateral plasma membrane. The Golgi complex is located api-

SKATE RENAL TUBULE: NECK AND PROXIMAL SEGMENT

123

Fig. 4. Varying aspects of the apical projections of the non-flagellar cells in the proximal segment a)

Px-I;b) late Px-11; c) Px-111;and d) Px-IV.The apical projections are short, irregularly shaped, and scattered in Px-1 and IV; longer in the late Px-11; and longer and tightly packed in Px-111.all, x 8,000.

cally, between this network and the nucleus; autophagosomes, dense bodies, and mitochondria are not numerous (Fig. 5). The transition from Px-I1 to Px-I11 (Fig. 6) does not occur abruptly but rather over a region whose cells are characterized by two distinct changes in ultrastructural organization. The first change is the dramatic increase in the number of coated pits and vesicles between or close to nearly every microvillus and the presence of membrane-bound apical tubules packed into the apical cytoplasm. These tubules contain a thick layer of electron dense material but are smooth on their cytoplasmic surface (Figs. 6, 7a, 8d, e). The content of the coated pits and vesicles as well as that of the apical tubules is characterized in the skate (Fig. 7b,d) as well as in the Atlantic sharpnose shark (Fig. 7c) by faint striations that have a diagonal orientation and a periodicity of about 15 nm. If the limiting membrane of coated pits and vesicles and of apical tubules of the specimens fixed with the glutaraldehyde-osmium mixture (Hatae et al., 1986) is cross sectioned, an incomplete layer of minute dots is observed with the same position and periodicity as that of the striations, suggesting a relationship between these two structures (Fig. 7e). Coated pits and vesicles (not the tubules) occur not only along and close to the apical plasma membrane but towards the lateral and basal cell surface as well. They are located in regions of the cell which are devoid of baso-lateral folds or inside thin cytoplasmic projections as also observed in the intermediate and distal segments (Fig. 8a-c). The second distinctive change in the cells of the Px11-Px-I11 transition region is the presence of large, nearly lucent membrane-bound vesicles immediately below the terminal web and the apical tubules (Figs. 6, 8d). The vesicles measure about 0.05 Fm in diameter; occasionally apical tubules and these vesicles are in continuity (Fig. 8d,e). Furthermore, in the same re-

gion, large vacuoles (up to 1 km in diameter) containing lucent material, lipid remnants, or granular material are present (Fig. 6). The Golgi apparatus of these cells is prominent as are dense bodies, mainly located in the basal cytoplasm (Fig. 6). Neighboring nonflagellar cells have numerous tightly interdigitating microfolds and rnicrovilli-like projections along the lateral borders (Fig. 6). The Px-I11epithelial cells beyond the transition area show a high brush border with tightly packed microvilli (Fig. 4c). The mitochondria are numerous and large, with extremely rare and short cristae and extensive matrices (compare Fig. 6 with Fig, 9). The lateral borders are more complex than those of the preceding segment (Fig. 9). The intercellular spaces between adjacent non-flagellar cells and their large primary and secondary baso-lateral processes are filled by tertiary projections that are comparable in size, number, and tightness to those of the microvillar brush border (Fig. 9). The large size of the mitochondria obviated their being located in these microvilli-like projections of the lateral plasma membrane. The border of the high columnar brush-border cells of Px-I11 with the flagellar cells is devoid of interdigitations (Figs. 6, 10a). The mitochondria of the flagellar cells are typical of those found in other segments of the nephron (Fig. 10a). Freeze-fracture replicas of Px-111 non-flagellar cells reveal a peculiar arrangement of the pores of the nuclear envelope (Fig. lob). They are numerous but are assembled in clusters of from 2 to 6 with individual pores in each cluster evenly spaced from one another. Furthermore, the intramembranous particles on the E fracture face are more densely distributed around the pore clusters than in the spaces between them. The zonulae occludentes between Px-I11 cells (Fig. l l a ) composed of several superimposed strands which frequently circumscribe gap junctions as well as desmosomes (Fig. l l a ) . Other gap junctions are located along

124

E.R. LACY AND E. REALE

Fig. 5. Non-flagellar cells of Px-11. Between nucleus and terminal web there is an extensive smooth surfaced endoplasmic reticulum. Note the undulating basal plasma membrane and accompanying basement membrane. x 12,000

the basolateral plasma membranes, below the zonula seen inside and below the terminal web. Abundant mioccludens. crofilaments oriented parallel to the basal plasma In the last portion of the proximal tubule (Px-IV),the membrane are observed in most non-flagellar cells of cells return to a low cuboidal shape with short, irreg- Px-IV. However, there are some regions where the ularly distributed, apical projections (Fig. 4d) and a basal plasma membrane and associated cytoplasm, rich paucity of lateral plasma membrane interdigitations. in microfilaments, abruptly protrudes deeper into the When present, the latter are confined to the basal re- peritubular connective tissue. These protrusions are algion of the cells where numerous invaginations of the ways in close relationship with or directly below flagelbasal plasma membrane (basal infoldings) occur. lar cells, suggesting a functional significance of changDense bodies, ranging from multivesicular bodies to ing epithelial cell shape in the basal region. large electron dense, almost homogenous structures As seen in freeze-fracture replicas, several, mostly (Fig. lOc), are numerous. In Px-IV, there are few mito- parallel strands of occluding junctions seal the interchondria but their cristae have the more classic dispo- cellular spaces from the Px-IV lumen (Fig. l l b ) . Gap sition extending across the organelle. An extensive junctions are not observed in Px-IV. network of smooth-surfaced endoplasmic reticulum is In summary, from the beginning to the end of the

SKATE R E N A L TUBULE: NECK AND PROXIMAL SEGMENT

125

Fig. 6. Transitio.1 region from Px-I1to Px-111.Lateral, tightly packed interdigitations are highly developed between non-flagellar cells (arrows) but absent between non-flagellar and flagellar cells (arrowheads). Below the high brush border, the cytoplasm contains numerous coated pits and vesicles, apical tubules, small vesicles, and large vacuoles, Dense bodies are present in the lower half of the non-flagellar cells. x 10,000.

changes from the typical crista type (Px-I, Px-11, and Px-IV) to large organelles with abundant matrix and few, markedly attenuated cristae (Px-111). 8. Gap junctions are observed by freeze-fracture in Px-I, IT, and 111, but not in Px-IV. The zonulae occlu1. The shape changes from low cuboidal in Px-I to dentes are composed both in Nk and Px of several sucuboidal-columnar in Px-I1 t o high columnar in Px-I11 perimposed strands. and back to low cuboidal in Px-IV. DISCUSSION 2. Short, scattered apical projections are present in The results of the present and subsequent investigaPx-I; further development of these occurs in Px-11, with maximal development in Px-111, and regression in Px- tions show, for the first time, the ultrastructural characteristics of the nephron of a marine elasmobranch IV. 3. An extensive tubular network of smooth-surfaced fish. The occurrence of the five major segments and endoplasmic reticulum in the apical and lateral re- their 16 subdivisions (Lacy and Reale, 1985a,b) in the gions, especially in Px-I1 segment, is occasionally open little skate has been confirmed by the present electronmicroscopic studies. Our findings elucidate the cellular towards the lateral intercellular space. 4. The plasma membrane elaborations are extensive components of this highly unique nephron which is laterally in Px-111, but only moderate in Px-I, Px-11, characterized by its extreme length, having one of the and Px-IV; Px-IV showed prominent basal infoldings. most complex configurations among vertebrates; its ar5. Numerous coated pits and vesicles, apical tubules, rangement into a countercurrent multiplier system, and subapical, lucent small vesicles and large vacuoles the presence of a dual (venous and arterial) blood supare present at the transition region from Px-I1 t o Px- ply; and its ability to reabsorb nearly all filtered urea despite production of an isotonic urine (Nash, 1931; 111. Dense bodies lie in the basal third of the cells. 6. Coated pits and vesicles also occur along and close Kempton, 1939, 1943, 1953, 1962; Borghese, 1966; to the basal plasma membrane in all segment subdivi- Ghouse et al., 1968; Hickman and Trump, 1969; Thurau and Acquisto, 1969; Deetjen and Ankowiak, sions. 7. The substructural aspect of the mitochondria 1970; Lacy et al., 1975, 1985a,b; Stolte et al., 1977;

proximal tubule (Px-I to Px-IV), the flagellar cells do not change their general morphology, which is similar to that of flagellar cells in the neck segment. The nonflagellar cells have the following characteristics:

Fig. 7. a,b,d: Skate; c,e: Atlantic sharpnose shark. Transition region, Px-I1 to Px-111.a: In the apical region of two brush-border cells, coated pits (vertical arrowheads) and apical tubules in both longitudinal and transverse sections (horizontal arrowheads) can be seen. sER, Smooth surfaced endoplasmic reticulum (see also Fig. Xd). b e :

The electron-dense material inside the apical tubules has a linear periodic arrangement (arrows) best observed in grazing sections. In cross sections (e),electron-dense particles can be observed (arrowheads). a, X 40,000; b, 80,000; c, x 100,000; d, X 120,000; e, X 160,000.

SKATE RENAL TUBULE: NECK AND PROXIMAIA S E ( X E N T

127

Fig, 8.Transition region, Px-I1 to Px-111. Coated pit (arrowhead in a) and vesicle (arrowhead in b) inside basal cytoplasmic projections of cells of the distal tubule: the same structures (arrowheads in c ) occur in the intermediate tubule (sixth subdivision). BM, basement membrane. d,e: Apical region of non-flagellar cells. In d, smooth-surfaced

endoplasmic reticulum (sER), coated pits, and apical tubules occur close to and surrounding small apical lucent vesicles. In e, apical tubules emerge from a small vesicle, partially filled by lipid remnants. a, b, X 36,000; c, X 10,000;d, X 40,000; e, x 116,000.

Lacy and Reale, 1981,1986; Ogawa and Hirano, 1982). The salient differences between each segment and its subdivisions have been summarized in the Results. The relevance of specific structures to epithelial function and urine production are discussed here. The predominance of flagellar cells in Nk-I and the paucity of structures usually associated with known functions of the renal tubule in the non-flagellar cells suggest that the main function of this subdivision probably consists of glomerular filtrate propulsion into the successive tubule segments. This speculation is further substantiated by the presence of similar flagellar cells lining the urinary pole of the renal

corpuscle (Lacy et al., 1987, 1989a). Furthermore, in vitro optical analyses of nephron tubular segments from marine elasmobranchs have shown that flagella beat in the same direction and extremely rapidly, giving additional credence to the ideas about the function of these cells (Lacy et al., 1989b). In Nk-11, the non-flagellar cells possess autophagosomes and dense bodies as do Px-I and IV cells, which is indicative of another function related to a lysosomal activity. As soon as the proximal segment exits the bundle to become Px-111, several notable morphological changes occur, in particular an increase in the cell surface area

128

E.R. LACY AND E. REALE

Fig. 9. Non+flagellar cells of Px-111. The high columnar cell has a brush border (left top corner). Asterisks indicate primary and secondary baso-lateral interdigitatingprojections. The basolateral spaces are filled with tightly packed, microvilli-like, tertiary projections (arrows). Mitochondria have scanty cristae and large extensive matrices (see also Fig. 10a). x 8,000.

SKATE RENAL TUBULE: N E C K A N D PROXIMAL SEGMENT

129

Fig. 10. a: Flagellar cell of Px-111 (on the right) is flanked by one brush border cell. Note the different structures and sizes of the mitochondria in the two cell types. b: (freeze-fracture replica). Characteristic clustered arrangement of the nuclear pores in a Px-I11 brush border cell. c: Transition from Px-IV (on the right) to In-I. In the

Px-IV cell, there are numerous dense bodies and loosely packed basal interdigitations. In the granulated cell of In-I (on the left), the variegated granules characterize this subdivision. a, x 20,000; b, x 24,000; c, x 8,000.

and in the cell components associated with a remarkable endocytic activity.

plifications are also missing from the flagellar cells, which throughout the length of the nephron have nearly straight borders with adjacent cells, few microvilli, but numerous extremely long flagella bound into a ribbon (Lacy et al., 1989a,b). The greatest cell-surface amplification is found in the proximal segment. The number, length, and density of packing of the apical projections per unit area increase in the skate renal tubule from Px-I t o Px-111, where they reach their greatest development but then decrease again in Px-IV. The progressive microvillar increase from Px-I to Px-I11 followed by a decrease in Px-IV demonstrates that, in the little skate like other

fncrease in the cell surface area: brush border; basolateral plasma membrane

According to Kriz (1984) and Koushanpour and Kriz (1986),an increase in the cell surface can be achieved through elaboration of plasma membranes into microvilli and microplicae, basal infoldings, and cellular interdigitations. All these membrane amplifications can be found in various segments of the little skate renal tubule, with the exception of the non-flagellar cells in both parts of the Nk segment. Membrane am-

130

E.R. LACY AND E. REALE

vertebrates, the brush border is heterogenous along the length of the proximal tubule (reviewed by Koushanpour and Kriz, 1986). The functional significance of a segment with a reduced brush border following segments of progressively better developed apical projections in the elasmobranch is not known.

The basolateral plasma membranes of the nonflagellar cells undergo a n amplification comparable to that of the brush border. Whereas invaginations of the basal plasma membrane (basal infoldings) and cellular interdigitations gradually increase from Px-I to Px-I1 and are mainly located close to the base of the epithelium, in Px-I11 they are extremely numerous, fingerlike, and tightly packed from the junctional complex to the basement membrane. Furthermore, these projections are in the spaces among the large primary and secondary interdigitating basolateral processes of the cells. We do not know any other epithelia showing such a n amplification of the basolateral plasma membrane a s that in the Px-I11 subdivision of the skate renal tubule. This amplification is further enhanced by a n increase of both the size of the cells (which are low cuboidal in Px-I, cuboidal-columnar in Px-I1 and tall columnar in Px-111) and the outer tubular diameter (from about 44 pm in Px-I to about 86 pm in Px-111; Lacy and Reale, 198513). Structures associated with endocytosis-exocytosis: smooth-surfacedpits and vesicles; coated pits and vesicles; dense apical tubules; apical vesicles and vacuoles; dense bodies

As in the cells of the mammalian proximal tubule (Rhodin, 1954; Ericsson, 1964; Fawcett, 1964; Maunsbach, 1966; Latta et al., 1967), at least two types of pits and vesicles are recognizable in all the cells of the elasmobranch tubule: the smooth-surfaced (or uncoated) type and the coated variety. It is not known if, as in most of the cells of other animals (Pearse, 1975, 1987), the coating contains the protein clathrin. Given the preponderance of this association, i t seems likely (Salisbury et al., 1980). Further investigations are necessary to support this assumption, however, since pits and vesicles devoid of antigenically detectable clathrin have been described (Brown and Orci, 1986; Orci et al., 1986; Malhotra et al., 1989) as have pits and vesicles with a coat showing three-dimensional characteristics different from those of the clathrin baskets (the “studlike” coat believed to be components of proton pumps) (Brown e t al., 1987; reviewed by Brown, 1989). There are few coated and uncoated pits and vesicles in the flagellar cells, but in the non-flagellar cells their density changes characteristically from one tubule segment to another. This suggests that in areas where pits and vesicles occur in “normal” numbers they may have a general, cell-related function such as performing nonselective (uncoated vesicles) and selective (coated vesicles) endocytosis for minimal transport across the cells. In areas where pits and vesicles occur in high density, they could have a very specific function related to that segment subdivision. Thus, the coated pits and vesicles in the brush-border cells a t the transition region from Px-I1 to Px-I11 could participate in protein reabsorption, as repeatedly demonstrated for morphologically comparable structures in the mammalian proximal convoluted tubule (reviewed by Maunsbach, 1973, 1976; Tisher and Madsen, 1986; Koushanpour ~

~

Fig. 11 Freeze-fracture replicas of Px-111(a)and Px-IV (b).Strands of occluding junctions can be seen in both subdivisions. In a, there are

numerous gap junctions (arrowheads) and desmosomes (arrows) intermingled within the strands of the occluding junction. BB, Brush border. a, x 72,000; b, X 65,000.

SKATE R E N A L TUBULE: NECK AND PROXIMAL SEGMENT

and Kriz, 1986). A receptor-mediated endocytotic function for these coated pits and vesicles is supported by the presence of “apical tubules” in the apical region of the cells. These membrane-bound tubules are similar in some fine structural details to the “apical tubules” recently described by Hatae et al. (1986) and by Ohtsuki et al. (1986) in the proximal tubule cells of the rat and of the mouse nephron. In the elasmobranch nephron, the apical tubules show a smooth cytoplasmic surface and a diagonal or helicoidal arrangement of the electron-dense tubular contents. These apical structures have been found in continuity with pits, vesicles, and vacuoles participating in the reabsorption and transport of proteins from the tubule lumen to the lysosomes (Miller, 1960) or in the transport of glycoproteins from the Golgi apparatus to the plasma membrane (Haddad e t al., 1977). Graham and Karnovsky (19661, using horseradish peroxidase to study mechanisms of cellular uptake, recognized the apical tubules as being different from other structures in the rat proximal tubule. According to Maunsbach (1976) and Christensen (19821, the apical tubules originate from the endosomes and are involved in the recycling of the plasma membrane. Hatae et al. (1986) share this opinion and suggest a relationship between apical tubules and tubular structures recycling (transporting) receptors from the endosomes to the plasma membrane. The possible function of the apical tubules in elasmobranchs as well as the possible composition of their contents await further investigation but their similarity in mammals and fish suggests similar roles in both phylogenetic groups. Whereas we noted the occasional confluence of apical tubules with the small lucent vesicles characteristic of CURL (compartment of uncoupling receptor and ligands; Geuze et al., 19831, the possible relationship of the large vacuoles with other cytoplasmic components is not clear. They could originate from coalescence of the small lucent vacuoles, as suggested by similarities in their content (see Fig. 6), and thus represent a n “intermediate compartment” (Grifiths e t al., 1988; Pfeffer, 1988; Schmid et al., 1988; Geuze et al., 1988). Accordingly, lucent vesicles and large vacuoles would belong to the prelysosomal compartment, and the apical tubules could transfer membrane and receptors from the endocytic vesicles to the cell surface. The secondary lysosomes, towards which endocytosed material converges (van Deurs and Christensen, 1984; Farquhar, 1985; Mellman et al., 1987), could be represented by the dense bodies we observed mainly in the basal region of the cell. Thus, several components of the endosomal system operating during the receptor-mediated endocytosis would occur a t the transition region from Px-I1 to Px-111 in the skate renal tubule. However, Kerjaschki et al. (1984) and Rodman et al. (19861, among others, have demonstrated that in the rat renal tubule the membranes of microvilli, coated pits, endosomes, and lysosomes do not share the same antigenic properties. Therefore, suggestions of relationships between the apical cytoplasmic components based solely on morphological similarities may be, at the moment, highly speculative. Structures morphologically identical to the apically located coated vesicles and pits were identified along the basolateral surface in all nephron segments of the

131

little skate. While i t is generally accepted that proteins filtered across the glomerular wall are retrieved in the nephron by endocytosis along the apical plasma membrane of proximal tubule cells (Maunsbach, 1973, 1976), recent evidence has shown t h a t abluminal protein uptake also occurs via endocytotic invaginations, vesicles, and vacuoles in rabbit proximal tubule cells (Nielsen and Christensen, 1985). Furthermore, extracellular tracer molecules were also found in lysosomes and multivesicular bodies in these cells, suggesting a degradative pathway for abluminal protein uptake. Considering that Px-I11 and IV subdivisions of the little skate tubule lie in a large blood sinus which receives both arterial and venous blood from the renal portal system, the basolateral uptake of proteins may be more important in these fish than in higher vertebrates such as mammals. ACKNOWLEDGMENTS

Supported in part by The National Science Foundation (DCB-8903369) and by the Deutsche Forschungsgemeinschaft (SFB 146 and RE 257/7-1). We thank Mrs. Kathy Cowart, Ms. Marion Hinson, and Mr. Hans Heidrich for the excellent technical assistance. LITERATURE CITED Borghese, E. 1966 Studies on the nephrons of an elasmobranch fish Scyliorhinus ste1Zari.s (L.).Z. Zellforsch., 72:88-99. Brown, D. 1989 Vesicle recycling and cell-specific function in kidney epithelial cells. Annu. Rev. Physiol., 51t771-784. Brown, D., and L. Orci 1986 The “coat” of kidney intercalated cell tubulovesicles does not contain clathrin. Am. J. Physiol., 250: C605-C608. Brown, D., S. Gluck, and J. Hartwig 1987 Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification ofan H’ ATPase. J. Cell Biol., 105t1637-1648. Christensen, E.I. 1982 Rapid membrane recycling in renal proximal tubule cells. Eur. J. Cell Biol., 29t43-49. Deetjen, P., and D. Ankowiak 1970 The nephron of the skate, Raja erinacea. Bull. Mt. Desert 161. Biol. Lab., 1 0 5 - 7 . Ericsson, J.L.E. 1964 Absorption and decomposition of homologous hemoglobin in renal proximal tubular cells. An experimental light and electron microscopic study. Acta Path. Microbiol. Scand., Suppl. 168. Farquhar, M.G. 1985 Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol., 1:447-488. Fawcett, D.W. 1964 Surface specialization of absorbing cells. J. Histochem. Cytochem., 13:75-98. Friedman, P.A., and S.C. Hebert 1990 Diluting segment in kidney of dogfish shark. I. Localization and characterization of chloride absorvption. Am. J. Physiol., 258 (Regulatory Integrative Comp. Physiol. 27):R398-R408. Geuze, H.J., J.W. Slot, and G.J.A.M. Strous 1983 lntracellular site of asialoglycoprotein receptor-ligand uncoupling: double label immunoelectron microscopy during receptor-mediated endocytosis. Cell, 32277-287. Geuze, H.J., W. Stoorvogel, G.J. Strous, J.W. Slot, J.E. Bleekemolen, and I. Mellman 1988 Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J. Cell Biol., 107:2491-2501. Ghouse, A.M., B. Parsa, J.W. Boylan, and J.C. Brennan 1968 The anatomy, micro-anatomy, and ultrastructure of the kidney of the dogfish, Squalus ncanthias. Bull. Mt. Desert Isl. Biol. Lab., 8; 22-29. Graham, R.C., Jr., and M.J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14391-302. Griffths, G., B. Hoflack, K. Simons, I. Mellman, and S. Kornfeld 1988 The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell, 52.329-341. Haddad, A., G. Bennett, and C.P. Leblond 1977 Formation and turnover of plasma membrane glycoproteins in kidney tubules of

132

E.R. LACY A.NU E. REALE

young rats and adult mice, as shown by radioautography after a n Marshall, E.K. 1934 The comparative physiology of the kidney in relation to theories of renal secretion. Physiol. Rev., 14r133-159. injection of 3H-fucose. Am. J. Anat., 148t241-274. Hatae, T., M. Fujita, and H. Sagara 1986 Helical structure in the Maunsbach, A.B. 1966 Absorption of '251-labelled homologous albumin by rat kidney proximal tubule cells. A study of microperfused apical tubules of several absorbing epithelia. Cell Tissue Res., single proximal tubules by electron microscopic autoradiography 244:39 -46. and histochemistry. J. Ultrastruct. Res., 15:197-241. Hebert, S.C., and P.A. Friedman 1990 Diluting segment in kidney of dogfish shark. 11. Electrophysiology of apical membranes and cel- Maunsbach, A.B. 1973 Ultrastructure of the proximal tubule. In: Handbook of Physiology, Section 8: Renal Physiology. J . Orloff lular resistances. Am. J. Physiol., 258 (Regulatory Integrative and R.W. Berliner, eds. Williams and Wilkins, Baltimore, pp. Comp. Physiol. 27):R409-R417. 31-79. Hickman, C.P., and B.F. Trump 1969 The kidney. In: Fish Physiology, Maunsbach, A.B. 1976 Cellular mechanism of tubular protein transVol. 1. W.S. Hoar, and D.J. Randall, eds. Academic Press, New port. Int. Rev. Physiol., 2r145-167. York, pp. 91-239. Karnovsky, M.J. 1965 A formaldehyde-glutaraldehydefixative of Mellman, I., C. Howe, and A. Helenius 1987 The control of membrane traffic on the endocytic pathway. Current Topics Membranes high osmolarity for use in electron microscopy. J. Cell Biol., 27; Transport, 29:255-288. 137A-138A. Karnovsky, M.J. 1971 Use of ferrocyanide-reduced osmium tetroxide Miller, F. 1960 Hemoglobin absorption by the cells of the proximal convoluted tubule in mouse kidney. J. Biophys. Cytol., 8t689in electron microscopy. Am. SOC.Cell Biol., Proc. 11th Annual 718. Meeting, New Orleans, p. 146. Kempton, R.T. 1939 The morphology of the dogfish renal tubule. Bull. Nash, J . 1931 The number and size of glomeruli in the kidneys of fishes with observations on the morphology. Am. J. Anat., 47: Mt. Desert Is. Biol. Lab., 42:28-34. 425-445. Kempton, R.T. 1943 Studies on the elasmobranch kidney. 1. The strucNielsen, J.T., and E.I. Christensen 1985 Basolateral endocytosis of ture of the renal tubule of the spiny dogfish (Syualus acanthias). protein in isolated perfused proximal tubules. Kidney Int., 27: J . Morphol., 73:247-263. 39-45. Kempton, R.T. 1953 Studies on the elasmobranch kidney. 11. Reabsorption of urea by the smooth dogfish, Mustelus canis. Biol. Ogawa, M., and T. Hirano 1982 Studies on the nephron of a fiesh water stingray, Potarnotrygon rnagdalenae. Zool. Mag., 91 t101Bull., 104;45-56. 105. Kempton, R.T. 1962 Studies on the elasmobranch kidney. 111. The Ohtsuki, M., A. Fujioka, M. Nagano, and S.Mori 1986 Membrane kidney of the lesser ray Narcine brasiliensis. J. Morphol., 111: specialization in the proximal tubule cells of the mouse kidney. J. 217-225. Electron Micros., 35292-297. Kegaschki, D., L. Noronha-Blob, B. Sacktor, and M.G. Farquhar 1984 Orci, L., B.S. Glick, and J.E. Rothman 1986 A new type of coated * Microdomains of distinctive glycoprotein composition in the kidvesicular carrier that appears not to contain clathrin: its possible ney proximal tubule brush border. J. Cell Biol., 98:1505-1513. role in protein transport within the Golgi stack. Cell, 46:171-184. Ktushanpour, E., and W. Kriz 1986 Renal Physiology. Principles, Pearse, B.M.F. 1975 Coated vesicles from pig brain: purification and Structure, and Function, 2nd ed. Springer-Verlag, Heidelberg. biochemical characterization. J. Mol. Biol., 97:93-98. Kriz, W. 1984 Der Bau transportierender Epithelien. Verh. Anat. Pearse, B.M.F. 1987 Clathrin and coated vesicles. EMBO J., 6:2507Ges., 78:25-40. 2512. Lacy, E.R., and E. Reale 1981 The brush border segment of the nephron in an elasmobranch. Thin section and freeze fracture obser- Pfeffer, S.R. 1988 Mannose 6-phosphate receptors and their role in vations. Verh. Anat. Ges., 75t6ll-612. targeting proteins to lysosomes. J . Membrane Biol., 103:7-16. Lacy, E.R., and E. Reale 1985a The elasmobranch kidney. I. Gross Rhodin, J. 1954 Correlation of ultrastructural organization and funcanatomy and general distribution of the nephrons. Anat. Emtion in normal and experimentally changed proximal convoluted bryol., 173.93-34. tubule cells of the mouse kidney. An electron microscopic study Lacy, E.R., and E. Reale 1985b The elasmobranch kidney. 11. Seincluding an experimental analysis of the conditions for fixation quence and structure of the nephrons. Anat. Embryol., 173:163of the renal tissue for high resolution electron microscopy. Ak186. tiebolaget Godvil, Stockholm, pp. 7-76. Lacy, E.R., and E. Reale 1986 The elasmobranch kidney. 111. Fine Rodman, J.S., L. Seidman, and M.G. Farquhar 1986 The membrane structure of the peritubular sheath. Anat. Embryol., 173t299composition of coated pits, microvilli, endosomes, and lysosomes 305. is distinctive in the rat kidney proximal tubule cell. J. Cell Biol., Lacy, E.R., B. Schmidt-Nielsen, E. Swenson, and T. Maren 1975 The 102:77-87. urinary bladder in the little skate, Ruja erinacea. Bull. Mt. Salisbury, J.L., J.S. Condeelis, and P. Satir 1980 Role of coated vesiDesert Isl. Biol. Lab., I5t56-58. cles, microfilaments, and calmodulin in receptor-mediated enLacy, E.R., E. Reale, D.S. Schlusselberg, W.K. Smith, and D.J. Wooddocytosis by cultured B lymphoblastoid cells. J. Cell Biol., 87: ward 1985 A renal countercurrent system in marine elasmo132-141. branch fish: a computer assisted reconstruction. Science, 227; Schmid, S.L., R. Fuchs, P. Male, and I. Mellman 1988 Two distinct 1351-1354. subpopulations of endosomes involved in membrane recycling Lacy, E.R., M. Castellucci, and E. Reale 1987 The elasmobranch renal and transport by lysosomes. Cell, 52:73-83. corpuscle: fine structure of Bowman's capsule and the glomerular Smith, H.S. 1936 The retention and physiological role of urea in Elascapillary wall. Anat. Rec., 218:294-305. mobranchii. Biol. Rev., 11r49-82. Lacy, E.R., L. Luciano, and E. Reale 1989a Flagellar cells and ciliary Stoke, H., R.G. Galaske, G.M. Eisenbach, C. Lechene, B. Schmidtcells in the renal tubule of elasmobranchs. J. Exp. Zool., Suppl. Nielsen, and J.W. Boylan 1977 Renal tubule ion transport and 2:186-192. collecting duct function in the elasmobranch little skate, Rqja Lacy, E.R., J.C. Williams, Jr., R. Rivers, K.J. Karnaky, Jr., L.A. Macerznacea. J . Exp. Zool., 199r403-410. kanos, G.M. Grabowski, L. Luciano, and E. Reale 198915 Motion Thurau, K., and P. Acquisto 1969 Localization of the diluting segment analysis of flagellar ribbon and ciliary beat cycles in teleost and in the dogfish nephron: a micropuncture study. Bull. Mt. Desert elasmobranch fish nephrons. Eur. J. Cell Biol., 49 (Suppl. 27):54. I d . Biol. Lab., 9:60-63. Latta, H., A.B. Maunsbach, and L. Osvaldo 1967 The fine structure of Tisher, C.C., and K.M. Madsen 1986 Anatomy of the kidney. In: The renal tubules in cortex and medulla. In: Ultrastructure of the Kidney, B.M. Brenner and F.C. Rector, Jr., eds. Saunders, PhilKidney, Vol. 2. A.J. Dalton and F. Haguenau, eds. Academic adelphia, pp. 3-60. Press, New York, pp. 1-56. van Deurs, B., and E.J. Christensen 1984 Endocytosis in kidney proxMalhotra, V., T. Serafini, L. Orci, J.C. Shepherd, and J.E. Rothman imal tubule cells and cultured fibroblasts: a review of the struc1989 Purification of a novel class of coated vesicles mediating tural aspects of membrane recycling between the plasma membiosynthetic protein transport through the Golgi stack. Cell, 58; brane and endocytic vacuoles. Eur. J . Cell Biol., 33:163-173. 329-336.