Renal Morphology of Freshwater Trout BETTINA G. ANDERSON AND RICHARD D. LOEWEN Department of Anatomy, W e s t e r n College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
ABSTRACT The nephron of the euryhaline freshwater salmonids is composed of the renal corpuscle and the renal tubules. Throughout much of the renal corpuscle, only the lamina densa separates the fenestrated processes of the endothelial cells from the foot processes of the visceral epithelium. The renal tubule consists of five distinct segments. The neck segment is short and intermittently ciliated; it lacks the mucous cells which appear in the neck segment of some teleosts. The proximal segment bears a dense brush border and is both structurally and functionally divisible into a first and a second segment. The first portion is typified by the presence of short apical tubules, variously sized apical vacuoles, and numerous lysosomes. The second proximal segment is distinguished by the abundance and distribution of mitochondria throughout the cytoplasm. Infoldings of the basilar plasmalemma are especially prominent in this region. A ciliated intermediate segment intervenes briefly between the proximal and distal portions of the tubule. The distal segment consists of cuboidal cells which bear scattered, short microvilli, small vesicles and multivesicular bodies. Renal tissue from several species of trout was examined in order to establish the basis for a common pattern of histological and ultrastructural characteristics within the familv Salmonidae. I n all sDecies examined, renal structure was very similar and could readily be compared with that previously described in other freshwater and marine species.
Structural variations resulting from en- prey (Petromyzon marinus: Youson and vironmental adaptation and species special- McMillan, '70a,b; '7la,b,c) and the Atlantic ization are frequently an obstacle to the hagfish (Myxine glutinosa: Heath-Eves study of fish morphology and physiology. and McMillan, '74). Renal morphology of a limited number The use of trout as laboratory animals of representatives from environments of has become more common with the advent varied salinity has been presented by previ- of fish farming, lake and stream stocking ous investigators. Both histological and and pollution monitoring. Structural alterultrastructural features of the kidney of ation in those systems which are rapidly the English sole (Parophrys v e t u l u s ) , a affected by stress may be considered an marine species, were described originally effective diagnostic tool both in disease by Bulger and Trump ('68), those of a control and pollutant detection. Its usefulfreshwater species of bluegill (Lepomis ness, however, is dependent upon a knowlmacrochirus) and a marine euryhaline edge of morphology in healthy animals. species, the Southern flounder (ParaThe objective of this study was to establichthys b t h o s t i g m a ) by Hickman and lish a common pattern of renal morphology Trump ('69). The work of other investi- among fresh-water trout, and to compare gators involving additional species was this with kidney structure as described by also reviewed by these last-named authors. others in fresh-water, euryhaline and maThe general microanatomy of the pink rine species. and coho salmon kidney (Oncorhynchus MATERIALS AND METHODS gorbuscha and 0. hisuteh) has also been Histological sections from each of four described (Newstead and Ford, '60; Hendricks, '71 ) . Excellent studies have species were examined; these were Salmo been made most recently on the sea lam- aguabonita, S. gairdneri, S . trutta and AM. J. ANAT., 143: 93-114.
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BETTINA G . ANDERSON AND RICHARD D. LOEWEN
Salvelinus fontinalis. These fish were obtained both in the wild and from hatcheries i n the Unite1 States and Canada.' Portions of both the head and trunk kidney2 were collected from 12 fish. After severance of the spinal cord the body cavity was opened along the ventral midline; renal tissue was removed and immediately immersed in Bouin's fluid for fixation. Paraffin sections were stained by a variety of methods. Routinely, hematoxylin-phloxin-safran (HPS) (Luna, '68) stain was applied, but occasionally sections were stained with Gomori's method for chromaffin bodies, Cain's method for mitochondria or hematoxylin and eosin for general staining . Subsequent to the histological studies, two species, Salmo gairdneri and Salvelinus fontinalis, were selected for examination at the ultrastructural level. I n each of six specimens the spinal cord was severed immediately after removal of the fish from a holding tank. The body cavity was opened and three blocks of kidney tissue removed from the region between the midbody and the anal oriface. Each tissue block was divided into several smaller blocks and these fixed by one of several methods: (a) 6.25% phosphate-buffered glutaraldehyde with postfixation in 1% osmium tetroxide at pH 8.1, ( b ) 1% glutaraldehyde with postfixation i n osmium tetroxide, and ( c ) 2% osmium tetroxide in sodium bicarbonate. These tissues were dehydrated in ethanol and embedded in Epon (Luft, '61). One micron sections were cut from each block, stained with 1% toluidine blue in 1% borax and examined so that specific portions of the nephron could be isolated for thin sectioning. Thin sections from the trimmed blocks were stained with saturated uranyl acetate and Reynold's lead citrate and examined with a n Hitachi HU-12 electron microscope. OBSERVATIONS
The kidneys of these trout were fused, appearing a s one organ rather than two. They occupied a dorsal retroperitoneal position along the entire length of the body cavity, bounded by the vertebrae dorsally, the ribs laterally and the swim bladder
ventrally. On the basis of morphological differences as well as location, they were divided into a cranial or head kidney and a caudal or trunk kidney. The cranial portion of the kidney was composed primarily of hemopoietic and lymphoid tissue interspersed with pigment granules. Renal tubules were sparsely placed in the head kidney but rapidly increased in number toward the more caudal regions. Mesonephric ducts extended from the interior portion to the ventral aspect of the caudal kidney, where they joined to form a single duct which terminated ultimately at the urogenital orifice. Interrenal tissue, homologous to the mammalian adrenal cortex, was found scattered along blood vessel walls. The caudal kidney contained numerous nephrons and ducts surrounded by or embedded in hemopoietic tissue. Chromaffin tissue, homologous to mammalian adrenal medullary tissue, was found in strips along the dorsal surface of the kidney. As described here, the nephron was composed of the renal corpuscle and renal tubules. These tubules were subdivided into: (1) a short neck segment, (2) the proximal convoluted segment, divided both structurally and functionally into a first and second portion, ( 3 ) a n intermediate segment and ( 4 ) the distal segment (fig. 1). Following the nephron were the collecting and mesonephric ducts. Renal corpuscle. Several structural characteristics appeared consistently among the species examined (figs. 2, 3). The parietal layer of Bowman's capsule was composed of a basement membrane surrounded by squamous cells with flattened nuclei. Elongate mitochondria were present throughout the cytoplasm of the parietal epithelium, The surrounding basement membrane was continuous with the base1 Specimens were obtained from the following sources: a. Colorado Game and Fish Department; Bellevue, Colorado Rearing Unit. b. Wvomine Game and Fish Department; Lzramie Wyoming R&earch Laboratory. c . Department of Ngtural Resources; Fort Qu'Appelle, Saskatchewan. 2 In previous publications the terms anterior or head kidney and posterior or trunk kidney have been used to describe the two structurally distinct regions of the kidney. These terms lead to confusion because of their application in human anatomy. In reference to quadrupeds they have been replaced in the Nomina Anatomica Veterinaria, 1973, by the terms cranial and caudal. The authors suggest that these terms should be used i n reference to the piscine kidney.
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RENAL MORPHOLOGY OF FRESHWATER TROUT
RC
YM -c
\IS
\
1 Fig. 1 Schematic drawing of the nephric tubule and duct system in the trout. Distinctions between segments are made on the basis of characteristic morphology as well as luminal and tubular diameter. This figure has been modified to demonstrate the trout nephron, from a diagram originally published in relation to the flounder nephron (with permission of C. P. Hickman and B. F. Trump). Renal corpuscle (RC), neck ( N ) , first portion of the proximal segment (PI), second portion of the proximal segment (P2), intermediate segment (IS), distal tubule (DT), collecting duct ( C D ) , and mesonephric duct (MD).
ment membrane of the visceral layer as well as that of the afferent arteriole (fig. 4). The podocytes of the visceral epithelial layer had large, cuboidal nuclei in their central portion. Near the mesangial area, these cells were packed rather closely together with little space for the foot processes to extend peripherally. Away from this point, however, the large complex foot
processes were readily identifiable. The smaller, interdigitating pedicels completed the extensions of the podocytes around the capillary loop (fig. 5). A basement membrane of approximately one-half the thickness of that surrounding the parietal layer was reflected along the complete layer of podocytic celIs and their extensions. Mitochondria were numerous throughout the
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BETTINA G. ANDERSON AND RICHARD D. LOEWEN
cytoplasmic extensions of the visceral epithelial cells. They were rather small and frequently circular, or narrow and elongated, in profile. The endothelium of the capillary loops was quite thin in some portions, and i n these areas lay adjacent to the basement membrane of the visceral layer of the capsule; there was no intervening layer of mesangium. Thus, mesangial cells did not completely encircle the capillary loops (fig. 5). The mesangial area of cells and matrix, as well as the capillary loops, were separated by the lamina densa from the visceral epithelium. T h e neck. In the area where the parietal layer of Bowman’s capsule approached the tubule neck, approximately 4-6 rows of cuboidal cells were found. Abruptly, the cells became columnar with basally located nuclei, marking the beginning of the neck region. The most prominent feature of this area was the clumps of cilia extending from some of these cells (‘fig. 6). Elongate mitochondria, multivesicular bodies and lipid droplets were scattered in the cytoplasm of the neck cells. Both rough- and smooth-surfaced endoplasmic reticulum were well developed and free ribosomes were abundant. These ciliated cells were found interspersed with a second cell type, similar i n structure to those to be described below in the first portion of the proximal tubule. The zonula occludens and zonula adherens portions of the junctional complex were prominent (fig. 7 ) . PToximal tubule. The transition from neck segment to the proximal tubule occurred with an additional increase i n cell height, as well as a n increase in tubular lumen diameter and outside diameter. The cells were columnar, with basal or subcentrally located spherical nuclei. The cytoplasm was relatively light i n staining, especially i n the highly vacuolated mica1 region (fig. 8). At the ultrastructural level, microvilli covering the apical surface of the cells appeared to be quite long and densely packed (fig. 9). These cells were characterized by organelles of the apical zone: dense apical tubules, vacuoles, small vesicles and numerous large vesicular structures with a moderately dense matrix. Mitochondria were scattered through threefourths of each cell but were absent from
the apical zone (fig. 9). Small vacuoles were present throughout the cell, as were smooth and rough endoplasmic reticulum. The junctional complex was prominent in the apical regions between adjacent cells, with zonula occludens and zonula adherens of approximately the same depth. The second portion of the proximal segment differed markedly from the first. The presence of even taller columnar epithelial cells produced a n increase in outer tubular diameter as well as a decrease in luminal diameter. The cells of this region had centrally located oval nuclei, and mitochondria were dispersed throughout the cytoplasm (fig. 10). The brush border remained prominent although the large vesicular structures and dense apical tubules characteristic of the first proximal segment were lacking (fig. 11). Large lipid droplets and dense-cored vesicles were also regularly distributed throughout this portion. An extensive system of smooth-surfaced endoplasmic reticulum was found throughout the cells. I n the basal portion of the cell the filamentous mitochondria were lined up parallel to the cell axis with infoldings of the basal plasmalemma interspersed between them (fig. 12). Intermediate segment. Our observations a t the ultrastructural level indicated that there was a very short ciliated portion of the tubule at the termination of the second proximal segment (fig. 13). ‘The cells were cuboidal i n shape; many had only a single cilium, others numerous cilia, extending from the apical portion (fig. 14). Microvilli were short and inconspicuous. Clumps of sectioned cells, apparently from the intermediate segment, were found i n the tubule lumen close to the termination of the second proximal portion. The nuclei were centrally located and the somewhat scanty cytoplasm was occupied by rounded mitochondria, smooth-surfaced endoplasmic reticulum, small clear vacuoles and rosettes of free ribosomes. Junctional complexes were distinctive in that the macula occludens or desmosomal portion was clearly defined, and frequently more than one desmosome was present between adjacent cells. Distal tubule. Cell height was less in the distal segment, in addition to a de-
RENAL MORPHOLOGY OF FRESHWATER TROUT
crease i n both inner and outer tubular diameter. Throughout this segment, the cells were low cuboidal and stained lightly and uniformly with eosin (fig. 15). The microvilli were short and evenly placed a t the initial portion of the segment, but subsequently became more random and inconspicuous in their appearance. Mitochondria were scattered throughout each cell, but tended to be perpendicular to the basement membrane and parallel to plasmalemma1 infoldings (fig. 16). The quantity of small vacuoles, multivesicular bodies and free ribosomes was increased above previously described segments. Ducts. The collecting duct system consisted of tubules joining small ducts, which in turn united with larger ducts until the mesonephric ducts were reached. Characteristically, layers of smooth muscle and connective tissue surrounded the basement membrane of the collecting ducts in increasing quantities as the mesonephric duct was approached (figs. 15, 17). Cells of the collecting tubules changed from cuboidal to columnar with basally located nuclei; those of the collecting ducts were tall columnar. The size of the mesonephric duct depended upon the area in which it was observed. The duct became larger caudally as it received increasing numbers of collecting ducts (fig. 17). The cells were tall columnar and became pseudostratified. The lumen of the duct was characteristically irregular in appearance. DISCUSSION
A basis for comparative ultrastructural study of the nephron among fishes has previously been established (Bulger and Trump, '68; Hickman and Trump, '69), relating to both renal structure and function in fresh-water and marine species. Still, the innumerable variatiops found among different species of bony fishes belie the assumption that renal structure of one fresh-water species is identical to that of any other. The glomerulus in fresh-water trout is highly vascular in nature. The visceral epithelial cells are located in close apposition to one another, with their pedicular extensions applied i n varying thickness to the lamina densa. When compared with the
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hagfish (Heath-Eves and McMillan, '74), the podocytes and capillary endothelium of the trout form a very slim structural barrier. That portion of the capillary loops not enclosed by lamina densa is bounded by mesangial cells, whose extensions are interposed between the basement membrane and the fenestrated endothelial cells. As i n the bluegill and other fresh-water species (Hickman and Trump, '69), the mesangial cells do not extend completely around the capillary loop. Ciliation of cells in the neck segment appears to be a highly variable characteristic. Two fresh-water species, Platypoecilus maculatus and Xiphophorus helleri, have been described as completely lacking ciliary apical extensions (Edwards, '35), while the neck segment in the bluegill is described as ciliated (Hickman and Trump, '69). Trout appear to be most like the English sole in that cilia emerge in groups from some cells. However, unlike the sole, the neck cells are not mucus-secreting in either Salmo gairdneri or Salvelinus fontinalis. Cells of the entire proximal tubule are characterized by a prominent brush border, unlike some marine species. Single, ciliated cells lacking microvilli, similar to those described by Gritzka ('63) in Fundulus, are occasionally observed in the proximal segment. Cells of the first proximal segment are distinguished from all others of the nephron by the presence of dense apical tubules, small vacuoles, and variously sized vesicular structures. Similar characteristics of corresponding segmental portions have been described in some mammalian species (Stone et al., '61; Latta et al., '67; Bulger et al., '74). Each of these organelles has also been identified in relation to the first proximal segment in other fishes (agnathans, Youson and McMillan, '70; fresh-water and marine teleosts, Hickman and Trump, '69). It is the presence of these signs of ingestion which suggests a function for the first proximal segment in the uptake of large molecules into the tubular cells. Attachment zones are extensive throughout the apical portions of cells in the renal tubular system. Evidence has been provided by a number of studies (Latta et al., '67) to indicate that in addition to holding
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BETTINA G . ANDERSON AND RICHARD D. LOEWEN
the renal epithelial cells together the attachment zones act as barriers to intercellular diffusion. In the trout proximal segment, tight and intermediate junctions are of nearly equal depth. Desmosomes occur generally in series of two or three along the apical third of the cell. This appearance is somewhat different from that in the rat, in which only the intermediate junctions are prominent in this segment (Farquhar and Palade, ’63). Cells of a similar nature have been described in the archinephric duct of the hagfish (Ericsson and Trump, ’69; Heath-Eves and McMillan, ’74), suggesting a homology with the first proximal segment i n higher fishes and perhaps with the proximal convoluted tubules of mammals (Trump and Bulger, ’68). As is the case with other species, the second proximal segment is quite different in appearance from the first. Mitochondria, cytomembranes and plasma membranes are the major structures. Infoldings of the basal plasmalemma are present but are not extensive when compared with those of the mammalian renal corpuscle. Connections of some membranes with the basal plasmalemma are distinctly obvious following glutaraldehyde fixation as shown by Bulger and Trump (’68) in the English sole. More elaborate arrangements of the membrane system are found in the toadfish, Opsanus tau, and in the English sole after osmium tetroxide fixation (Bulger, ’65; Bulger and Trump, ’68). The membrane systems are found in close association with the numerous elongate mitochondria of the second segment. The arrangement of the mitochondria in this segment gives it the striated appearance at the light microscopic level described as the “Stabchen of Heidenhain” (1884). I n trout, as i n the bluegill, these filamentous mitochondria which parallel the long axis of the cell are prominent only in the second portion of the proximal tubule. The presence of a ciliated terminal portion of the proximal tubule i n trout has been suggested previously (Trump, ’68) although not demonstrated. The segment was not identified by Newstead and Ford (’60) in their study of the closely related Pacific pink salmon (Oncorhynchus gor-
buscha). The intermediate segment in the trout appears to be comparable in structure to that of the fresh-water species Lepomis macrochirus (Hickman and Trump, ’69). Cells of the intermediate segment in trout, however, are high cuboidal rather than low cuboidal and only a few of them are ciliated. Those cells which are ciliated probably assist with the propulsion of fluid along the nephron. I n the distal segment, the number and size of the mitochondria are markedly decreased. Plasmalemmal systems remain associated with the mitochondria as in the second proximal segment. Attachment zones are generally narrower than those of the proximal segment. Single cilia are frequently associated with the apical portion of the cells of the distal tubule and are perhaps made more evident by the lack of a brush border. Similar long cilia have been described projecting into the lumen a t frequent intervals in the rat kidney (Bulger et al., ’74). The structure of the kidney in all species examined was found to be quite similar. There were no apparent differences at the ultrastructural level between the nephrons of Salmo gairdneri and Salvelinus fontinalis. The trout kidney showed few variations from the general pattern of the freshwater species Lepomis macrochirus. The most significant differences occur in the number of ciliated cells in the neck and intermediate segments, the location of infoldings of the basal plasmalemma and the height of epithelial cells throughout the nephron. In order to provide a more complete picture of the renal morphology of the family Salmonidae, the whitefish and grayling groups should also be examined. LITERATURE CITED Bulger, R. E. 1965 The fine structure of the aglomerular nephron of t h e toadfish, Opsunus tau. Am. J. Anat., 117: 171-192. Bulger, R. E., R. L. Siege1 a n d R. Peiidergrass 1974 Scanning and transmission electron microscopy of t h e r a t kidney. Am. J. Anat., 139: 483-502. Bulger, R. E., a n d B. F. Trump 1968 Renal morphology of t h e English sole ( P m o p h r y s vetulus). Am. J. Anat., 123: 195-226. Edwards, J. G. 1935 The epithelium of t h e renal tubule in bony fish. Anat. Rec., 63: 263279. Ericsson, J. L. E., and B. F. Trump 1969 Elec-
RENAL MORPHOLOGY OF FRESHWATER TROUT tron microsccpy of the uriniferous tubules. In: The Kidney, Morphology, Biochemistry and Physiology. C. Rouiller and A. Muller, eds. Vol. 1. Academic Press Inc., New York, pp. 351-440. Farquhar, M. G., and G. E. Palade 1963 Junctional complexes in various epithelia. J. Cell. Biol., 17: 375-412. Gritzka, T. L. 1963 The ultrastructure of the proximal convoluted tubule of a euryhaline teleost, Fundulus heteroclitus. Anat. Rec., 145: 235-236. Heath-Eves, M. J., and D. B. McMillan 1974 The morphology of the kidney of the Atlantic hagfish, Myxine glutinosa (L.). Am. J. Anat., 139: 309-334. Heidenhain, R. 1874 Mikroskopishe Beitrage zur Anatomie und Physiologie der Nieren. Arch. Mikroskop. Anat. Entwieklungs mech., 10: 1-50. Hendricks, J. D. 1971 Histological and histochemical changes occurring in the kidneys of coho salmon smolts (Oncorhynchus kisutch) upon transfer to hypertonic and hypotonic salt solutions. Ph.D. Thesis. Colorado State University. Hickman, C. P., qnd B. F. Trump 1969 The kidney. In: Fish Physiology. Vol. 1. W. S . Hoar and D. J. Randall, ~ 7 s .Academic Press Inc., New York, pp. 91-240. Latta, H., A. B. Maunsbach and L. Osvaldo 1967 The fine structure of renal tubules in cortex and medulla. In: Ultrastructure in Biological Systems Vol. 2. Ultrastructure of the Kidney. A. J . Dalton and F. Haguenae, eds. Academic Press Inc., New York, pp. 1-56. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409414. Luna, L. G., ed. 1968 Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third ed. The Blakiston Division, McGraw-Hill Book Company, New York. 258 pp.
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Newstead, J. D., and P. Ford 1960 Studies on the development of the kidney of the Pacific pink salmon (Oncorhynchus gorbuscha (Walbaus) ). 111. The development of the mesonephIons with particular reference to the mesonephric tubules. Can. J. Zool., 38: 1-7. Stone, R. S . , S. A. Bencosme, H. Latta and S. C. Madden 1961 Renal tubular fine structure studied during reaction to acute uranium injury. Arch. Pathol., 71: 160-174. Trump, B. F. 1968 Unpublished data. Cited by: Hickman, C. P., and B. F. Trump 1969 The kidney. In: Fish Physiology. Vol. 1. W. S. Hoar and D. J. Randall, eds. Academic Press Inc., New York, pp. 91-240. Trump, B. F., and R. E. Bulger 1968 The morphology of the kidney. In: The Structural Basis of Renal Disease. E. L. Becker, ed. Harper (Hoeber), New York, pp. 1-92. Youson, J. H., and D. B. McMillan 1970a The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). I. The renal corpuscle. Am. J. Anat., 127: 207-232. 1970b The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.).11. Neck and proximal segments of the tubular nephron. Am. J. Anat., 127: 233-25 8. 1971a The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). 111. Intermediate, distal and collecting segments of the ammocoete. Am. J. Anat., 130: 55-72. __ 1971b The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). IV. Intermediate, distal and collecting segments of the adult. Am. J. Anat., 130: 281-304. 1971c The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). V. The archinephric duct. Am. J. Anat., 131: 289-314.
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PLATE 1 EXPLANATION OF FIGURES
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2
Schematic drawing of the glomerulus of fresh-water trout (modified with permission of C. P. Hickman and B. F. Trump). The parietal epithelium (PE) and basment membrane (BM) form Bowman’s capsule. At the hilar region, the mesangid matrix and cells ( M e ) predominate. Throughout the major portion of the capillary tuft the lamina densa (LD) is sandwiched between the attenuated endothelial cells ( E n ) and the complex foot processes of the visceral epithelium ( V E ) . Bowman’s space (BS) becomes continuous with the lumen of the neck segment ( N ) . Outlined square indicates the location of figure 4.
3
Photomicrograph of the trout glomerulus from which figure 2 was drawn. Basement membrane (BM), Eowman’s space ( B S ) , endothelial cell ( E n ) , mesangial cells and matrix (Me), short neck segment ( N ) , parietal (PE) and visceral epithelium ( V E ) , lamina densa (LD) and pigment granules (PG). Gomori for chromaffin. X 1,540.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
4 Part of a glomerulus from the Rainbow trout, Salmo gairdneri. The parietal epithelium ( P E ) and basement membrane (BM) of Bowman’s capsule are shown as they approach the hilum of the glomerulus. The continuity of each with the visceral epithelium (VE) and the lami-la densa (LD), respectively, is demonstrated. x 6,000. 5
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Electron micrograph of a portion of the trout glomerulus. The capillary walls are quite thin in regions where the endothelium (En) and lamina densa (LD) are in close association with the podocytes of the visceral epithelial cells (VE). Erythrocytes ( E ) are found within the capillary lumen. Mesangial cell (Me). X 5,250.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G . Anderson and Richard D. Loewen
PLATE 2
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PLATE 3 EXPLANATION O F FIGURES
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6
Neck segment of the Brook trout, Salvelinus fontinalis. Note the numerous cilia within the tubular lumen ( C ) . Not all of the cells of the neck segment are ciliated ( N ) . Some cells appear to have only microvilli extending from the apical region into the lumen. x 7,875.
7
Neck segment of the nephron in the Brook trout, Saluelinus fontinalis. The nonciliated cells have characteristics typical of the adjacent proximal segment (PI). Ciliated neck cell ( N ) . X 10,800.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G . Anderson and Richard D. Loewen
PLATE 3
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PLATE 4 EXPLANATION O F FIGURES
8 Photomicrograph of kidney tubules in the Brook trout, Salvelinus fontinalis. Cells of the proximal segment bear a prominent brush border. Cells of the first proximal segment ( P I ) appear nearly clear in the apical region, while those of the second proximal segment ( P 2 ) show a dispersion of stained elements throughout the cell. HPS. X 1,025. 9 First proximal segment of the nephron of the Rainbow trout, Salmo gairdneri. Microvilli forming the brush border (BB) are long and densely packed. Junctional complexes ( J C ) are located at the apical ends of the cells. The segment is characterized in part by the presence of numerous secondary lysosomes ( L ) and apical vacuoles ( A V ) . The mitochondria ( M ) tend to be central or basal in location. X 11,370.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
10 Photomicrograph of 1 p section of the second proximal segment of the Rainbow trout nephron. The nuclei are centrally located, the brush border prominent, and mitochondria are distributed throughout the cell. Richardson. X 1,025. 11
Second proximal segment from the Rainbow trout, Salmo gairdaeri. The brush border (BB) is formed of closely packed microvilli. Junctional complexes ( J C ) join adjacent cells at the apical end. Mitochondria are both numerous and large, generally containing several mitochondria1 bodies (MB). Infolding of the plasmalemma (arrows) is common. Both smooth- and rough-surfaced endoplasmic reticulum are distributed throughout the cell. X 12,250.
12 The apical region of the second proximal segment is markedly different from the first; both apical tubules and vacuoles are lacking in the second segment. Lipid ( L ) droplets are occasionally found in the apical portions of cells. The elongate mitochondria (M) appear to be related to plasmalemma infoldings (arrows). X 10,800.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 5
PLATE 6 EXPLANATION O F FIGURES
13
The terminal portion of the second proximal segment. The intermediate segment is quite short, but is distinctive from both proximal and distal segments. Cells of the proximal segment ( P z ) are low columnar and have a brush border (BB) of short microvilli. Cells of the intermediate segment are cuboidal and generally ciliated. Cilia (C) are numerous in the tubular lumen. Junctional complexes (JC) are present at the apical cell boundaries. These are distinctive in that frequently more than one desmosome ( D ) is present. Secondary lysosomes ( L ) are scattered in the cytoplasm, as are numerous infoldings of the plasmalemma (arrows). x 6,000.
14 Cells of the intermediate segment have scattered, small mitochondria and cilia ( C ) protruding on the luminal surface. Brush border (BE) of adjacent cell. x 12,000. 15 Photomicrograph showing a part of the collecting system in the Rainbow trout. The distal tubules (DT) are formed of cuboidal cells with basal nuclei. This segment is followed by the collecting tubules (CT), which are lined by columnar epithelial cells with basally located nuclei. Connective tissue and smooth muscle cells line the outer limits of the tubule. The tubules terminate in still larger collecting ducts (CD) in which the epithelial cells are still taller columnar. Collecting ducts are surrounded by one or more layers of smooth muscle. Interstitial hemopoietic tissue (IH). HPS. X 770.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 6
111
PLATE 7 EXPLANATION O F FIGURES
16 The general intracellular relationships of the distal segment in the trout. The microvilli which extend from the apical portion of the cell are short and inconspicuous. The mitochondria are elongate and tend to be placed perpendicular to the basement membrane (BM) of the cell. In addition they appear to be associated with infoldings of the plasmalemma (arrows). In the more basal portion of the cell are the Golgi apparatus (G), multivesicular bodies (MB) and several lysosomes ( L ) . x 9,600.
17 Photomicrograph of the collecting system i n the Brook trout, Salvelilzus f o n t i n a h . The collecting tubule ( C T ) is distinguished by tall columnar epithelium. The collecting ducts (CD) have the addition of some smooth muscle and connective tissue surrounding the tubule. Ultimately the collecting ducts terminate in mesonephric ducts (MD) which are distinguished by the presence of pseudostratified epithelium and several layers of smooth muscle applied to the outer outer aspect of the tubule. HPS. x 300.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 7
113
ABSTRACT The nephron of the euryhaline freshwater salmonids is composed of the renal corpuscle and the renal tubules. Throughout much of the renal corpuscle, only the lamina densa separates the fenestrated processes of the endothelial cells from the foot processes of the visceral epithelium. The renal tubule consists of five distinct segments. The neck segment is short and intermittently ciliated; it lacks the mucous cells which appear in the neck segment of some teleosts. The proximal segment bears a dense brush border and is both structurally and functionally divisible into a first and a second segment. The first portion is typified by the presence of short apical tubules, variously sized apical vacuoles, and numerous lysosomes. The second proximal segment is distinguished by the abundance and distribution of mitochondria throughout the cytoplasm. Infoldings of the basilar plasmalemma are especially prominent in this region. A ciliated intermediate segment intervenes briefly between the proximal and distal portions of the tubule. The distal segment consists of cuboidal cells which bear scattered, short microvilli, small vesicles and multivesicular bodies. Renal tissue from several species of trout was examined in order to establish the basis for a common pattern of histological and ultrastructural characteristics within the familv Salmonidae. I n all sDecies examined, renal structure was very similar and could readily be compared with that previously described in other freshwater and marine species.
Structural variations resulting from en- prey (Petromyzon marinus: Youson and vironmental adaptation and species special- McMillan, '70a,b; '7la,b,c) and the Atlantic ization are frequently an obstacle to the hagfish (Myxine glutinosa: Heath-Eves study of fish morphology and physiology. and McMillan, '74). Renal morphology of a limited number The use of trout as laboratory animals of representatives from environments of has become more common with the advent varied salinity has been presented by previ- of fish farming, lake and stream stocking ous investigators. Both histological and and pollution monitoring. Structural alterultrastructural features of the kidney of ation in those systems which are rapidly the English sole (Parophrys v e t u l u s ) , a affected by stress may be considered an marine species, were described originally effective diagnostic tool both in disease by Bulger and Trump ('68), those of a control and pollutant detection. Its usefulfreshwater species of bluegill (Lepomis ness, however, is dependent upon a knowlmacrochirus) and a marine euryhaline edge of morphology in healthy animals. species, the Southern flounder (ParaThe objective of this study was to establichthys b t h o s t i g m a ) by Hickman and lish a common pattern of renal morphology Trump ('69). The work of other investi- among fresh-water trout, and to compare gators involving additional species was this with kidney structure as described by also reviewed by these last-named authors. others in fresh-water, euryhaline and maThe general microanatomy of the pink rine species. and coho salmon kidney (Oncorhynchus MATERIALS AND METHODS gorbuscha and 0. hisuteh) has also been Histological sections from each of four described (Newstead and Ford, '60; Hendricks, '71 ) . Excellent studies have species were examined; these were Salmo been made most recently on the sea lam- aguabonita, S. gairdneri, S . trutta and AM. J. ANAT., 143: 93-114.
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BETTINA G . ANDERSON AND RICHARD D. LOEWEN
Salvelinus fontinalis. These fish were obtained both in the wild and from hatcheries i n the Unite1 States and Canada.' Portions of both the head and trunk kidney2 were collected from 12 fish. After severance of the spinal cord the body cavity was opened along the ventral midline; renal tissue was removed and immediately immersed in Bouin's fluid for fixation. Paraffin sections were stained by a variety of methods. Routinely, hematoxylin-phloxin-safran (HPS) (Luna, '68) stain was applied, but occasionally sections were stained with Gomori's method for chromaffin bodies, Cain's method for mitochondria or hematoxylin and eosin for general staining . Subsequent to the histological studies, two species, Salmo gairdneri and Salvelinus fontinalis, were selected for examination at the ultrastructural level. I n each of six specimens the spinal cord was severed immediately after removal of the fish from a holding tank. The body cavity was opened and three blocks of kidney tissue removed from the region between the midbody and the anal oriface. Each tissue block was divided into several smaller blocks and these fixed by one of several methods: (a) 6.25% phosphate-buffered glutaraldehyde with postfixation in 1% osmium tetroxide at pH 8.1, ( b ) 1% glutaraldehyde with postfixation i n osmium tetroxide, and ( c ) 2% osmium tetroxide in sodium bicarbonate. These tissues were dehydrated in ethanol and embedded in Epon (Luft, '61). One micron sections were cut from each block, stained with 1% toluidine blue in 1% borax and examined so that specific portions of the nephron could be isolated for thin sectioning. Thin sections from the trimmed blocks were stained with saturated uranyl acetate and Reynold's lead citrate and examined with a n Hitachi HU-12 electron microscope. OBSERVATIONS
The kidneys of these trout were fused, appearing a s one organ rather than two. They occupied a dorsal retroperitoneal position along the entire length of the body cavity, bounded by the vertebrae dorsally, the ribs laterally and the swim bladder
ventrally. On the basis of morphological differences as well as location, they were divided into a cranial or head kidney and a caudal or trunk kidney. The cranial portion of the kidney was composed primarily of hemopoietic and lymphoid tissue interspersed with pigment granules. Renal tubules were sparsely placed in the head kidney but rapidly increased in number toward the more caudal regions. Mesonephric ducts extended from the interior portion to the ventral aspect of the caudal kidney, where they joined to form a single duct which terminated ultimately at the urogenital orifice. Interrenal tissue, homologous to the mammalian adrenal cortex, was found scattered along blood vessel walls. The caudal kidney contained numerous nephrons and ducts surrounded by or embedded in hemopoietic tissue. Chromaffin tissue, homologous to mammalian adrenal medullary tissue, was found in strips along the dorsal surface of the kidney. As described here, the nephron was composed of the renal corpuscle and renal tubules. These tubules were subdivided into: (1) a short neck segment, (2) the proximal convoluted segment, divided both structurally and functionally into a first and second portion, ( 3 ) a n intermediate segment and ( 4 ) the distal segment (fig. 1). Following the nephron were the collecting and mesonephric ducts. Renal corpuscle. Several structural characteristics appeared consistently among the species examined (figs. 2, 3). The parietal layer of Bowman's capsule was composed of a basement membrane surrounded by squamous cells with flattened nuclei. Elongate mitochondria were present throughout the cytoplasm of the parietal epithelium, The surrounding basement membrane was continuous with the base1 Specimens were obtained from the following sources: a. Colorado Game and Fish Department; Bellevue, Colorado Rearing Unit. b. Wvomine Game and Fish Department; Lzramie Wyoming R&earch Laboratory. c . Department of Ngtural Resources; Fort Qu'Appelle, Saskatchewan. 2 In previous publications the terms anterior or head kidney and posterior or trunk kidney have been used to describe the two structurally distinct regions of the kidney. These terms lead to confusion because of their application in human anatomy. In reference to quadrupeds they have been replaced in the Nomina Anatomica Veterinaria, 1973, by the terms cranial and caudal. The authors suggest that these terms should be used i n reference to the piscine kidney.
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RENAL MORPHOLOGY OF FRESHWATER TROUT
RC
YM -c
\IS
\
1 Fig. 1 Schematic drawing of the nephric tubule and duct system in the trout. Distinctions between segments are made on the basis of characteristic morphology as well as luminal and tubular diameter. This figure has been modified to demonstrate the trout nephron, from a diagram originally published in relation to the flounder nephron (with permission of C. P. Hickman and B. F. Trump). Renal corpuscle (RC), neck ( N ) , first portion of the proximal segment (PI), second portion of the proximal segment (P2), intermediate segment (IS), distal tubule (DT), collecting duct ( C D ) , and mesonephric duct (MD).
ment membrane of the visceral layer as well as that of the afferent arteriole (fig. 4). The podocytes of the visceral epithelial layer had large, cuboidal nuclei in their central portion. Near the mesangial area, these cells were packed rather closely together with little space for the foot processes to extend peripherally. Away from this point, however, the large complex foot
processes were readily identifiable. The smaller, interdigitating pedicels completed the extensions of the podocytes around the capillary loop (fig. 5). A basement membrane of approximately one-half the thickness of that surrounding the parietal layer was reflected along the complete layer of podocytic celIs and their extensions. Mitochondria were numerous throughout the
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BETTINA G. ANDERSON AND RICHARD D. LOEWEN
cytoplasmic extensions of the visceral epithelial cells. They were rather small and frequently circular, or narrow and elongated, in profile. The endothelium of the capillary loops was quite thin in some portions, and i n these areas lay adjacent to the basement membrane of the visceral layer of the capsule; there was no intervening layer of mesangium. Thus, mesangial cells did not completely encircle the capillary loops (fig. 5). The mesangial area of cells and matrix, as well as the capillary loops, were separated by the lamina densa from the visceral epithelium. T h e neck. In the area where the parietal layer of Bowman’s capsule approached the tubule neck, approximately 4-6 rows of cuboidal cells were found. Abruptly, the cells became columnar with basally located nuclei, marking the beginning of the neck region. The most prominent feature of this area was the clumps of cilia extending from some of these cells (‘fig. 6). Elongate mitochondria, multivesicular bodies and lipid droplets were scattered in the cytoplasm of the neck cells. Both rough- and smooth-surfaced endoplasmic reticulum were well developed and free ribosomes were abundant. These ciliated cells were found interspersed with a second cell type, similar i n structure to those to be described below in the first portion of the proximal tubule. The zonula occludens and zonula adherens portions of the junctional complex were prominent (fig. 7 ) . PToximal tubule. The transition from neck segment to the proximal tubule occurred with an additional increase i n cell height, as well as a n increase in tubular lumen diameter and outside diameter. The cells were columnar, with basal or subcentrally located spherical nuclei. The cytoplasm was relatively light i n staining, especially i n the highly vacuolated mica1 region (fig. 8). At the ultrastructural level, microvilli covering the apical surface of the cells appeared to be quite long and densely packed (fig. 9). These cells were characterized by organelles of the apical zone: dense apical tubules, vacuoles, small vesicles and numerous large vesicular structures with a moderately dense matrix. Mitochondria were scattered through threefourths of each cell but were absent from
the apical zone (fig. 9). Small vacuoles were present throughout the cell, as were smooth and rough endoplasmic reticulum. The junctional complex was prominent in the apical regions between adjacent cells, with zonula occludens and zonula adherens of approximately the same depth. The second portion of the proximal segment differed markedly from the first. The presence of even taller columnar epithelial cells produced a n increase in outer tubular diameter as well as a decrease in luminal diameter. The cells of this region had centrally located oval nuclei, and mitochondria were dispersed throughout the cytoplasm (fig. 10). The brush border remained prominent although the large vesicular structures and dense apical tubules characteristic of the first proximal segment were lacking (fig. 11). Large lipid droplets and dense-cored vesicles were also regularly distributed throughout this portion. An extensive system of smooth-surfaced endoplasmic reticulum was found throughout the cells. I n the basal portion of the cell the filamentous mitochondria were lined up parallel to the cell axis with infoldings of the basal plasmalemma interspersed between them (fig. 12). Intermediate segment. Our observations a t the ultrastructural level indicated that there was a very short ciliated portion of the tubule at the termination of the second proximal segment (fig. 13). ‘The cells were cuboidal i n shape; many had only a single cilium, others numerous cilia, extending from the apical portion (fig. 14). Microvilli were short and inconspicuous. Clumps of sectioned cells, apparently from the intermediate segment, were found i n the tubule lumen close to the termination of the second proximal portion. The nuclei were centrally located and the somewhat scanty cytoplasm was occupied by rounded mitochondria, smooth-surfaced endoplasmic reticulum, small clear vacuoles and rosettes of free ribosomes. Junctional complexes were distinctive in that the macula occludens or desmosomal portion was clearly defined, and frequently more than one desmosome was present between adjacent cells. Distal tubule. Cell height was less in the distal segment, in addition to a de-
RENAL MORPHOLOGY OF FRESHWATER TROUT
crease i n both inner and outer tubular diameter. Throughout this segment, the cells were low cuboidal and stained lightly and uniformly with eosin (fig. 15). The microvilli were short and evenly placed a t the initial portion of the segment, but subsequently became more random and inconspicuous in their appearance. Mitochondria were scattered throughout each cell, but tended to be perpendicular to the basement membrane and parallel to plasmalemma1 infoldings (fig. 16). The quantity of small vacuoles, multivesicular bodies and free ribosomes was increased above previously described segments. Ducts. The collecting duct system consisted of tubules joining small ducts, which in turn united with larger ducts until the mesonephric ducts were reached. Characteristically, layers of smooth muscle and connective tissue surrounded the basement membrane of the collecting ducts in increasing quantities as the mesonephric duct was approached (figs. 15, 17). Cells of the collecting tubules changed from cuboidal to columnar with basally located nuclei; those of the collecting ducts were tall columnar. The size of the mesonephric duct depended upon the area in which it was observed. The duct became larger caudally as it received increasing numbers of collecting ducts (fig. 17). The cells were tall columnar and became pseudostratified. The lumen of the duct was characteristically irregular in appearance. DISCUSSION
A basis for comparative ultrastructural study of the nephron among fishes has previously been established (Bulger and Trump, '68; Hickman and Trump, '69), relating to both renal structure and function in fresh-water and marine species. Still, the innumerable variatiops found among different species of bony fishes belie the assumption that renal structure of one fresh-water species is identical to that of any other. The glomerulus in fresh-water trout is highly vascular in nature. The visceral epithelial cells are located in close apposition to one another, with their pedicular extensions applied i n varying thickness to the lamina densa. When compared with the
97
hagfish (Heath-Eves and McMillan, '74), the podocytes and capillary endothelium of the trout form a very slim structural barrier. That portion of the capillary loops not enclosed by lamina densa is bounded by mesangial cells, whose extensions are interposed between the basement membrane and the fenestrated endothelial cells. As i n the bluegill and other fresh-water species (Hickman and Trump, '69), the mesangial cells do not extend completely around the capillary loop. Ciliation of cells in the neck segment appears to be a highly variable characteristic. Two fresh-water species, Platypoecilus maculatus and Xiphophorus helleri, have been described as completely lacking ciliary apical extensions (Edwards, '35), while the neck segment in the bluegill is described as ciliated (Hickman and Trump, '69). Trout appear to be most like the English sole in that cilia emerge in groups from some cells. However, unlike the sole, the neck cells are not mucus-secreting in either Salmo gairdneri or Salvelinus fontinalis. Cells of the entire proximal tubule are characterized by a prominent brush border, unlike some marine species. Single, ciliated cells lacking microvilli, similar to those described by Gritzka ('63) in Fundulus, are occasionally observed in the proximal segment. Cells of the first proximal segment are distinguished from all others of the nephron by the presence of dense apical tubules, small vacuoles, and variously sized vesicular structures. Similar characteristics of corresponding segmental portions have been described in some mammalian species (Stone et al., '61; Latta et al., '67; Bulger et al., '74). Each of these organelles has also been identified in relation to the first proximal segment in other fishes (agnathans, Youson and McMillan, '70; fresh-water and marine teleosts, Hickman and Trump, '69). It is the presence of these signs of ingestion which suggests a function for the first proximal segment in the uptake of large molecules into the tubular cells. Attachment zones are extensive throughout the apical portions of cells in the renal tubular system. Evidence has been provided by a number of studies (Latta et al., '67) to indicate that in addition to holding
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BETTINA G . ANDERSON AND RICHARD D. LOEWEN
the renal epithelial cells together the attachment zones act as barriers to intercellular diffusion. In the trout proximal segment, tight and intermediate junctions are of nearly equal depth. Desmosomes occur generally in series of two or three along the apical third of the cell. This appearance is somewhat different from that in the rat, in which only the intermediate junctions are prominent in this segment (Farquhar and Palade, ’63). Cells of a similar nature have been described in the archinephric duct of the hagfish (Ericsson and Trump, ’69; Heath-Eves and McMillan, ’74), suggesting a homology with the first proximal segment i n higher fishes and perhaps with the proximal convoluted tubules of mammals (Trump and Bulger, ’68). As is the case with other species, the second proximal segment is quite different in appearance from the first. Mitochondria, cytomembranes and plasma membranes are the major structures. Infoldings of the basal plasmalemma are present but are not extensive when compared with those of the mammalian renal corpuscle. Connections of some membranes with the basal plasmalemma are distinctly obvious following glutaraldehyde fixation as shown by Bulger and Trump (’68) in the English sole. More elaborate arrangements of the membrane system are found in the toadfish, Opsanus tau, and in the English sole after osmium tetroxide fixation (Bulger, ’65; Bulger and Trump, ’68). The membrane systems are found in close association with the numerous elongate mitochondria of the second segment. The arrangement of the mitochondria in this segment gives it the striated appearance at the light microscopic level described as the “Stabchen of Heidenhain” (1884). I n trout, as i n the bluegill, these filamentous mitochondria which parallel the long axis of the cell are prominent only in the second portion of the proximal tubule. The presence of a ciliated terminal portion of the proximal tubule i n trout has been suggested previously (Trump, ’68) although not demonstrated. The segment was not identified by Newstead and Ford (’60) in their study of the closely related Pacific pink salmon (Oncorhynchus gor-
buscha). The intermediate segment in the trout appears to be comparable in structure to that of the fresh-water species Lepomis macrochirus (Hickman and Trump, ’69). Cells of the intermediate segment in trout, however, are high cuboidal rather than low cuboidal and only a few of them are ciliated. Those cells which are ciliated probably assist with the propulsion of fluid along the nephron. I n the distal segment, the number and size of the mitochondria are markedly decreased. Plasmalemmal systems remain associated with the mitochondria as in the second proximal segment. Attachment zones are generally narrower than those of the proximal segment. Single cilia are frequently associated with the apical portion of the cells of the distal tubule and are perhaps made more evident by the lack of a brush border. Similar long cilia have been described projecting into the lumen a t frequent intervals in the rat kidney (Bulger et al., ’74). The structure of the kidney in all species examined was found to be quite similar. There were no apparent differences at the ultrastructural level between the nephrons of Salmo gairdneri and Salvelinus fontinalis. The trout kidney showed few variations from the general pattern of the freshwater species Lepomis macrochirus. The most significant differences occur in the number of ciliated cells in the neck and intermediate segments, the location of infoldings of the basal plasmalemma and the height of epithelial cells throughout the nephron. In order to provide a more complete picture of the renal morphology of the family Salmonidae, the whitefish and grayling groups should also be examined. LITERATURE CITED Bulger, R. E. 1965 The fine structure of the aglomerular nephron of t h e toadfish, Opsunus tau. Am. J. Anat., 117: 171-192. Bulger, R. E., R. L. Siege1 a n d R. Peiidergrass 1974 Scanning and transmission electron microscopy of t h e r a t kidney. Am. J. Anat., 139: 483-502. Bulger, R. E., a n d B. F. Trump 1968 Renal morphology of t h e English sole ( P m o p h r y s vetulus). Am. J. Anat., 123: 195-226. Edwards, J. G. 1935 The epithelium of t h e renal tubule in bony fish. Anat. Rec., 63: 263279. Ericsson, J. L. E., and B. F. Trump 1969 Elec-
RENAL MORPHOLOGY OF FRESHWATER TROUT tron microsccpy of the uriniferous tubules. In: The Kidney, Morphology, Biochemistry and Physiology. C. Rouiller and A. Muller, eds. Vol. 1. Academic Press Inc., New York, pp. 351-440. Farquhar, M. G., and G. E. Palade 1963 Junctional complexes in various epithelia. J. Cell. Biol., 17: 375-412. Gritzka, T. L. 1963 The ultrastructure of the proximal convoluted tubule of a euryhaline teleost, Fundulus heteroclitus. Anat. Rec., 145: 235-236. Heath-Eves, M. J., and D. B. McMillan 1974 The morphology of the kidney of the Atlantic hagfish, Myxine glutinosa (L.). Am. J. Anat., 139: 309-334. Heidenhain, R. 1874 Mikroskopishe Beitrage zur Anatomie und Physiologie der Nieren. Arch. Mikroskop. Anat. Entwieklungs mech., 10: 1-50. Hendricks, J. D. 1971 Histological and histochemical changes occurring in the kidneys of coho salmon smolts (Oncorhynchus kisutch) upon transfer to hypertonic and hypotonic salt solutions. Ph.D. Thesis. Colorado State University. Hickman, C. P., qnd B. F. Trump 1969 The kidney. In: Fish Physiology. Vol. 1. W. S . Hoar and D. J. Randall, ~ 7 s .Academic Press Inc., New York, pp. 91-240. Latta, H., A. B. Maunsbach and L. Osvaldo 1967 The fine structure of renal tubules in cortex and medulla. In: Ultrastructure in Biological Systems Vol. 2. Ultrastructure of the Kidney. A. J . Dalton and F. Haguenae, eds. Academic Press Inc., New York, pp. 1-56. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409414. Luna, L. G., ed. 1968 Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third ed. The Blakiston Division, McGraw-Hill Book Company, New York. 258 pp.
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Newstead, J. D., and P. Ford 1960 Studies on the development of the kidney of the Pacific pink salmon (Oncorhynchus gorbuscha (Walbaus) ). 111. The development of the mesonephIons with particular reference to the mesonephric tubules. Can. J. Zool., 38: 1-7. Stone, R. S . , S. A. Bencosme, H. Latta and S. C. Madden 1961 Renal tubular fine structure studied during reaction to acute uranium injury. Arch. Pathol., 71: 160-174. Trump, B. F. 1968 Unpublished data. Cited by: Hickman, C. P., and B. F. Trump 1969 The kidney. In: Fish Physiology. Vol. 1. W. S. Hoar and D. J. Randall, eds. Academic Press Inc., New York, pp. 91-240. Trump, B. F., and R. E. Bulger 1968 The morphology of the kidney. In: The Structural Basis of Renal Disease. E. L. Becker, ed. Harper (Hoeber), New York, pp. 1-92. Youson, J. H., and D. B. McMillan 1970a The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). I. The renal corpuscle. Am. J. Anat., 127: 207-232. 1970b The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.).11. Neck and proximal segments of the tubular nephron. Am. J. Anat., 127: 233-25 8. 1971a The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). 111. Intermediate, distal and collecting segments of the ammocoete. Am. J. Anat., 130: 55-72. __ 1971b The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). IV. Intermediate, distal and collecting segments of the adult. Am. J. Anat., 130: 281-304. 1971c The opisthonephric kidney of the sea lamprey of the Great Lakes, Petromyzon marinus (L.). V. The archinephric duct. Am. J. Anat., 131: 289-314.
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PLATE 1 EXPLANATION OF FIGURES
100
2
Schematic drawing of the glomerulus of fresh-water trout (modified with permission of C. P. Hickman and B. F. Trump). The parietal epithelium (PE) and basment membrane (BM) form Bowman’s capsule. At the hilar region, the mesangid matrix and cells ( M e ) predominate. Throughout the major portion of the capillary tuft the lamina densa (LD) is sandwiched between the attenuated endothelial cells ( E n ) and the complex foot processes of the visceral epithelium ( V E ) . Bowman’s space (BS) becomes continuous with the lumen of the neck segment ( N ) . Outlined square indicates the location of figure 4.
3
Photomicrograph of the trout glomerulus from which figure 2 was drawn. Basement membrane (BM), Eowman’s space ( B S ) , endothelial cell ( E n ) , mesangial cells and matrix (Me), short neck segment ( N ) , parietal (PE) and visceral epithelium ( V E ) , lamina densa (LD) and pigment granules (PG). Gomori for chromaffin. X 1,540.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
4 Part of a glomerulus from the Rainbow trout, Salmo gairdneri. The parietal epithelium ( P E ) and basement membrane (BM) of Bowman’s capsule are shown as they approach the hilum of the glomerulus. The continuity of each with the visceral epithelium (VE) and the lami-la densa (LD), respectively, is demonstrated. x 6,000. 5
102
Electron micrograph of a portion of the trout glomerulus. The capillary walls are quite thin in regions where the endothelium (En) and lamina densa (LD) are in close association with the podocytes of the visceral epithelial cells (VE). Erythrocytes ( E ) are found within the capillary lumen. Mesangial cell (Me). X 5,250.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G . Anderson and Richard D. Loewen
PLATE 2
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PLATE 3 EXPLANATION O F FIGURES
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6
Neck segment of the Brook trout, Salvelinus fontinalis. Note the numerous cilia within the tubular lumen ( C ) . Not all of the cells of the neck segment are ciliated ( N ) . Some cells appear to have only microvilli extending from the apical region into the lumen. x 7,875.
7
Neck segment of the nephron in the Brook trout, Saluelinus fontinalis. The nonciliated cells have characteristics typical of the adjacent proximal segment (PI). Ciliated neck cell ( N ) . X 10,800.
RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G . Anderson and Richard D. Loewen
PLATE 3
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PLATE 4 EXPLANATION O F FIGURES
8 Photomicrograph of kidney tubules in the Brook trout, Salvelinus fontinalis. Cells of the proximal segment bear a prominent brush border. Cells of the first proximal segment ( P I ) appear nearly clear in the apical region, while those of the second proximal segment ( P 2 ) show a dispersion of stained elements throughout the cell. HPS. X 1,025. 9 First proximal segment of the nephron of the Rainbow trout, Salmo gairdneri. Microvilli forming the brush border (BB) are long and densely packed. Junctional complexes ( J C ) are located at the apical ends of the cells. The segment is characterized in part by the presence of numerous secondary lysosomes ( L ) and apical vacuoles ( A V ) . The mitochondria ( M ) tend to be central or basal in location. X 11,370.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
10 Photomicrograph of 1 p section of the second proximal segment of the Rainbow trout nephron. The nuclei are centrally located, the brush border prominent, and mitochondria are distributed throughout the cell. Richardson. X 1,025. 11
Second proximal segment from the Rainbow trout, Salmo gairdaeri. The brush border (BB) is formed of closely packed microvilli. Junctional complexes ( J C ) join adjacent cells at the apical end. Mitochondria are both numerous and large, generally containing several mitochondria1 bodies (MB). Infolding of the plasmalemma (arrows) is common. Both smooth- and rough-surfaced endoplasmic reticulum are distributed throughout the cell. X 12,250.
12 The apical region of the second proximal segment is markedly different from the first; both apical tubules and vacuoles are lacking in the second segment. Lipid ( L ) droplets are occasionally found in the apical portions of cells. The elongate mitochondria (M) appear to be related to plasmalemma infoldings (arrows). X 10,800.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 5
PLATE 6 EXPLANATION O F FIGURES
13
The terminal portion of the second proximal segment. The intermediate segment is quite short, but is distinctive from both proximal and distal segments. Cells of the proximal segment ( P z ) are low columnar and have a brush border (BB) of short microvilli. Cells of the intermediate segment are cuboidal and generally ciliated. Cilia (C) are numerous in the tubular lumen. Junctional complexes (JC) are present at the apical cell boundaries. These are distinctive in that frequently more than one desmosome ( D ) is present. Secondary lysosomes ( L ) are scattered in the cytoplasm, as are numerous infoldings of the plasmalemma (arrows). x 6,000.
14 Cells of the intermediate segment have scattered, small mitochondria and cilia ( C ) protruding on the luminal surface. Brush border (BE) of adjacent cell. x 12,000. 15 Photomicrograph showing a part of the collecting system in the Rainbow trout. The distal tubules (DT) are formed of cuboidal cells with basal nuclei. This segment is followed by the collecting tubules (CT), which are lined by columnar epithelial cells with basally located nuclei. Connective tissue and smooth muscle cells line the outer limits of the tubule. The tubules terminate in still larger collecting ducts (CD) in which the epithelial cells are still taller columnar. Collecting ducts are surrounded by one or more layers of smooth muscle. Interstitial hemopoietic tissue (IH). HPS. X 770.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 6
111
PLATE 7 EXPLANATION O F FIGURES
16 The general intracellular relationships of the distal segment in the trout. The microvilli which extend from the apical portion of the cell are short and inconspicuous. The mitochondria are elongate and tend to be placed perpendicular to the basement membrane (BM) of the cell. In addition they appear to be associated with infoldings of the plasmalemma (arrows). In the more basal portion of the cell are the Golgi apparatus (G), multivesicular bodies (MB) and several lysosomes ( L ) . x 9,600.
17 Photomicrograph of the collecting system i n the Brook trout, Salvelilzus f o n t i n a h . The collecting tubule ( C T ) is distinguished by tall columnar epithelium. The collecting ducts (CD) have the addition of some smooth muscle and connective tissue surrounding the tubule. Ultimately the collecting ducts terminate in mesonephric ducts (MD) which are distinguished by the presence of pseudostratified epithelium and several layers of smooth muscle applied to the outer outer aspect of the tubule. HPS. x 300.
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RENAL MORPHOLOGY OF FRESHWATER TROUT Bettina G. Anderson and Richard D. Loewen
PLATE 7
113
