Cell Tiss. Res. 163, 15--28 (1975) 9 b y Springer-Verlag 1975
In vitro Culture of the Proximal Tubule
of the Bovine Nephron Transmission
and Scanning Electron
Microscopy*
Denise Cade-Treyer and Shigeru Tsuji Laboratoires de Biologic Animale V1 et IV. Universit6 Pierre et Marie Curie, Paris, France Received March 21, 1975
Summary. By means of a previously described method, viable pure tubules of the nephron were isolated in high yield from the outer cortex of the near-term foetal bovine kidney. The tubular suspension obtained was constituted almost exclusively of proximal segments (about 95%), whose cells were dispersed and grown as confluent primary cultures. The cultured proximal cells were shown to m a i n t a i n in vitro, on glass or plastic surfaces, the same orientation as on the tubular basement membrane in vivo, with interdigitations extending from the base of the cells and along their full height. Numerous mitochondria and the typical cytoplasmic bodies of the proximal cell were retained in cells grown in vitro. A flagellum was seen in every cultured cell and was shown to be present in the proximal cell in vivo. There is a progressive change, in vitro, of the microvilli of the brush border, from a close-packed to a sparse distribution and to a decrease in height a n d a reduction in number. This in vitro regression to an earlier embryonic state was correlated with the ability of the proximal cells to synthesize in vitro a n ~-foetoprotein and with the loss in vitro of histiospecific antigen synthesis, confined in vivo to the brush border area. The confluent proximal cells became filled with microfilaments a n d microtubules, the significance of which is discussed. Key words: Kidney - - Proximal tubule - - Cell culture - - Transmission and scanning electron microscopy. Rdsumd. Une m6thode, d6crite pr6c6demment, permet d'isoler, en grande quantit6 des tubules purs du nephron bovin, ~ partir du cortex externe du rein foetal s terme. La suspension tubulaire obtenue est constitu6e presqu'exclusivement de segments proximaux (de Fordre de 95 % ), dont on disperse les cellules pour les cultiver in vitro. Les cellules proximales, en cultures primaires confluentes, gardent la m~me orientation sur le verre ou le plastique, que sur la lame basale tubulaire in vivo. Elles s'engrbnent sur leurs faces lat6rales et s leur base, p a r des interdigitations. De nombreuses mitochondries et les inclusions cytoplasmiques de la cellule proximale se r e t r o u v e n t in vitro. Chaque cellule proximale in vitro porte un flagelle, qui existe d6js darts les cellules proximales du veau in vivo. Les microvillosit6s de la bordure en brosse se desserrent et s'6parpillent sur les cellules 6tal~es in vitro. Leur taille diminue et elles disparaissent m~me de certaines cellules. Ce retour in vitro, vers un 6tat embryonnaire plus pr6coce, est s mettre en rapport avec la propri6t6 des cellules proximales de synth6tiser in vitro unc a-foetoproteine; de m~me il dolt exister une corr61ation avec la perte de synthbse, dans ces cellules in vitro, des antig~nes histiosp6cifiques, localis6s in vivo, dans la zSne de la bordure en brosse. Des microtubules et des microfilaments envahissent les cellules proximales in vitro; leur signification est discut~e. Send o//print requests to: Dr. D. Cade-Treyer, Laboratoire de Biologic Animale VI, Universit6 Pierre et Marie Curie, 12 rue Cuvier, 75005 Paris, France. * We are indebted to Mrs. M. Brisorgueil and to Mrs. P. Cloup for skilful technical assistance.
16
D. Cade-Treyer and S. Tsuji Introduction
N e p h r o n s can be easily isolated from t h e k i d n e y with t h e help of e n z y m a t i c digestion. R e c e n t l y , one of us (Cade-Treyer, 1972b) devised a m e t h o d to s e p a r a t e t u b u l e s from glomeruli in high y i e l d a n d in a h i g h l y purified form. These t u b u l e s were isolated as v i a b l e f r a g m e n t s a n d were t h u s r e a d i l y c u l t u r e d in vitro (CadeTreyer, 1972 b). A n analysis of t h e t u b u l a r suspension, p r o v i d e d evidence t h a t t h e t u b u l e f r a g m e n t s o b t a i n e d with our m e t h o d from t h e o u t e r cortex of t h e calf k i d n e y , were a l m o s t e x c l u s i v e l y d e r i v e d from p r o x i m a l t u b u l e s ; we h a d t h u s a t hand, a m e t h o d to i n i t i a t e p r i m a r y cultures with a f a i r l y homogenous p o p u l a t i o n of h i s t i o t y p i c cells from t h e k i d n e y , which we h a v e called an " h i s t i o n " (CadeTreyer, 1972b). This " h i s t i o n " t h e p r o x i m a l t u b u l e of t h e nephron, has been characterized b y histiospecific antigens, which were specifically localized to its cells in vivo with i m m u n o f l u o r e s c e n t techniques. (Cade-Treyer, 1972 a.) The phenot y p i c shift of these antigens in vitro, was followed in p r i m a r y cultures of t h e p r o x i m a l cells, with i m m u n o c h e m i c a l a n d i m m u n o r a d i o g r a p h i c m e t h o d s (CadeTreyer, 1975). Moreover, t h e neosynthesis of a bovine ~-foetoprotein (Cade-Treyer, 1974) was shown to occur in cultures of the crude o u t e r cortex (Cade-Treyer, 1973) a n d to be a p r o p e r t y of p r o x i m a l t u b u l e cells alone (Cade-Treyer, 1975). These i m m u n o c h e m i c a l studies, r e v e a l i n g a double p h e n o t y p i c shift in t h e p r o x i m a l cells in vitro: t h e switching off of t h e histiospecific antigens a n d the switching on of ~-foetoprotein synthesis p r o m p t e d us to s t u d y t h e fate of t h e well-defined morphological m a r k e r s of t h e p r o x i m a l cells in p r i m a r y cultures in vitro, a t t h e level of t h e t r a n s m i s s i o n a n d t h e scanning electron microscope.
Material and Methods
Primary Cultures o/Proximal Tubular Cells The method for isolating a pure tubule fraction from the outer renal cortex from near-term calf foetuses has been described (Cade-Treyer, 1972b). After gentle trypsinization of the outer cortex, the isolated nephrons are freed of interstitial cells by repeated settling, and the supernatant, with isolated cells, discarded. The nephrons, after further disruption by pipetting, are then submitted to sieving and successive settling to separate pure tubules in the supernatant from glomeruli. Confirmation of the proximal nature of the pure tubule fragments obtained will be described in the Results. The tubules were further disrupted by pipetting to obtain tiny tubular fragments, or clusters of cells, or isolated cells, which were seeded in plastic "Falcon" flasks or on cover-slips of Leighton tubes. One ml of packed cells was diluted in 200 ml of culture medium, which consisted of casein hydrolysate in Earle's buffered salt solution with penicillin 200 iu/ml, streptomycin 50 ~zg/ml, and 3 % calf serum. After 24 to 48 hours the nonadherent cells were discarded with the medium change. A confluent sheet of cells was obtained within 5 to 7 days.
Light Microscopy The cells grown on cover-slips were observed with a Leitz ortholux microscope fitted with Zeruicke phase contrast equipment.
Transmission Electron Microscopy ( T E M ) The confluent cells, on glass cover-slips, were fixed for 2 hour at room temperature in 1% glutaraldehyde and 1% paraformaldehyde in 0.12 M phosphate buffer (pH 7.4). They were then rinsed three times with the same buffer and postfixed for 1 hour in phosphate buffered 2% osmium textroxide. The postfixed cells were then processed conventionally and embedded in Araldite, directly on the cover-slips. These were split from the embedding capsule,
Cultures of Kidney Proximal Tubules: Electron Microscopy
17
by dipping into liquid nitrogen, thus exposing at the surface of the capsule, the basal part of the cell monolayer originally in contact with the cover-slip. a) Ultrathin sections were cut parallel to the plane of the monolayer. They were stained with uranyl acetate and lead citrate for subsequent study with a Philips 300 electron microscope. b) Ultrathin sections were aslo cut perpendicular to the plane of the monolayer. In this case, cells were grown in Falcon flasks and small fragments of the flasks covered with cells were directly embedded in Araldite. A diamond knife easily cut the embedded plastic of the flask along with the sheet of ceils grown on it. c) Ultrathin sections were also cut from isolated proximal tubules in suspension, embedded as a pellet.
Scanning Electron Microscopy ( S E M ) Cells grown on cover-slips were fixed as for TEM. Air-drying was performed from absolute ethanol, as described by Boyde et al. (1972) or from propylene oxide; the latter method gave the better results. The dried cells on their cover-slip, were fixed to aluminium rivets and given a conducting coating of 150 /~ gold and palladium. They were examined at a tilt angle of 38 ~ at 20 KV with a CAMECA 07 SEM.
Results 1. Our m e t h o d of isolation of p u r e t u b u l e s requires k i d n e y cortex from neart e r m foetuses (see Cade-Treyer, 1972b). Therefore, we first verified t h a t the histogenesis o/ the proximal tubules was complete at this stage, this is n o t t h e case in o t h e r species such as t h e mouse, where t h e p r o x i m a l cell continues d i f f e r e n t i a t i n g a f t e r birth, b y progressive a c c u m u l a t i o n of apical microvilli (Clark, 1957). I n t h e calf foetal p r o x i m a l cell n e a r birth, we o b s e r v e d a good apical a l k a l i n e phosp h a t a s e a c t i v i t y and, a t t h e u l t r a s t r u c t u r a l level, a well-developed brush border. Likewise, t h e apical histiospecific a n t i g e n s of t h e p r o x i m a l cell, were r e v e a l e d with t h e same i n t e n s i t y in the foetal p r o x i m a l cell n e a r birth, as in the calf. (Cade-Treyer, 1972b.), 2. The Constitution o/ the Suspension o] the Pure Tubules i s o l a t e d from t h e bovine o u t e r cortex was s t u d i e d a t t h e u l t r a s t r u c t u r a l level on two series of suspensions, purified s e p a r a t e l y , each from 4 foetal k i d n e y s n e a r t e r m ; 150 t u b u l a r f r a g m e n t s were o b s e r v e d in each case. The results showed t h a t 93% of the fragm e n t s were of t h e p r o x i m a l t y p e , with t y p i c a l brush borders; t h e r e m a i n d e r were c o m p o s e d of collecting ducts a n d d i s t a l t u b u l e fragments. 3. The Growing Cell Population. The v e r y high p e r c e n t a g e of p r o x i m a l t u b u l a r f r a g m e n t s o b t a i n e d was n o t surprising, since we s t a r t e d with o u t e r cortex, which is k n o w n to be p a r t i c u l a r l y rich in p r o x i m a l t u b u l e s ( L i e b e r m a n a n d Ore, 1962). W e o b s e r v e d t h a t the primary cultures arising from such a t u b u l a r suspension y i e l d e d an homogenous population o/proximal cells. The confluent cultures arose from t h e fusion of islands, each s t a r t i n g from a cluster of cells, a f t e r t h e breakdown of t h e t u b u l e s (Fig. 1 a). A s t u d y of these islands a f t e r t h e first two d a y s in v i t r o (Fig. 1 a, b) d i d n o t r e v e a l a n y b u t p r o x i m a l t u b u l e islands. A t confluence, t h e initial clusters of cells are still visible as swirling groups of closely p a c k e d cells (Fig. l c ) . U n d e r t h e phase c o n t r a s t microscope, e i t h e r p o l y g o n a l , m i t o c h o n d r i a rich cells (Fig. I c, d), or e l o n g a t e d spindle-like cells (Fig. I c) are seen. The l a t t e r were easily i d e n t i f i e d as p r o x i m a l cells u n d e r t h e electron microscope, t h a n k s to t h e specific f e a t u r e of these cells (see below). 2 Cell Tiss.Res.
18
D. Cade-Treyer and S. Tsuji
Cultures of Kidney Proximal Tubules: Electron Microscopy
19
4. The Flagellum o] the Proximal Cell in vitro. SEM seemed especially appropriate to study the fate of the microvilli of the brush border of the proximal cells in vitro. But, the first surprising result was that at the time of confluence each cell was endowed with a long flagellum (Fig. 2a-f), which was present on the first day in vitro. This flagellum was still present after 2 months' subculture in vitro. In cultured cells, transverse sections of the flagellum, observed by TEM, displayed a typical structure of 2 central fibrils and 9 peripheral doublets (Fig. 3a-c). A transverse section through a cultured proximal cell in Fig. 4a shows the fl~gellar rootlet and the kinetosome. Phase contrast study of live cultured proximal cells, revealed spasmodic fl~gellar beating. The few indications in the literature of the existence of a flagellum in the proximal tubular cell, prompted us to reinvestigate the question in kidney sections and in isolated tubular suspensions. We found a single cilium (or flagellum; its length could not be determined in sections) in the proximal cells (Fig. 3 a) of the foetal calf nephron near term. In suspensions of isolated proximal tubules, we frequently observed a single flagellum, associated with a typical centriole, emerging from between the microvilli of the brush border (Fig. 3b). Moreover, a few atypical proximal cells were observed in sections of the cortex and in isolated proximal tubules. They displayed from 6 to 10 cilia (or flagella) among the microvilli. In the cytoplasm of such cells the associated centriolar structures of the kinetosomes could be identified (Fig. 3c). As adult kidney proximal cells were not investigated at the ultrastructural level, it is not possible to state whether such atypical cells are found only in foetal kidney. 5. The Brush Border. Scanning electron microscopy was especially suitable for studying the brush border cells in vitro (Fig. 2a-f). In the proximal cells at confluence, the microvilli are seen at the free cell surface, extending into the culture medium. From this it can be concluded that, in culture, the proximal cell is oriented on the glass surface as it is in vivo on the basement membrane of the tubule. In vitro, the microvilli are not as closely packed as in vivo, but they are sparsely distributed on almost every fully-spread confluent cell (Fig. 2a, b). Moreover a marked decrease in the length of the microvilli by comparison with those in vivo is evident (Figs. 2a-c, e, f, 4b, 5b), although it is possible to find adjacent cells with long microvilli (Fig. 2d) and with short microvilli
Fig. 1a--d. Phase contrast micrographs of the proximal tubular cell cultures. Fig. 1a and b. 2nd day in vitro (a, • 300, b, X 2100). Several islands of proximal cells are visible (Fig.1 a). At their centres the initial clusters of cells from which they arose are indicated by arrows. Numerous rod-like mitochondria are visible in the cytoplasm. The clear spaces between cells (Fig. lb) indicate the zone of interdigitations (zi), clearly visible in Fig. 5a. Active nuclcoli (n) are conspicuous. Fig. 1c--d. 7th day in vitro (c, • 300, d, x 3 000). Confluent cells (Fig. 1c) with the initial clusters visible as cores of crowded cells (arrows). Fully-spread cells, whether polygonal or spindle-shaped contain numerous rod-like mitoehondria. Lipid globules are also seen (arrows) (Fig. 1d)
20
D. Cade-Treyer and S. Tsuii
Fig. 2 a - - f . SEM's of the proximal cells at the 7th day in vitro (a, x 1000, b, e, x 5000, c, d, X 10000, f, x 2000). A flagellum is conspicuous on every cell (arrows). The flagellar root is visible in Fig. 2 b and Fig. 2e. The microvilli of the brush border are no longer close-packed but sparse and smaller. Cells with very short microvilli (Fig. 2c) and cells with longer micro~ villi (Fig. 2 d) occur side b y side. Cells rich in microvilli or poor (Fig. 2 e) or even without microvilli (Fig. 2 f) are found intermingled. Intercellular spaces where interdigitations are known to occur are seen in Fig. 2f (double arrow). The prominent spheres visible in Fig. 2a, f m i g h t be lipid globules
Cultures of Kidney Proximal Tubules: Electron Microscopy
21
(Fig. 2c). Likewise, it is possible to see proximal cells rich in microvilli adjacent to others that are poor in microvilli (Fig. 2e) or even almost devoid of them (Fig. 2f). But the flagellum is always present (Fig. 2c-f). We have kept intact, fragments of proximal tubules and studied the cultures starting from these fragments at the first or second day in vitro. I t then became evident that, as soon as the cells spill out of the proximal tubule, spread, increase their surface area and divide, the microvilli lose their close-packing and decrease in height. But, the progressive impoverishment in microvilli of some cells suggests that in addition to the spreading effect a true phenotypic change is occurring. 6. The other Characteristic Features o/ the Proximal Cell Studied by T E M . Sections cut perpendicular to the surface of the culture (Fig. 4a, b), confirmed the SEM observation that the orientation of the proximal cell on the glass surface was the same as in vivo. In Fig. 4a and 4b it is also evident that the cells do not grow as a true monolayer: two or three cells are seen to overlap, at least partially. The interdigitating processes typical of the proximal cell and shown by Bulger (1965) to extend not only at the bottom, but along the full extent of the lateral cell membranes, were also observed in vitro, on the lateral walls and the basal surface of neighbouring ceils (Fig. 4a, b). In sections parallel to the plane of the monolayer the same interdigitations are visible (Fig. 5a). With phase microscopy a clear space is seen between adjacent cells and corresponds to these interdigitations (see Fig. 1 b). The nucleoli of the cultured cells display a highly active appearance (Fig. 5a). This correlates with the active ribosomal RNA synthesis shown by Lee et al. (1970) in kidney cortical cells in vitro. The typical cytoplasmic bodies described by Maunsbach (1966) in the proximal cell, were also observed in vitro. Dense bodies (Figs. 4b, 5a), limited by a unit membrane (Fig. 6a) with layered material (Fig. 6a, b) or multivesicular bodies (Fig. 6c) were frequently found, some adherent to lipid globules (Fig. 6d), and some containing mitochondria or ribosomes. As acid phosphatase activity was not studied, a correlation with lysosomes cannot be established. 7. Micro/ilaments and Microtubules. A general feature in almost every cell of the confluent population was the presence of microfilaments and microtubules. These were present even in early cultures, at the second day in vitro. An ultrastructural cell by cell study, of fields of 100 to 200 or more confluent cells, often revealed a core of 10 to 30 cells with numerous, crowded mitochondria, comparable to the polygonal, mitochondria-rich cells seen with phase microscopy (Fig. I d). Even in such cells, which have retained the typical proximal cell ultrastructure, fibrils (Fig. 5b) or bundles of filaments can be seen between the packed mitochondria (Fig. 7a). Adjacent to them, or intermingled with them, are spindlelike proximal cells crowded with bundles of filaments, microtubules, and irregularly distributed fibrils (Fig. 7b). Transitional stages of this fibrillar transformation have been observed in different parts of the same proximal cell and in different parts of the same colony. Discussion Our method of isolating pure tubules from the outer cortex of the kidney (Cade-Treyer, 1972b), yielded a connective tissue cell-free suspension, constituted almost exclusively of pure proximal tubular fragments. These, produced, after
22
D. Cade-Treyer ~nd S. Tsuji
Cultures of Kidney Proximal Tubules: Electron Microscopy
23
Fig. 4a and b. TEM's of proximal cells at the 5th day in vitro, cut perpendicular to the substrate (a, X 14000, b, x 22000). Two cells are seen to overlap in (a) and (b). Interdigitations (i) are seen on both cell sides and even at the bottom. A section through the kinetosome (k) and through the root of the flagellum (/) is visible in Fig. 4a. The microvilli (m) are short and sparse in vitro (Fig. 4b). Cytoplasmic dense bodies (b) are seen in Fig. 4b. n, nucleus; mi, mitochondria
confluence, a n h o m o g e n o u s p o p u l a t i o n of p r o x i m a l cells, which r e t a i n e d t h e s a m e o r i e n t a t i o n o n t h e glass surface i n v i t r o as t h e y h a d i n v i v o o n t h e t u b u l a r basem e n t m e m b r a n e , w i t h t h e i r i n t e r d i g i t a t i o n s e x t e n d i n g a l o n g t h e cell sides a n d a t t h e b o t t o m . I n t e r d i g i t a t i o n s were also s h o w n to be m a i n t a i n e d i n v i t r o i n t h e r a b b i t k i d n e y e p i t h e l i o i d cell line s t u d i e d b y D i m m o c k (1970). A n o t h e r different i a t e d s t r u c t u r e of t h e p r o x i m a l cell, m a i n t a i n e d i n vitro, is t h e flagellum, w h i c h
Fig. 3a--c. TEM's (a, x 26000, b, • 35000, c, x 33000). (a) A proximal cell of foetal kidney near term. ~Tell-differentiated, closely packed microvilli (m) with apical canaliculi (ac) at their base. In between the microvilli is a flagellum (/) (or cilium), with an associated centriole (c). (b) A portion of a proximal cell from the suspension of isolated tubules. A flagellum (/) and its root is visible. The brush border microvilli (m) are cut transversely. Numerous apical vacuoles (v) are present. (c) An atypical proximal cell from a foetal calf kidney near term, with 8 flagella (/) (or cilia) each with 2 central fibrils and 9 peripheral doublets, and with corresponding centriolar structures (c) in the cytoplasm
24
D. Cade-Treyer and S. Tsuji
Fig. 5a and b. TEM's of proximal cells at the 7th day of culture, cut parallel to the substrate (a, x 5800, b, x 16000). Interdigitations (i) are visible in the space between the cells (Fig. 5a). Active nucleoli (n). Dense bodies (b). The brush border microvilli (m) are directed upwards on the undulating free cell surface and become visible in horizontal sections,when hollows are cut as in Fig. 5 b. Numerous microfilaments (m/) are seen, running through the cytoplasm (Fig. 5 b)
Cultures of Kidney Proximal Tubules: Electron Microscopy
25
Fig. 6a--d. Different types of dense bodies are found in the cytoplasm of the cultured proximal cells: with a unit membrane visible (a, • 33000), with layered material (a, b, • 20000); multivesicular bodies (c, • 11200); dense bodies associated with a lipid globule (d, • 11200) was found in primary cultures and also in 2 months old subcultures; Dimmock (1970) also, mentioned the existence of flagellar structures in her rabbit kidney cell line. The presence of the flagellum in every proximal cell in vitro suggests the permanent existence of flagella in proximal cells in vivo. Only occasional evidence for this has been found by TEM (Latta et al., 1961 ; Trump and Ericcson, 1965). A recent SEM study of the nephron in vivo revealed a flagellum in the proximal cells (Andrews and Porter, 1974). We could confirm the existence of a flagellum in proximal cells in vivo with our bovine tubular suspension where it could easily be observed with TEM. The typical proximal cells, with ciliary structures in great profusion which we sometimes found in the prenatal bovine nephron resemble those observed by Mannweiler and Bernard (1957) in a kidney tumour arising from proximal cells and by Rossmann and Galle (1968) in proximal tubules of humans with nephropathies. SEM was particulary useful in studying the development of the microvilli of the brush border in vitro. The microvilli on cells spreading out from proximal tubules and maintained on these cells when fully-spread are characteristic features of this differentiated cell type and cannot be mistaken for the transient microvilli of rounded and dividing cells of certain cell lines in vitro, which are linked to certain stages of the cell cycle (Follet and O'Neill, 1969; Follet and Goldman, 1970).
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D. Cade-Trcyer and S. Tsuji
Fig. 7 a and b. (a, • 21000, b, • 36000). The cultured proximal ceils show cytoplasmic microfilaments (m/) and microtubules (mr). (a) In a cell of typical proximal tubule appearance (5th day in vitro), a bundle of filaments is visible between numerous mitochondria; other randomly disposed microfilaments and microtubules are also visible. (b) A proximal cell (5th day in vitro) containing many interlacing microfilaments
The progressive change in vitro, of t h e microvilli of t h e p r o x i m a l cells, from a close-packed to a sparse d i s t r i b u t i o n a n d t o w a r d s a decrease in h e i g h t a n d even t o w a r d s a r e d u c t i o n in n u m b e r , seems to be due o n l y in p a r t to the cell s p r e a d i n g a n d to t h e increase in t h e free cell surface. These changes do in fact show a phenot y p i c t r a n s f o r m a t i o n which a p p e a r s to be t h e reverse of w h a t h a p p e n s when the p r o x i m a l cell differentiates d u r i n g n o r m a l histogenesis. I n t h e mouse foetus n e a r t e r m for instance, t h e p r o x i m a l cells b e a r few small microvilli which increase in n u m b e r a n d become close-packed after b i r t h (Clark, 1957). I n t h e bovine foetus,
Cultures of Kidney Proximal Tubules: Electron Microscopy
27
the brush border is well-differentiated near birth, but the proximal cells, after several days in vitro revert to an earlier embryonic state, at least so far as the microvilli are concerned. In connection with this reversion the property of the proximal cells to synthesize cr in vitro (Cade-Treyer, 1973, 1975) should be remembered. Furthermore, the morphological change observed on the microvi]li of the proximal cells in vitro, might be related to a phenotypic change in the histiospecific antigens of the proximal cells. These were shown to decrease in vitro and their synthesis to be suppressed at confluence (Cade-Treyer, 1975). An immunofluorescent study has localized the antigens to the brush border area and to the apical part of the proximal cell in vivo (Cadc-Treyer, 1972a). Therefore it will be useful to refine their topography with immunocytochemical methods at the ultrastructura] level, to investigate the relationship between these phenomena. Another feature of the proximal cells in vitro, was the proliferation of microtubules and microfilaments often packed in bundles, or randomly distributed. We could not detect any filaments in the proximal cell cytoplasm in vivo. But, the ciliary neck of the proximal tubule of the amphibian was shown to contain cytoplasmic bundles of filaments (Bargmann, 1955), perhaps related to the profusion of cilia borne by these cells. The question therefore arises: is the fibrillar activity in our cultured ceils related to the flagellar and brush border structures and to their development in vitro ? Microfilaments have already been shown in primary cultures of rhesus kidney cells (Malinin, 1973) and also in kidney cell lines (Goldman and Follet, 1969; Kisch et al., 1973); and a link with contact i1~hibition (Heaysman and Pegrum, 1973) and with cell movement has been suggested (Goldman and Follet, 1969). A ruffling activity of kidney ceils in vitro has, in fact, been demonstrated (Price, 1972). Further experimental work is needed to understand the fibrillar activity in our proximal cell cultures. References Andrews, P., Porter, K. : A scanning electron microscopic study of the nephron. Am. J. Anat. 14O, 81-116 (1974) Bargmann, W. : Histologische, cytochemische und elektronenmikroskopische Untersuchungen am Nephron. Z. Zellforsch. 42, 386-422 (1955) Boyde, A., Weiss, R., Vesely, P. : Scanning electron microscopy of cells in culture. Exp. Cell Res. 71, 313-324 (1972) Bulger, R. : Shape of rat kidney tubular cells. Amer. J. Anat. 116, 237-247 (1965) Cade-Treycr, D. : Immunofluorescent localization of calf kidney-specific antigens with monospecific antisera and with specific antigen-antibody immunoprecipitation lines. Ann. Inst. Pasteur 122, 251-262 (1972a) Cade-Treyer, D. : Isolation of pure fractions of viable calf kidney tubules and glomeruli. In vitro culture, immunochemical and esterase zymogram analysis. Ann. Inst. Pasteur 122, 263-271 (1972b) Cade-Treyer, D. : Neosynthesis of an a-foetoprotein in calf kidney cells cultured in vitro. Immunochemical and autoradiographic evidence. Ann. Immunol. 124 C, 27-43 (1973) Cade-Treyer, D. : Deux foetoproteines bovines distinctes de la f6tuine. In "~-foetoprotein" Proc. Int. Confer. Saint Paul de Venee France (Inserm colloquium) 37-45, (1974) Cade-Treyer, D. : In vitro culture of the proximal tubule of the bovine nephron: The fate of the histiospecific antigens, neosynthesis of an a-foetoprotein. Ann. Immunol. 126 C, 201-218, (1975)
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D. Cade-Treyer and S. Tsuji
Clark, S. : Cellular differentiation in the kidneys of new-born mice studied with the electron microscope. J. biophys, biochem. Cytol. 8, 349-362 (1957) Dimmock, E. : The surface structure of cultured rabbit kidney cells as revealed by electron microscopy. J. Cell Sci. 7, 719-737 (1970) Follet, E., Goldman, t~. : The occurrence of microvilli during spreading and growth of B H K 21/C 13 fibroblasts. Exp. Cell l~es. 159, 124-136 (1970) Follett, E., O'Neill, C. : The distribution of microvilli on B H K 21/C fibroblasts. Exp. Cell Res. 55, 136-138 (1969) Goldman, R., Follet, E. : The structure of the major cell processes of isolated B H K 21 fibroblasts. Exp. Cell Res. 57, 263-276 (1969) Heasman, J., Pegrum, S. : Early contacts between fibroblasts. Exp. Cell Res. 78, 71-78 (1973) Kisch, A., Kelley, R., Crissman, It. L. : DMSO-induced reversion of several features of polyoma transformed baby hamster kidney cells (BHK 21). J. Cell Biol. 57, 38-53 (1973) Latta, H., Maunsbach, A., Madden, S.: Cilia in different segments of the rat nephron. J. biophys, biochem. Cytol. 11, 248-251 (1961) Lee, M., Vaugham, M., Abrams, R. : Nature of the I~NA formed prior to DNA synthesis in kidney cortex cells cultured directly from the rabbit. J. biol. Chem. 245, 45254533 (1970) Lieberman, I., Ove, P. : DNA synthesis and its inhibition in mammalian cells cultured from the animal. J. biol. Chem. 237, 1634-1642 (1962) Malinin, G. : Cytotoxic effect of DMSO on the ultrastructure of cultured rhesus kidney cells. Cryobiology 10, 22-32 (1973) Mannweiller, K., Bernard, W.: Recherches ultrastructurales sur une tumeur r@nale exp~rimentale du hamster. J. Ultrastruct. Res. 1, 158-169 (1957) Maunsbach, A. : Observations on the ultrastrncture and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubule cells. J. Ultrastruct. Res. 16, 197-238 (1966) Price, Z. : A three-dimensional model of membrane ruffling from transmission and scanning electron microscopy of cultured monkey kidney cells (LLCMK2). J. Microscopy 95,493-505 (1972) Rossmann, P., Galle, P. : Mise en 6vidence de cellules r~nales cili@es chez l'homme. Nephron 5, 426436 (1968) Trump, B., Ericcson, J. :Effect of fixative solution on ultrastrueture of cells and tissues. The proximal convoluted tubule of the rat kidney. Lab. Invest. 14, 1245-1323 (1965)
In vitro Culture of the Proximal Tubule
of the Bovine Nephron Transmission
and Scanning Electron
Microscopy*
Denise Cade-Treyer and Shigeru Tsuji Laboratoires de Biologic Animale V1 et IV. Universit6 Pierre et Marie Curie, Paris, France Received March 21, 1975
Summary. By means of a previously described method, viable pure tubules of the nephron were isolated in high yield from the outer cortex of the near-term foetal bovine kidney. The tubular suspension obtained was constituted almost exclusively of proximal segments (about 95%), whose cells were dispersed and grown as confluent primary cultures. The cultured proximal cells were shown to m a i n t a i n in vitro, on glass or plastic surfaces, the same orientation as on the tubular basement membrane in vivo, with interdigitations extending from the base of the cells and along their full height. Numerous mitochondria and the typical cytoplasmic bodies of the proximal cell were retained in cells grown in vitro. A flagellum was seen in every cultured cell and was shown to be present in the proximal cell in vivo. There is a progressive change, in vitro, of the microvilli of the brush border, from a close-packed to a sparse distribution and to a decrease in height a n d a reduction in number. This in vitro regression to an earlier embryonic state was correlated with the ability of the proximal cells to synthesize in vitro a n ~-foetoprotein and with the loss in vitro of histiospecific antigen synthesis, confined in vivo to the brush border area. The confluent proximal cells became filled with microfilaments a n d microtubules, the significance of which is discussed. Key words: Kidney - - Proximal tubule - - Cell culture - - Transmission and scanning electron microscopy. Rdsumd. Une m6thode, d6crite pr6c6demment, permet d'isoler, en grande quantit6 des tubules purs du nephron bovin, ~ partir du cortex externe du rein foetal s terme. La suspension tubulaire obtenue est constitu6e presqu'exclusivement de segments proximaux (de Fordre de 95 % ), dont on disperse les cellules pour les cultiver in vitro. Les cellules proximales, en cultures primaires confluentes, gardent la m~me orientation sur le verre ou le plastique, que sur la lame basale tubulaire in vivo. Elles s'engrbnent sur leurs faces lat6rales et s leur base, p a r des interdigitations. De nombreuses mitochondries et les inclusions cytoplasmiques de la cellule proximale se r e t r o u v e n t in vitro. Chaque cellule proximale in vitro porte un flagelle, qui existe d6js darts les cellules proximales du veau in vivo. Les microvillosit6s de la bordure en brosse se desserrent et s'6parpillent sur les cellules 6tal~es in vitro. Leur taille diminue et elles disparaissent m~me de certaines cellules. Ce retour in vitro, vers un 6tat embryonnaire plus pr6coce, est s mettre en rapport avec la propri6t6 des cellules proximales de synth6tiser in vitro unc a-foetoproteine; de m~me il dolt exister une corr61ation avec la perte de synthbse, dans ces cellules in vitro, des antig~nes histiosp6cifiques, localis6s in vivo, dans la zSne de la bordure en brosse. Des microtubules et des microfilaments envahissent les cellules proximales in vitro; leur signification est discut~e. Send o//print requests to: Dr. D. Cade-Treyer, Laboratoire de Biologic Animale VI, Universit6 Pierre et Marie Curie, 12 rue Cuvier, 75005 Paris, France. * We are indebted to Mrs. M. Brisorgueil and to Mrs. P. Cloup for skilful technical assistance.
16
D. Cade-Treyer and S. Tsuji Introduction
N e p h r o n s can be easily isolated from t h e k i d n e y with t h e help of e n z y m a t i c digestion. R e c e n t l y , one of us (Cade-Treyer, 1972b) devised a m e t h o d to s e p a r a t e t u b u l e s from glomeruli in high y i e l d a n d in a h i g h l y purified form. These t u b u l e s were isolated as v i a b l e f r a g m e n t s a n d were t h u s r e a d i l y c u l t u r e d in vitro (CadeTreyer, 1972 b). A n analysis of t h e t u b u l a r suspension, p r o v i d e d evidence t h a t t h e t u b u l e f r a g m e n t s o b t a i n e d with our m e t h o d from t h e o u t e r cortex of t h e calf k i d n e y , were a l m o s t e x c l u s i v e l y d e r i v e d from p r o x i m a l t u b u l e s ; we h a d t h u s a t hand, a m e t h o d to i n i t i a t e p r i m a r y cultures with a f a i r l y homogenous p o p u l a t i o n of h i s t i o t y p i c cells from t h e k i d n e y , which we h a v e called an " h i s t i o n " (CadeTreyer, 1972b). This " h i s t i o n " t h e p r o x i m a l t u b u l e of t h e nephron, has been characterized b y histiospecific antigens, which were specifically localized to its cells in vivo with i m m u n o f l u o r e s c e n t techniques. (Cade-Treyer, 1972 a.) The phenot y p i c shift of these antigens in vitro, was followed in p r i m a r y cultures of t h e p r o x i m a l cells, with i m m u n o c h e m i c a l a n d i m m u n o r a d i o g r a p h i c m e t h o d s (CadeTreyer, 1975). Moreover, t h e neosynthesis of a bovine ~-foetoprotein (Cade-Treyer, 1974) was shown to occur in cultures of the crude o u t e r cortex (Cade-Treyer, 1973) a n d to be a p r o p e r t y of p r o x i m a l t u b u l e cells alone (Cade-Treyer, 1975). These i m m u n o c h e m i c a l studies, r e v e a l i n g a double p h e n o t y p i c shift in t h e p r o x i m a l cells in vitro: t h e switching off of t h e histiospecific antigens a n d the switching on of ~-foetoprotein synthesis p r o m p t e d us to s t u d y t h e fate of t h e well-defined morphological m a r k e r s of t h e p r o x i m a l cells in p r i m a r y cultures in vitro, a t t h e level of t h e t r a n s m i s s i o n a n d t h e scanning electron microscope.
Material and Methods
Primary Cultures o/Proximal Tubular Cells The method for isolating a pure tubule fraction from the outer renal cortex from near-term calf foetuses has been described (Cade-Treyer, 1972b). After gentle trypsinization of the outer cortex, the isolated nephrons are freed of interstitial cells by repeated settling, and the supernatant, with isolated cells, discarded. The nephrons, after further disruption by pipetting, are then submitted to sieving and successive settling to separate pure tubules in the supernatant from glomeruli. Confirmation of the proximal nature of the pure tubule fragments obtained will be described in the Results. The tubules were further disrupted by pipetting to obtain tiny tubular fragments, or clusters of cells, or isolated cells, which were seeded in plastic "Falcon" flasks or on cover-slips of Leighton tubes. One ml of packed cells was diluted in 200 ml of culture medium, which consisted of casein hydrolysate in Earle's buffered salt solution with penicillin 200 iu/ml, streptomycin 50 ~zg/ml, and 3 % calf serum. After 24 to 48 hours the nonadherent cells were discarded with the medium change. A confluent sheet of cells was obtained within 5 to 7 days.
Light Microscopy The cells grown on cover-slips were observed with a Leitz ortholux microscope fitted with Zeruicke phase contrast equipment.
Transmission Electron Microscopy ( T E M ) The confluent cells, on glass cover-slips, were fixed for 2 hour at room temperature in 1% glutaraldehyde and 1% paraformaldehyde in 0.12 M phosphate buffer (pH 7.4). They were then rinsed three times with the same buffer and postfixed for 1 hour in phosphate buffered 2% osmium textroxide. The postfixed cells were then processed conventionally and embedded in Araldite, directly on the cover-slips. These were split from the embedding capsule,
Cultures of Kidney Proximal Tubules: Electron Microscopy
17
by dipping into liquid nitrogen, thus exposing at the surface of the capsule, the basal part of the cell monolayer originally in contact with the cover-slip. a) Ultrathin sections were cut parallel to the plane of the monolayer. They were stained with uranyl acetate and lead citrate for subsequent study with a Philips 300 electron microscope. b) Ultrathin sections were aslo cut perpendicular to the plane of the monolayer. In this case, cells were grown in Falcon flasks and small fragments of the flasks covered with cells were directly embedded in Araldite. A diamond knife easily cut the embedded plastic of the flask along with the sheet of ceils grown on it. c) Ultrathin sections were also cut from isolated proximal tubules in suspension, embedded as a pellet.
Scanning Electron Microscopy ( S E M ) Cells grown on cover-slips were fixed as for TEM. Air-drying was performed from absolute ethanol, as described by Boyde et al. (1972) or from propylene oxide; the latter method gave the better results. The dried cells on their cover-slip, were fixed to aluminium rivets and given a conducting coating of 150 /~ gold and palladium. They were examined at a tilt angle of 38 ~ at 20 KV with a CAMECA 07 SEM.
Results 1. Our m e t h o d of isolation of p u r e t u b u l e s requires k i d n e y cortex from neart e r m foetuses (see Cade-Treyer, 1972b). Therefore, we first verified t h a t the histogenesis o/ the proximal tubules was complete at this stage, this is n o t t h e case in o t h e r species such as t h e mouse, where t h e p r o x i m a l cell continues d i f f e r e n t i a t i n g a f t e r birth, b y progressive a c c u m u l a t i o n of apical microvilli (Clark, 1957). I n t h e calf foetal p r o x i m a l cell n e a r birth, we o b s e r v e d a good apical a l k a l i n e phosp h a t a s e a c t i v i t y and, a t t h e u l t r a s t r u c t u r a l level, a well-developed brush border. Likewise, t h e apical histiospecific a n t i g e n s of t h e p r o x i m a l cell, were r e v e a l e d with t h e same i n t e n s i t y in the foetal p r o x i m a l cell n e a r birth, as in the calf. (Cade-Treyer, 1972b.), 2. The Constitution o/ the Suspension o] the Pure Tubules i s o l a t e d from t h e bovine o u t e r cortex was s t u d i e d a t t h e u l t r a s t r u c t u r a l level on two series of suspensions, purified s e p a r a t e l y , each from 4 foetal k i d n e y s n e a r t e r m ; 150 t u b u l a r f r a g m e n t s were o b s e r v e d in each case. The results showed t h a t 93% of the fragm e n t s were of t h e p r o x i m a l t y p e , with t y p i c a l brush borders; t h e r e m a i n d e r were c o m p o s e d of collecting ducts a n d d i s t a l t u b u l e fragments. 3. The Growing Cell Population. The v e r y high p e r c e n t a g e of p r o x i m a l t u b u l a r f r a g m e n t s o b t a i n e d was n o t surprising, since we s t a r t e d with o u t e r cortex, which is k n o w n to be p a r t i c u l a r l y rich in p r o x i m a l t u b u l e s ( L i e b e r m a n a n d Ore, 1962). W e o b s e r v e d t h a t the primary cultures arising from such a t u b u l a r suspension y i e l d e d an homogenous population o/proximal cells. The confluent cultures arose from t h e fusion of islands, each s t a r t i n g from a cluster of cells, a f t e r t h e breakdown of t h e t u b u l e s (Fig. 1 a). A s t u d y of these islands a f t e r t h e first two d a y s in v i t r o (Fig. 1 a, b) d i d n o t r e v e a l a n y b u t p r o x i m a l t u b u l e islands. A t confluence, t h e initial clusters of cells are still visible as swirling groups of closely p a c k e d cells (Fig. l c ) . U n d e r t h e phase c o n t r a s t microscope, e i t h e r p o l y g o n a l , m i t o c h o n d r i a rich cells (Fig. I c, d), or e l o n g a t e d spindle-like cells (Fig. I c) are seen. The l a t t e r were easily i d e n t i f i e d as p r o x i m a l cells u n d e r t h e electron microscope, t h a n k s to t h e specific f e a t u r e of these cells (see below). 2 Cell Tiss.Res.
18
D. Cade-Treyer and S. Tsuji
Cultures of Kidney Proximal Tubules: Electron Microscopy
19
4. The Flagellum o] the Proximal Cell in vitro. SEM seemed especially appropriate to study the fate of the microvilli of the brush border of the proximal cells in vitro. But, the first surprising result was that at the time of confluence each cell was endowed with a long flagellum (Fig. 2a-f), which was present on the first day in vitro. This flagellum was still present after 2 months' subculture in vitro. In cultured cells, transverse sections of the flagellum, observed by TEM, displayed a typical structure of 2 central fibrils and 9 peripheral doublets (Fig. 3a-c). A transverse section through a cultured proximal cell in Fig. 4a shows the fl~gellar rootlet and the kinetosome. Phase contrast study of live cultured proximal cells, revealed spasmodic fl~gellar beating. The few indications in the literature of the existence of a flagellum in the proximal tubular cell, prompted us to reinvestigate the question in kidney sections and in isolated tubular suspensions. We found a single cilium (or flagellum; its length could not be determined in sections) in the proximal cells (Fig. 3 a) of the foetal calf nephron near term. In suspensions of isolated proximal tubules, we frequently observed a single flagellum, associated with a typical centriole, emerging from between the microvilli of the brush border (Fig. 3b). Moreover, a few atypical proximal cells were observed in sections of the cortex and in isolated proximal tubules. They displayed from 6 to 10 cilia (or flagella) among the microvilli. In the cytoplasm of such cells the associated centriolar structures of the kinetosomes could be identified (Fig. 3c). As adult kidney proximal cells were not investigated at the ultrastructural level, it is not possible to state whether such atypical cells are found only in foetal kidney. 5. The Brush Border. Scanning electron microscopy was especially suitable for studying the brush border cells in vitro (Fig. 2a-f). In the proximal cells at confluence, the microvilli are seen at the free cell surface, extending into the culture medium. From this it can be concluded that, in culture, the proximal cell is oriented on the glass surface as it is in vivo on the basement membrane of the tubule. In vitro, the microvilli are not as closely packed as in vivo, but they are sparsely distributed on almost every fully-spread confluent cell (Fig. 2a, b). Moreover a marked decrease in the length of the microvilli by comparison with those in vivo is evident (Figs. 2a-c, e, f, 4b, 5b), although it is possible to find adjacent cells with long microvilli (Fig. 2d) and with short microvilli
Fig. 1a--d. Phase contrast micrographs of the proximal tubular cell cultures. Fig. 1a and b. 2nd day in vitro (a, • 300, b, X 2100). Several islands of proximal cells are visible (Fig.1 a). At their centres the initial clusters of cells from which they arose are indicated by arrows. Numerous rod-like mitochondria are visible in the cytoplasm. The clear spaces between cells (Fig. lb) indicate the zone of interdigitations (zi), clearly visible in Fig. 5a. Active nuclcoli (n) are conspicuous. Fig. 1c--d. 7th day in vitro (c, • 300, d, x 3 000). Confluent cells (Fig. 1c) with the initial clusters visible as cores of crowded cells (arrows). Fully-spread cells, whether polygonal or spindle-shaped contain numerous rod-like mitoehondria. Lipid globules are also seen (arrows) (Fig. 1d)
20
D. Cade-Treyer and S. Tsuii
Fig. 2 a - - f . SEM's of the proximal cells at the 7th day in vitro (a, x 1000, b, e, x 5000, c, d, X 10000, f, x 2000). A flagellum is conspicuous on every cell (arrows). The flagellar root is visible in Fig. 2 b and Fig. 2e. The microvilli of the brush border are no longer close-packed but sparse and smaller. Cells with very short microvilli (Fig. 2c) and cells with longer micro~ villi (Fig. 2 d) occur side b y side. Cells rich in microvilli or poor (Fig. 2 e) or even without microvilli (Fig. 2 f) are found intermingled. Intercellular spaces where interdigitations are known to occur are seen in Fig. 2f (double arrow). The prominent spheres visible in Fig. 2a, f m i g h t be lipid globules
Cultures of Kidney Proximal Tubules: Electron Microscopy
21
(Fig. 2c). Likewise, it is possible to see proximal cells rich in microvilli adjacent to others that are poor in microvilli (Fig. 2e) or even almost devoid of them (Fig. 2f). But the flagellum is always present (Fig. 2c-f). We have kept intact, fragments of proximal tubules and studied the cultures starting from these fragments at the first or second day in vitro. I t then became evident that, as soon as the cells spill out of the proximal tubule, spread, increase their surface area and divide, the microvilli lose their close-packing and decrease in height. But, the progressive impoverishment in microvilli of some cells suggests that in addition to the spreading effect a true phenotypic change is occurring. 6. The other Characteristic Features o/ the Proximal Cell Studied by T E M . Sections cut perpendicular to the surface of the culture (Fig. 4a, b), confirmed the SEM observation that the orientation of the proximal cell on the glass surface was the same as in vivo. In Fig. 4a and 4b it is also evident that the cells do not grow as a true monolayer: two or three cells are seen to overlap, at least partially. The interdigitating processes typical of the proximal cell and shown by Bulger (1965) to extend not only at the bottom, but along the full extent of the lateral cell membranes, were also observed in vitro, on the lateral walls and the basal surface of neighbouring ceils (Fig. 4a, b). In sections parallel to the plane of the monolayer the same interdigitations are visible (Fig. 5a). With phase microscopy a clear space is seen between adjacent cells and corresponds to these interdigitations (see Fig. 1 b). The nucleoli of the cultured cells display a highly active appearance (Fig. 5a). This correlates with the active ribosomal RNA synthesis shown by Lee et al. (1970) in kidney cortical cells in vitro. The typical cytoplasmic bodies described by Maunsbach (1966) in the proximal cell, were also observed in vitro. Dense bodies (Figs. 4b, 5a), limited by a unit membrane (Fig. 6a) with layered material (Fig. 6a, b) or multivesicular bodies (Fig. 6c) were frequently found, some adherent to lipid globules (Fig. 6d), and some containing mitochondria or ribosomes. As acid phosphatase activity was not studied, a correlation with lysosomes cannot be established. 7. Micro/ilaments and Microtubules. A general feature in almost every cell of the confluent population was the presence of microfilaments and microtubules. These were present even in early cultures, at the second day in vitro. An ultrastructural cell by cell study, of fields of 100 to 200 or more confluent cells, often revealed a core of 10 to 30 cells with numerous, crowded mitochondria, comparable to the polygonal, mitochondria-rich cells seen with phase microscopy (Fig. I d). Even in such cells, which have retained the typical proximal cell ultrastructure, fibrils (Fig. 5b) or bundles of filaments can be seen between the packed mitochondria (Fig. 7a). Adjacent to them, or intermingled with them, are spindlelike proximal cells crowded with bundles of filaments, microtubules, and irregularly distributed fibrils (Fig. 7b). Transitional stages of this fibrillar transformation have been observed in different parts of the same proximal cell and in different parts of the same colony. Discussion Our method of isolating pure tubules from the outer cortex of the kidney (Cade-Treyer, 1972b), yielded a connective tissue cell-free suspension, constituted almost exclusively of pure proximal tubular fragments. These, produced, after
22
D. Cade-Treyer ~nd S. Tsuji
Cultures of Kidney Proximal Tubules: Electron Microscopy
23
Fig. 4a and b. TEM's of proximal cells at the 5th day in vitro, cut perpendicular to the substrate (a, X 14000, b, x 22000). Two cells are seen to overlap in (a) and (b). Interdigitations (i) are seen on both cell sides and even at the bottom. A section through the kinetosome (k) and through the root of the flagellum (/) is visible in Fig. 4a. The microvilli (m) are short and sparse in vitro (Fig. 4b). Cytoplasmic dense bodies (b) are seen in Fig. 4b. n, nucleus; mi, mitochondria
confluence, a n h o m o g e n o u s p o p u l a t i o n of p r o x i m a l cells, which r e t a i n e d t h e s a m e o r i e n t a t i o n o n t h e glass surface i n v i t r o as t h e y h a d i n v i v o o n t h e t u b u l a r basem e n t m e m b r a n e , w i t h t h e i r i n t e r d i g i t a t i o n s e x t e n d i n g a l o n g t h e cell sides a n d a t t h e b o t t o m . I n t e r d i g i t a t i o n s were also s h o w n to be m a i n t a i n e d i n v i t r o i n t h e r a b b i t k i d n e y e p i t h e l i o i d cell line s t u d i e d b y D i m m o c k (1970). A n o t h e r different i a t e d s t r u c t u r e of t h e p r o x i m a l cell, m a i n t a i n e d i n vitro, is t h e flagellum, w h i c h
Fig. 3a--c. TEM's (a, x 26000, b, • 35000, c, x 33000). (a) A proximal cell of foetal kidney near term. ~Tell-differentiated, closely packed microvilli (m) with apical canaliculi (ac) at their base. In between the microvilli is a flagellum (/) (or cilium), with an associated centriole (c). (b) A portion of a proximal cell from the suspension of isolated tubules. A flagellum (/) and its root is visible. The brush border microvilli (m) are cut transversely. Numerous apical vacuoles (v) are present. (c) An atypical proximal cell from a foetal calf kidney near term, with 8 flagella (/) (or cilia) each with 2 central fibrils and 9 peripheral doublets, and with corresponding centriolar structures (c) in the cytoplasm
24
D. Cade-Treyer and S. Tsuji
Fig. 5a and b. TEM's of proximal cells at the 7th day of culture, cut parallel to the substrate (a, x 5800, b, x 16000). Interdigitations (i) are visible in the space between the cells (Fig. 5a). Active nucleoli (n). Dense bodies (b). The brush border microvilli (m) are directed upwards on the undulating free cell surface and become visible in horizontal sections,when hollows are cut as in Fig. 5 b. Numerous microfilaments (m/) are seen, running through the cytoplasm (Fig. 5 b)
Cultures of Kidney Proximal Tubules: Electron Microscopy
25
Fig. 6a--d. Different types of dense bodies are found in the cytoplasm of the cultured proximal cells: with a unit membrane visible (a, • 33000), with layered material (a, b, • 20000); multivesicular bodies (c, • 11200); dense bodies associated with a lipid globule (d, • 11200) was found in primary cultures and also in 2 months old subcultures; Dimmock (1970) also, mentioned the existence of flagellar structures in her rabbit kidney cell line. The presence of the flagellum in every proximal cell in vitro suggests the permanent existence of flagella in proximal cells in vivo. Only occasional evidence for this has been found by TEM (Latta et al., 1961 ; Trump and Ericcson, 1965). A recent SEM study of the nephron in vivo revealed a flagellum in the proximal cells (Andrews and Porter, 1974). We could confirm the existence of a flagellum in proximal cells in vivo with our bovine tubular suspension where it could easily be observed with TEM. The typical proximal cells, with ciliary structures in great profusion which we sometimes found in the prenatal bovine nephron resemble those observed by Mannweiler and Bernard (1957) in a kidney tumour arising from proximal cells and by Rossmann and Galle (1968) in proximal tubules of humans with nephropathies. SEM was particulary useful in studying the development of the microvilli of the brush border in vitro. The microvilli on cells spreading out from proximal tubules and maintained on these cells when fully-spread are characteristic features of this differentiated cell type and cannot be mistaken for the transient microvilli of rounded and dividing cells of certain cell lines in vitro, which are linked to certain stages of the cell cycle (Follet and O'Neill, 1969; Follet and Goldman, 1970).
26
D. Cade-Trcyer and S. Tsuji
Fig. 7 a and b. (a, • 21000, b, • 36000). The cultured proximal ceils show cytoplasmic microfilaments (m/) and microtubules (mr). (a) In a cell of typical proximal tubule appearance (5th day in vitro), a bundle of filaments is visible between numerous mitochondria; other randomly disposed microfilaments and microtubules are also visible. (b) A proximal cell (5th day in vitro) containing many interlacing microfilaments
The progressive change in vitro, of t h e microvilli of t h e p r o x i m a l cells, from a close-packed to a sparse d i s t r i b u t i o n a n d t o w a r d s a decrease in h e i g h t a n d even t o w a r d s a r e d u c t i o n in n u m b e r , seems to be due o n l y in p a r t to the cell s p r e a d i n g a n d to t h e increase in t h e free cell surface. These changes do in fact show a phenot y p i c t r a n s f o r m a t i o n which a p p e a r s to be t h e reverse of w h a t h a p p e n s when the p r o x i m a l cell differentiates d u r i n g n o r m a l histogenesis. I n t h e mouse foetus n e a r t e r m for instance, t h e p r o x i m a l cells b e a r few small microvilli which increase in n u m b e r a n d become close-packed after b i r t h (Clark, 1957). I n t h e bovine foetus,
Cultures of Kidney Proximal Tubules: Electron Microscopy
27
the brush border is well-differentiated near birth, but the proximal cells, after several days in vitro revert to an earlier embryonic state, at least so far as the microvilli are concerned. In connection with this reversion the property of the proximal cells to synthesize cr in vitro (Cade-Treyer, 1973, 1975) should be remembered. Furthermore, the morphological change observed on the microvi]li of the proximal cells in vitro, might be related to a phenotypic change in the histiospecific antigens of the proximal cells. These were shown to decrease in vitro and their synthesis to be suppressed at confluence (Cade-Treyer, 1975). An immunofluorescent study has localized the antigens to the brush border area and to the apical part of the proximal cell in vivo (Cadc-Treyer, 1972a). Therefore it will be useful to refine their topography with immunocytochemical methods at the ultrastructura] level, to investigate the relationship between these phenomena. Another feature of the proximal cells in vitro, was the proliferation of microtubules and microfilaments often packed in bundles, or randomly distributed. We could not detect any filaments in the proximal cell cytoplasm in vivo. But, the ciliary neck of the proximal tubule of the amphibian was shown to contain cytoplasmic bundles of filaments (Bargmann, 1955), perhaps related to the profusion of cilia borne by these cells. The question therefore arises: is the fibrillar activity in our cultured ceils related to the flagellar and brush border structures and to their development in vitro ? Microfilaments have already been shown in primary cultures of rhesus kidney cells (Malinin, 1973) and also in kidney cell lines (Goldman and Follet, 1969; Kisch et al., 1973); and a link with contact i1~hibition (Heaysman and Pegrum, 1973) and with cell movement has been suggested (Goldman and Follet, 1969). A ruffling activity of kidney ceils in vitro has, in fact, been demonstrated (Price, 1972). Further experimental work is needed to understand the fibrillar activity in our proximal cell cultures. References Andrews, P., Porter, K. : A scanning electron microscopic study of the nephron. Am. J. Anat. 14O, 81-116 (1974) Bargmann, W. : Histologische, cytochemische und elektronenmikroskopische Untersuchungen am Nephron. Z. Zellforsch. 42, 386-422 (1955) Boyde, A., Weiss, R., Vesely, P. : Scanning electron microscopy of cells in culture. Exp. Cell Res. 71, 313-324 (1972) Bulger, R. : Shape of rat kidney tubular cells. Amer. J. Anat. 116, 237-247 (1965) Cade-Treycr, D. : Immunofluorescent localization of calf kidney-specific antigens with monospecific antisera and with specific antigen-antibody immunoprecipitation lines. Ann. Inst. Pasteur 122, 251-262 (1972a) Cade-Treyer, D. : Isolation of pure fractions of viable calf kidney tubules and glomeruli. In vitro culture, immunochemical and esterase zymogram analysis. Ann. Inst. Pasteur 122, 263-271 (1972b) Cade-Treyer, D. : Neosynthesis of an a-foetoprotein in calf kidney cells cultured in vitro. Immunochemical and autoradiographic evidence. Ann. Immunol. 124 C, 27-43 (1973) Cade-Treyer, D. : Deux foetoproteines bovines distinctes de la f6tuine. In "~-foetoprotein" Proc. Int. Confer. Saint Paul de Venee France (Inserm colloquium) 37-45, (1974) Cade-Treyer, D. : In vitro culture of the proximal tubule of the bovine nephron: The fate of the histiospecific antigens, neosynthesis of an a-foetoprotein. Ann. Immunol. 126 C, 201-218, (1975)
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D. Cade-Treyer and S. Tsuji
Clark, S. : Cellular differentiation in the kidneys of new-born mice studied with the electron microscope. J. biophys, biochem. Cytol. 8, 349-362 (1957) Dimmock, E. : The surface structure of cultured rabbit kidney cells as revealed by electron microscopy. J. Cell Sci. 7, 719-737 (1970) Follet, E., Goldman, t~. : The occurrence of microvilli during spreading and growth of B H K 21/C 13 fibroblasts. Exp. Cell l~es. 159, 124-136 (1970) Follett, E., O'Neill, C. : The distribution of microvilli on B H K 21/C fibroblasts. Exp. Cell Res. 55, 136-138 (1969) Goldman, R., Follet, E. : The structure of the major cell processes of isolated B H K 21 fibroblasts. Exp. Cell Res. 57, 263-276 (1969) Heasman, J., Pegrum, S. : Early contacts between fibroblasts. Exp. Cell Res. 78, 71-78 (1973) Kisch, A., Kelley, R., Crissman, It. L. : DMSO-induced reversion of several features of polyoma transformed baby hamster kidney cells (BHK 21). J. Cell Biol. 57, 38-53 (1973) Latta, H., Maunsbach, A., Madden, S.: Cilia in different segments of the rat nephron. J. biophys, biochem. Cytol. 11, 248-251 (1961) Lee, M., Vaugham, M., Abrams, R. : Nature of the I~NA formed prior to DNA synthesis in kidney cortex cells cultured directly from the rabbit. J. biol. Chem. 245, 45254533 (1970) Lieberman, I., Ove, P. : DNA synthesis and its inhibition in mammalian cells cultured from the animal. J. biol. Chem. 237, 1634-1642 (1962) Malinin, G. : Cytotoxic effect of DMSO on the ultrastructure of cultured rhesus kidney cells. Cryobiology 10, 22-32 (1973) Mannweiller, K., Bernard, W.: Recherches ultrastructurales sur une tumeur r@nale exp~rimentale du hamster. J. Ultrastruct. Res. 1, 158-169 (1957) Maunsbach, A. : Observations on the ultrastrncture and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubule cells. J. Ultrastruct. Res. 16, 197-238 (1966) Price, Z. : A three-dimensional model of membrane ruffling from transmission and scanning electron microscopy of cultured monkey kidney cells (LLCMK2). J. Microscopy 95,493-505 (1972) Rossmann, P., Galle, P. : Mise en 6vidence de cellules r~nales cili@es chez l'homme. Nephron 5, 426436 (1968) Trump, B., Ericcson, J. :Effect of fixative solution on ultrastrueture of cells and tissues. The proximal convoluted tubule of the rat kidney. Lab. Invest. 14, 1245-1323 (1965)
