Experimental Physiology (1992), 77, 129-139 Printed in Great Britain
PRIMARY CULTURE OF COLLECTING DUCT CELL EPITHELIUM FROM NEONATE RABBIT KIDNEY IN MONOLAYER STANLEY WHITE AND HELEN REEVE Department of Physiology, Medical and Dental Building, University of Leeds, Leeds LS2 9NQ (MANUSCRIPT RECEIVED 9 JULY 1991, ACCEPTED 6 AUGUST 1991)
SUMMARY
Cortical collecting duct cells from rabbit neonate kidney capsula fibrosa explants were grown in low (0-1 %) serum medium on permeable culture supports. We found that over a period of 3-4 days in culture, the cells migrated from the explants, to form monolayers. The cells in monolayer displayed morphology similar to adult collecting duct cells. Approximately 24 % of cells grown in monolayer were found to bind a specific ,-cell-probe, peanut agglutinin. Single cells were isolated by brief exposure to 0 Ca2l-Mg2" solutions and the whole-cell clamp technique was used to measure macroscopic currents. The whole-cell conductance of 1 35 + 0-1 nS was reduced to 0 9 + 0-2 nS in the presence of 100 /LM-amiloride. We conclude that these cells express cell membrane properties consistent with those of the collecting duct and that it may be a useful model for the study of cellular transport mechanisms and the factors controlling development of collecting duct cell types. INTRODUCTION
Recently, there has been considerable interest in the use of primary cultures of renal tubule as models for the native epithelia (Horster, Fabritius & Schmolke, 1985; Bello-Reuss & Weber, 1987; Merot, Bidet, Gachot, Le Maout, Koechlin, Tauc & Poujeol, 1989). A culture of principal cells from neonate collecting duct maturation sites (Minuth, 1983, 1987) shows many of the differentiated properties of the adult collecting duct (CCD). However, a major disadvantage of this preparation is that, because the epithelium grows on top of a thick layer of fibroblasts and degenerating nephrogenic cells, it is opaque. The opacity of this preparation produces great difficulties for single-cell methods such as microelectrode impalement or patch clamping, and single-cell optical techniques using fluorescently labelled probes are not feasible. In an earlier report Minuth (1987) showed that if the epithelial cells were allowed to grow out from the explants onto plastic culture dishes, epithelial organization was lost and the epithelial cells soon de-differentiated. In this paper, we report our efforts to obtain monolayer cultures of CCD cells growing on transparent, permeable supports, and some of our experiments to ascertain whether or not the cells retain their differentiated characteristics. We found that these cultures were very easily prepared, and out results demonstrate that, when grown on permeable supports, the epithelial organization was maintained and the cells exhibited differentiated properties. However, unlike the explant culture, the monolayers were not composed solely of principal cells, but also contained some cells that expressed surface glycoproteins which are specific for the ,8-type intercalated cell.
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Primary culture One- to two-day-old New Zealand White rabbits of either sex were killed by chloroform inhalation. The kidneys were removed under aseptic conditions into ice-cold HEPES-buffered solution containing (mM): NaCl, 1180; KCI 47; CaCI2, 1 3; MgSO4, 1-3; KH2PO,, 1 2; sodium glucuronate, 200; glucose, 50; glycine, 50; HEPES, 50, buffered with NaOH to pH 74; penicillin, 100 U/ml; and streptomycin, 100 ,g/ml; hereafter referred to as 'HEPES solution'. After removal of the perirenal fat with forceps, the kidneys were cut into three or four parts and the pieces kept in HEPES solution on ice. Under a stereomicroscope (Wild M3), the kidney capsule and the adherent collecting duct maturation sites were dissected off the kidney cortex (Minuth, 1983). Each portion of capsule was then carefully cut with spring-loaded scissors into smaller (1-3 mm) pieces. On average, each kidney produced fifteen to twenty such explants. Three or four explants were placed onto 12 mm diameter rat-tail collagen-coated culture plate inserts (Millicell-CM) in which was 600,1u of Dulbecco's modified Eagle's medium (DMEM) containing IO foetal calf serum (FCS) and 25 mMNaHCO3. The inserts were enclosed in 24-well culture plates (Nunc-Nunclon) which were placed in an incubator (Leec MkII) at 37 °C in a 5 % CO2-95 % air atmosphere. Penicillin (100 U/ml) and streptomycin (100,ug/ml) were added to the medium for the first 24 h and then removed. After 24 h, the medium was changed for DMEM containing 5 % FCS, and then on the next day 2 5 % FCS. On day 4, medium was replaced by a 50:50 mixture of Ham's F-12 and DMEM supplemented with 2 5% 'Nu-Serum' (Collaborative Research Inc.) which is a low-serum, partially defined alternative to FCS; so that the final concentration of serum in the culture medium was 010%. Cultures were maintained thereafter in this medium. %
Fluorescence microscopy To assess apical membrane binding of peanut agglutinin (PNA), a marker for /3-type intercalated cells in the cortical collecting duct (Schwartz, Barasch & Al-Awqati, 1985) we incubated monolayers in the presence of fluoroscein isothiocyanate (FITC)-conjugated PNA (Vector Laboratories) at a concentration of 4,ug/ml in HEPES solution in the apical compartment of the culture well insert. min. The monolayers were then placed in Incubation was carried out in the dark at 4°C for HEPES solution in a glass cover-slip bath which was situated on the stage of a Nikon diaphot TMD inverted microscope equipped for epifluorescence observation. Monolayers were illuminatedfrom a 100 W xenon lamp at 490 nm excitation. The resulting emitted light was observed through a 520 nm barrier filter. 10
Electron microscopy Seven-day old monolayers were fixed in 250% glutaraldehyde at pH 74 in 15 mM-phosphatebuffered saline at an osmolality of 295 mosM. Thin sections were cut on a Reichert ultracut microtome, treated with osmium and examined with a Jeol1OOS electron microscope at 80 kV. Some monolayers were prepared for scanning electron microscopy, gold sputtered and viewed on a Jeol T20 scanning electron microscope. Transmonolayer potential difference (Vtm) and resistance (Rtm) Transmonolayer potential difference (VKm) and resistance (Rtm) were measured in culture by means of a volt/ohm meter ('EVOM', World Precision Instruments, New Haven, CT, USA). Vm was measured with a pair of spiral Ag-AgCl2 wire electrodes through which alternating square-wave ,uA at 12 5 Hz) were passed for measurement of Rt,. The measurement of Vt. was current pulses (20 referenced to the basolateral solution compartment. Whole-cell conductance measurements Single, isolated cells were obtained by 1-2 min exposure of the monolayers to 0 Ca2+_Mg2+ EGTAbuffered Hank's solution (Sigma) and gentle trituration through a pasteur pipette. Cells obtained in this way had a viability of at least 80% (Trypan Blue exclusion). Cells were viewed with Hoffman modulation contrast optics (HMC) at 600 x magnification on an inverting microscope (Olympus IMT-2). All studies were conducted at room temperature. Whole-cell clamp experiments were performed according to the methods of Hamill, Marty, Neher, Sakmann & Sigworth, 1981. Patch-clamp electrodes were fabricated from glass capillaries
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(Drummond Scientific Co., USA) of 2-4 MQ resistance when filled with saline. Experiments were carried out on cells in which the seal resistance (estimated by passing 20 mV pulses) was greater than 2 GQ). Recordings were made using a List L/M-EPC7 patch-clamp amplifier (List Medical, Darmstadt, Germany). Voltage step protocols were delivered through the computer using an Axon Instruments TL-I interface and pClamp 5-5 software. Records were filtered at 10 kHz bandwidth and stored directly on the computer hard disc. RESULTS
Mor'ph0olcgi' The typical morphology of the monolayers is illustrated in Fig. 1, which shows an HMC image of an 8-day-old monolayer. It can be seen that the majority of cells display a polygonal 'cobblestone' appearance, but there were also some cells in the monolayer with a circular profile. This is in keeping with the normal appearance of the mature CCD epithelium (O'Neil & Hayhurst, 1985), and also with CCD in culture (Bello-Reuss & Weber, 1987). The appearance of a round cell type may be consistent with expression of intercalated cells in these cultures which is also suggested by our observations of PNA binding to some cells in the monolayers (see below). At the ultrastructural level (Figs 2 and 3) the cells showed a morphology which was very similar to that reported for CCD principal cells (Kaissling & Kriz, 1979; Ridderstrale, Kashgarian, Koeppen, Giebisch, Stetson, Ardito & Stanton, 1988) with small apical projections and well-developed tight junctions (Fig. 3 B and C). Cells with 'dark' and with 'light' cytoplasmic staining were apparent (Fig. 2 B). The scanning electron micrographs
Fig. 1. HMC image of anl 8-day-old monolayer (mtagnification x 400; scale bar 10 /im). Most of the cells have a polygonall profile, but somiie cells (arrow-heads) have a round profile.
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Fig. 2. Transmission electron micrographs of 9-day-old monolayers. A, magnification x 5000; scale bar 1-0 /im. B, cell on the left shows 'dark' cytoplasm contrasting with 'light' cytoplasm of the cell on the right (magnification x 3000; scale bar 1 /im).
(Fig. 4) showed that the cells were of generally uniform appearance with short, apical projections, and that some cells possessed a single cilium. These results demonstrate that when the epithelial cells are allowed to grow out from the cortical explants onto a permeable growth support, they develop typical epithelial organization. These observations are in marked contrast to cells grown on plastic, which did not develop differentiated characteristics (Minuth, 1987). PNA binding The intercalated cell of the CCD can be distinguished by its distinctive morphology (O'Neil & Hayhurst, 1985) and by binding of PNA to the apical membrane (Le Hir, Kaissling, Koeppen & Wade, 1982). It appears that PNA is bound selectively by the fisubtype of intercalated cells, which are the cell type effecting HC03- secretion (Schwartz et al. 1985). Figure 5 shows a fluorescence micrograph of FITC-conjugated PNA binding to a 7-day monolayer. Two patterns of binding are apparent. Some cells show strong PNA binding. These cells constitute 23 ± 3 00 (n = 6) of cells in the monolayer. Other cells (unquantifled) exhibited a lower level of binding of the lectin. The reasons for the differential density of staining are unknown at present. Binding of PNA could be prevented by incubation in HEPES-solution containing 20 mm-galactose (data not shown) suggesting the presence of galactosyl (/1-1,3) N-acetyl galactosamine residues on these cells. These results are consistent with expression of /3-type intercalated cells in these cultures. This is
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B*N ffP~~
Flia. 3. TraImMISSIOn electroni microgra,phs of 7-dav old moniol ayecs shoW~ing tight junictions. A magnificationi x 5)0(0 sclale hatr omii B. hoxed dItcd fromi A (nidgnificdtion x 15000: sc aie hit- ami). C boxecd area fromi B (maignification x )00)) scale bar 02 urn). in contra,-Ist to an earlier study in which neonate CCD cells were maintained on their own kidnev-specific SuIpport (MinuLth., Gilbert, LaLter. Akteries & Gross, 1986) and in whilch
PNA
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absent.
Electrical properties By 7 davs monola-yers did niot achieve fuIll conflulence (a-ssessed visually) aLnd this was r-eflected by the laIck of a- signiflicanit 1, and low! R,,, (22-5 -V 1 Q cillui1= 24). However, we tested wheitheri cells expr-essed an amilloride-sensitixve conductanice. a pr-operty consistent withi pr-incipal cell funtCtionl. Figurec 6A shiows 'an examiple of a whole-cell current-voltage relaLtioniship recor-ded fr-omi anii isolated cell. The miean wxhole-cell current wxas 1-35 -1 0-1 nS (n 8; Fig. 6 B). Additioni of 100 pNi-amniloride to the bath caused a signiificanit inhibition of the mnw ard Cuirr-ent (Fig. 6 A anid B) to 0-9 + 0-2 niS (P < 0-05 paired ( test; n 7), and hiyperpolarized the cell memnbra--ne potential ; the mieani memiibrane potenitial uinder conitrol
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Fig. 4. Scanning electroin micrograph of a ninle day-old monolaver (magnification x 3500; scale balr 5 /m) showxing xiew of apical membrane of monolaver cells. Note short apical projections atnd occasionall central ciliumii.
conditions was -36+ 2 mV. aid -50+ 3 mV in the presence of amiloride (P < 0 01 1 = 7) as would be expected for inhibition of a catioin-selective conductaince. The eflects of amiloride were reversible (Fig. 6B). More thain half of the cells tested displayed low seal resistance ( < 0 5 GQ) rand were therefore not included in the analysis (see DiscussioIn). In summary. in spite of lack of trainsepithelial measui-emilenits, these experimenits show that these cells express armiloride-sensitiV e compoIneInts of the wrhole-cell conduIctaInce which is a characteristic of principal cells of the CCD. DlISCUSSI () N
The use of cultul-ed models of defined renal epithelia is cUrrently of great interest for a v ariety of reasons. Firstly. the 'flat sheet coInfigluatioin of cells grow n in imlonolayer is more convenient for electrophiysiological inxestigation than the isolated perfused tubule. Secondly, isolated tuibule peerfusion1 studies generally do not gener-ate suflicienit amounIts of imaterial to be able to corr-elate biochemical ecvents with electrophysiological responises. Fin -ally, the comparatively short time of xviability of most tubule SeKJIllellts in itio mlakes microperfusion ill-suited to study phenoimiena with a prolonged time COUrIse. Several different methlods hlaxe beein described ftor the cultuir-e of' cells which display propeities consistent with the CCD (Bello-Reuss & Weber, 1987: Fejes-Toth & Naray-Fejes-Toth, 1987: Minuth, 1987). An explaiit culture derixed fi-omil neonatal collecting duct (Minuth. 1987) can be grown13 on plistic ring supports aind used for transepitheliial electrophysiology. The properties of such cuIltuir-es are consisteint with ain homogeinous population of principal cells (Gross, MinutIl. Kri/ & Fromter, 1986). Paitch clalIlIp stuidies haxe also beeni conduc-
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CU'LTURF1
Fig. 5. Fluorescence image (see Methods) of FITC PNA binding to a 7-dcav-old monolaver (maignification sc,ale bar 10/om).
x
400:
ted (Gitter, Beyenbach, Christine, Gross, Minuth & Fromter, 1987), which describe K+ channels, similar to those described in CCD principal cells. However, a major disadvantage of the explant preparation, is that the epithelial cells grow on top of a thick (> 100 /im) layer of fibroblasts and other nephrogenic material, rendering visualization of the epithelial cells in an undistorted monolayer impossible. The opacity of the preparation makes the use of fluorescence techniques impracticable and microelectrode impalements or patch-clamp study extremely difficult (Laskowski, Christine, Gitter, Beyenbach, Gross & Fromter, 1990). Attempts to culture the epithelial cells away from their kidney-specific support on plastic culture dishes resulted in rapid loss of epithelial polarization and de-differentiation (Minuth, 1987). It is well known that a significant factor in maintaining cultured epithelial characteristics is the provision of growth factors from the basolateral aspect of the cells. This can only be provided by growing the cells on a permeable support (Handler, Perkins & Johnson, 1980; Handler, 1986; Steel, Preston, Johnson & Handler, 1986). It was with this in mind that the current study was undertaken. Groit/i th11(1 morphology, The growth of these cells is rather slow, so that even after I week in culture, monolayers were still not fully confluent over the 1 13 cm! area of the millicells. However, this slow rate of growth is probably related to the composition of the media used, and we are currently experimenting with different types of media to ascertain the optimum growth conditions for the epithelial cells. The slow growth rate of the monolayers notwithstanding, the morphology of the cells was highly similar to CCD cells of native rabbit kidney and in cultured monolayers
S. WHITE AND H. REEVE
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200
I (pA)
100
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I
I
50
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V (mV) -100 K
-200 L
B
Control 16r
100
pM-amiloride
-
c)
1-4
Q ct _
0
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1.2
-
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I
Fig. 6. A, whole-cell current-voltage relationship under control conditions (0) and in the presence of 100 /iMamiloride (0). Recordings were made at room temperature with a solution containing 140 mM-NaCl; 4-5 mMKCI in the bath and 140 mM-potassium gluconate (pH 7 1 with KOH and 10 mM-HEPES) in the pipette. B, mean (± S.E.M.) data for whole-cell conductance for control and in the presence of amiloride. Right-hand box is post-amiloride. * indicates P < 0 05 (two-tailed) for a Student's t test on paired data.
(Kaissling & Kriz, 1979; Bello-Reuss & Weber, 1987). In complete contrast to an earlier study (Minuth, 1987) where epithelial organization and morphology was lost when the epithelial cells were grown on plastic culture dishes, the cells grown on the collagen-coated permeable supports maintained their distinctive morphology and epithelial organization (Figs 1-3). The factors that determine differentiation of cells in culture are for the most part unknown. Primary cultures of CCD obtained from adult kidney grow to form an epithelial organization, with tight junctions and exhibit a polygonal cellular morphology (BelloReuss & Weber, 1987; S. White, unpublished). The reasons why cells derived from neonate kidney are not able to behave in a similar fashion (Minuth, 1987) are undetermined. However, our results illustrate very clearly the importance of polarized supply of growth factors to growing epithelial cells for development of the differentiated phenotype (Handler, 1986).
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binding Our finding that FITC-conjugated PNA bound to a proportion of the cells in monolayer is consistent with the presence of fl-type intercalated cells in the monolayers. The proportion of PNA-binding cells was found to be very similar to that reported for CCDs perfused in vitro and in other culture models (Le Hir et al. 1982; O'Neil & Hayhurst, 1985; Schwartz et al. 1985; Schwartz, Satlin & Bergmann, 1988; Emmons, Matsuzaki, Stokes & Schuster, 1991). In an earlier study (Minuth et al. 1986), binding of PNA could not be demonstrated in cells grown on explants. This difference in lectin binding may be due to the loss of some inhibitory factor(s) released by the nephrogenic material of the explant when the epithelial cells migrate from the floating explant, or may be related to differences in the age of the cultures in the two studies. The earlier study used cultures which were only 24 h, rather than the 7-day-old cultures used in the present work. In the neonate CCD functional intercalated cells are very sparse and increase in number with age (Satlin & Schwartz, 1987). Although we observed round-profile cells within the monolayers (Fig. 1), which is consistent with intercalated cell morphology, we could find no correlation of cell morphology with binding of PNA (Fig. 5). The factors controlling the development of PNA
intercalated cells are for the most part unknown. Indeed it is not clear whether or not
principal and intercalated cells may be derived from a common germ cell or arise from different cell types (Minuth, Gilbert, Rudolph & Spielman, 1989). It may be that the expression of cell surface binding sites for PNA precedes the development of mature cell morphology in these cultures, resulting in staining of cells which are at different stages of development. We have not yet determined whether the proportion of PNA-positive cells varies with time in culture. However, in preliminary experiments we have shown that the number of intercalated cells that are expressed can be influenced by the presence of aldosterone (White & Reeve, 1991). Since it is possible to visualize living cells in these monolayers, it seems likely that this monolayer preparation may be of great use as a model for investigating the factors involved in controlling cell expression in the developing collecting duct.
Electrophysiological properties The lack of confluence of these cultures was the reason for a lack of transepithelial electrical potential and the low resistance of the monolayers. Clearly, more experiments are required to determine the optimum growth conditions required to obtain areas of confluent cells which will be suitable for transepithelial electrophysiology. However, the presence of amiloride-sensitive whole-cell currents is a good indicator that some cells within the monolayer express properties consistent with those of principal cells. We found that not all cells isolated displayed amiloride-sensitive currents. All of these cells were rejected on the grounds of low seal resistance (see Methods). It is likely that this group included the 'intercalated-type' cells. Intercalated cells are characterized by low membrane potential and microelectrode impalements are often 'leaky' or unstable (Muto, Sansom & Giebisch, 1987). The absolute magnitude of the recorded currents was low in comparison to those recorded from principal cells of the rat CCD (Frindt, Sackin & Palmer, 1990). The difference may be due to the fact we used isolated single cells or, more likely, that the culture conditions play a major role in defining transport properties. In preliminary studies (White & Reeve, 1991), we have found that both the amiloride-sensitive and insensitive components of whole-cell conductance are increased after incubation of the monolayers for 24 h in media containing aldosterone.
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From the whole-cell clamp experiments, we cannot determine in which membrane the amiloride-sensitive conductance is situated. Patch-clamp studies of the apical membrane of the explant cultures (Laskowski et al. 1990) have failed to demonstrate amiloride-sensitive sodium channels of the type observed in the rat CCD (Palmer & Frindt, 1986). Likewise, we have not yet identified whether these monolayer cells contain amiloride-sensitive sodium channels in the apical membrane. In summary, we have found that when neonatal CCD explants are grown in culture on permeable supports, the cells form monolayers which exhibit morphological, histochemical and some electrophysiological properties consistent with the CCD. The ease with which these cultures can be propagated, and the excellent optical properties of these monolayers make them a significantly improved model over explant preparations for certain types of experiment. This work was supported by the Wellcome Trust, the Nuffield Foundation and the University of Leeds Research Fund. We wish to express our gratitude to Professor W. Minuth (University of Regensburg) for showing us the neonatal preparation and for continued helpful advice during the course of this study, and to Dr Malcolm Hunter for comments on an earlier version of the manuscript. REFERENCES
BELLO-REUSS, E. & WEBER, M. R. (1987). Electrophysiological studies of primary cultures of rabbit distal tubule cells. American Journal of Physiology 252, F899-909. EMMONS, C. L., MATSUZAKI, K., STOKES, J. B. & SCHUSTER, V. L. (1991). Axial heterogeneity of rabbit cortical collecting duct. American Journal of Ph/vsiologj' 260, F498-505. FEJES-TOTH, G & NARAY-FEJES-TOTH, A. (1987). Differentiated transport functions in primary cultures of rabbit collecting ducts. American Journal of Physiology 253, F1302-1307. FRINDT, G., SACKIN, H. & PALMER, L. G. (1990). Whole-cell currents in rat cortical collecting tubule: low-Na diet increases amiloride-sensitive conductance. American Journal of Physiology 258, F562-567. GITTER, A. H., BEYENBACH, K. W., CHRISTINE, C. W., GROSS, P., MINUTH, W. W. & FROMTER, E. (1987). High conductance K+ channel cultured from rabbit renal cortical collecting duct anlagen. Pflugers Archir 408, 282-290. GROSS, P., MINUTH, W. W., KRIZ, W. & FROMTER, E. (1986). Electrical properties of renal collecting duct principal cell epithelium in tissue culture. Pfluigers Archiv 406, 380-386. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluigers Archiv 391, 85-100. HANDLER, J. S. (1986). Studies of kidney cells in culture. Kidney International 30, 208-215. HANDLER, J. S., PERKINS, F. M. & JOHNSON, J. P. (1980). Studies of renal cell function using cell culture techniques. American Journal of Physiology 238, Fl-9. HORSTER, M. F., FABRITIUS, J. & SCHMOLKE, M. (1985). The study of epithelial function by in vitro culture of nephron cells. Pfluigers Archiv 405, suppI. 1, S158-162. KAISSLING, B. & KRIZ, W. (1979). Structural Analysis of the Rabbit Kidney, pp. 1-121. SpringerVerlag, Heidelberg. LASKOWSKI, F. H., CHRISTINE, C. W., GITTER, A. H., BEYENBACH, K. W., GROSS, P. & FROMTER, E. (1990). Cation channels in the apical membrane of collecting duct principal cell epithelium in culture. Renal Physiology and Biochemistry 13, 70-81. LE HIR, M., KAISSLING, B., KOEPPEN, B. M. & WADE, J. B. (1982). Binding of peanut lectin to specific epithelial cell types in kidney. American Journal of Physiology 242, 117-120. MEROT, J., BIDET, M., GACHOT, B., LE MAOUT, S., KOECHLIN, N., TAUC, M. & POUJEOL, P. (1989).
Electrical properties of rabbit early distal convoluted tubule in primary culture. American Journal of Physiology 257, F288-299. MINUTH, W. W. (1983). Induction and inhibition of outgrowth and development of renal collecting duct epithelium. Laboratory Investigation 48 (5), 543-548.
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MINUTH, W. W., (1987). Neonatal rabbit kidney cortex in culture as tool for the study of collecting duct formation and nephron differentiation. Differentiation 36, 12-22. MINUTH, W. W., GILBERT, P., LAUER, G., AKTERIES, K. & GROSS, P. (1986). Differentiation properties of renal collecting duct cells in culture. Differentiation 33, 156-167. MINUTH, W. W., GILBERT, P., RUDOLPH, U. & SPIELMAN, W. S. (1989). Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney. Difterentiation 93, 19-25. MUTO, S., GIEBISCH, G. & SANSOM, S. (1987). Effects of adrenalectomy on CCD: evidence for differential response of two cell types. American Journal of Physiology 253, F742-752. O'NEIL, R. G. & HAYHURST, R. A. (1985). Functional differentiation of cell types of cortical collecting duct. American Journal of Physiology 248, F449-453. PALMER, L. G. & FRINDT, G. (1986). Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proceedings of the National Academy of Sciences 83, 2767-2770. RIDDERSTRALE, Y., KASHGARIAN, M., KOEPPEN, B. M., GIEBISCH, G., STETSON, D., ARDITO, T. & STANTON, B. A. (1988). Morphological heterogeneity of the rabbit collecting duct. Kidney International 34, 655-670. SATLIN, L. M. & SCHWARTZ, G. J. (1987). Postnatal maturation of rabbit renal collecting duct: intercalated cell function. American Journal of Physiology 253, F622-635. SCHWARTZ, G. J., BARASCH, J. & AL-AWQATI, Q. (1985). Plasticity of epithelial polarity. Nature 318, 368-371. SCHWARTZ, G. J., SATLIN, L. M. & BERGMANN, J. E. (1988). Fluorescent characterization of collecting duct cells: a second H+-secreting type. American Journal of Physiology 255, F1003-1014. STEELE, R. E., PRESTON, A. S., JOHNSON, J. P. & HANDLER, J. S. (1986). Porous-bottom dishes for culture of polarized cells. American Journal of Physiology 251, C136-139. WHITE, S. J. & REEVE, H. (1991). Effects of aldosterone on cultured collecting duct cells from neonate rabbit kidney. In Aldosterone: Fundamental Aspects, ed. BONVALET, J.-P., FARMAN, N., LOMBES, M. & RAFESTIN-OBLIN, M.-E. p. 330. John Libbey, Paris.
PRIMARY CULTURE OF COLLECTING DUCT CELL EPITHELIUM FROM NEONATE RABBIT KIDNEY IN MONOLAYER STANLEY WHITE AND HELEN REEVE Department of Physiology, Medical and Dental Building, University of Leeds, Leeds LS2 9NQ (MANUSCRIPT RECEIVED 9 JULY 1991, ACCEPTED 6 AUGUST 1991)
SUMMARY
Cortical collecting duct cells from rabbit neonate kidney capsula fibrosa explants were grown in low (0-1 %) serum medium on permeable culture supports. We found that over a period of 3-4 days in culture, the cells migrated from the explants, to form monolayers. The cells in monolayer displayed morphology similar to adult collecting duct cells. Approximately 24 % of cells grown in monolayer were found to bind a specific ,-cell-probe, peanut agglutinin. Single cells were isolated by brief exposure to 0 Ca2l-Mg2" solutions and the whole-cell clamp technique was used to measure macroscopic currents. The whole-cell conductance of 1 35 + 0-1 nS was reduced to 0 9 + 0-2 nS in the presence of 100 /LM-amiloride. We conclude that these cells express cell membrane properties consistent with those of the collecting duct and that it may be a useful model for the study of cellular transport mechanisms and the factors controlling development of collecting duct cell types. INTRODUCTION
Recently, there has been considerable interest in the use of primary cultures of renal tubule as models for the native epithelia (Horster, Fabritius & Schmolke, 1985; Bello-Reuss & Weber, 1987; Merot, Bidet, Gachot, Le Maout, Koechlin, Tauc & Poujeol, 1989). A culture of principal cells from neonate collecting duct maturation sites (Minuth, 1983, 1987) shows many of the differentiated properties of the adult collecting duct (CCD). However, a major disadvantage of this preparation is that, because the epithelium grows on top of a thick layer of fibroblasts and degenerating nephrogenic cells, it is opaque. The opacity of this preparation produces great difficulties for single-cell methods such as microelectrode impalement or patch clamping, and single-cell optical techniques using fluorescently labelled probes are not feasible. In an earlier report Minuth (1987) showed that if the epithelial cells were allowed to grow out from the explants onto plastic culture dishes, epithelial organization was lost and the epithelial cells soon de-differentiated. In this paper, we report our efforts to obtain monolayer cultures of CCD cells growing on transparent, permeable supports, and some of our experiments to ascertain whether or not the cells retain their differentiated characteristics. We found that these cultures were very easily prepared, and out results demonstrate that, when grown on permeable supports, the epithelial organization was maintained and the cells exhibited differentiated properties. However, unlike the explant culture, the monolayers were not composed solely of principal cells, but also contained some cells that expressed surface glycoproteins which are specific for the ,8-type intercalated cell.
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Primary culture One- to two-day-old New Zealand White rabbits of either sex were killed by chloroform inhalation. The kidneys were removed under aseptic conditions into ice-cold HEPES-buffered solution containing (mM): NaCl, 1180; KCI 47; CaCI2, 1 3; MgSO4, 1-3; KH2PO,, 1 2; sodium glucuronate, 200; glucose, 50; glycine, 50; HEPES, 50, buffered with NaOH to pH 74; penicillin, 100 U/ml; and streptomycin, 100 ,g/ml; hereafter referred to as 'HEPES solution'. After removal of the perirenal fat with forceps, the kidneys were cut into three or four parts and the pieces kept in HEPES solution on ice. Under a stereomicroscope (Wild M3), the kidney capsule and the adherent collecting duct maturation sites were dissected off the kidney cortex (Minuth, 1983). Each portion of capsule was then carefully cut with spring-loaded scissors into smaller (1-3 mm) pieces. On average, each kidney produced fifteen to twenty such explants. Three or four explants were placed onto 12 mm diameter rat-tail collagen-coated culture plate inserts (Millicell-CM) in which was 600,1u of Dulbecco's modified Eagle's medium (DMEM) containing IO foetal calf serum (FCS) and 25 mMNaHCO3. The inserts were enclosed in 24-well culture plates (Nunc-Nunclon) which were placed in an incubator (Leec MkII) at 37 °C in a 5 % CO2-95 % air atmosphere. Penicillin (100 U/ml) and streptomycin (100,ug/ml) were added to the medium for the first 24 h and then removed. After 24 h, the medium was changed for DMEM containing 5 % FCS, and then on the next day 2 5 % FCS. On day 4, medium was replaced by a 50:50 mixture of Ham's F-12 and DMEM supplemented with 2 5% 'Nu-Serum' (Collaborative Research Inc.) which is a low-serum, partially defined alternative to FCS; so that the final concentration of serum in the culture medium was 010%. Cultures were maintained thereafter in this medium. %
Fluorescence microscopy To assess apical membrane binding of peanut agglutinin (PNA), a marker for /3-type intercalated cells in the cortical collecting duct (Schwartz, Barasch & Al-Awqati, 1985) we incubated monolayers in the presence of fluoroscein isothiocyanate (FITC)-conjugated PNA (Vector Laboratories) at a concentration of 4,ug/ml in HEPES solution in the apical compartment of the culture well insert. min. The monolayers were then placed in Incubation was carried out in the dark at 4°C for HEPES solution in a glass cover-slip bath which was situated on the stage of a Nikon diaphot TMD inverted microscope equipped for epifluorescence observation. Monolayers were illuminatedfrom a 100 W xenon lamp at 490 nm excitation. The resulting emitted light was observed through a 520 nm barrier filter. 10
Electron microscopy Seven-day old monolayers were fixed in 250% glutaraldehyde at pH 74 in 15 mM-phosphatebuffered saline at an osmolality of 295 mosM. Thin sections were cut on a Reichert ultracut microtome, treated with osmium and examined with a Jeol1OOS electron microscope at 80 kV. Some monolayers were prepared for scanning electron microscopy, gold sputtered and viewed on a Jeol T20 scanning electron microscope. Transmonolayer potential difference (Vtm) and resistance (Rtm) Transmonolayer potential difference (VKm) and resistance (Rtm) were measured in culture by means of a volt/ohm meter ('EVOM', World Precision Instruments, New Haven, CT, USA). Vm was measured with a pair of spiral Ag-AgCl2 wire electrodes through which alternating square-wave ,uA at 12 5 Hz) were passed for measurement of Rt,. The measurement of Vt. was current pulses (20 referenced to the basolateral solution compartment. Whole-cell conductance measurements Single, isolated cells were obtained by 1-2 min exposure of the monolayers to 0 Ca2+_Mg2+ EGTAbuffered Hank's solution (Sigma) and gentle trituration through a pasteur pipette. Cells obtained in this way had a viability of at least 80% (Trypan Blue exclusion). Cells were viewed with Hoffman modulation contrast optics (HMC) at 600 x magnification on an inverting microscope (Olympus IMT-2). All studies were conducted at room temperature. Whole-cell clamp experiments were performed according to the methods of Hamill, Marty, Neher, Sakmann & Sigworth, 1981. Patch-clamp electrodes were fabricated from glass capillaries
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(Drummond Scientific Co., USA) of 2-4 MQ resistance when filled with saline. Experiments were carried out on cells in which the seal resistance (estimated by passing 20 mV pulses) was greater than 2 GQ). Recordings were made using a List L/M-EPC7 patch-clamp amplifier (List Medical, Darmstadt, Germany). Voltage step protocols were delivered through the computer using an Axon Instruments TL-I interface and pClamp 5-5 software. Records were filtered at 10 kHz bandwidth and stored directly on the computer hard disc. RESULTS
Mor'ph0olcgi' The typical morphology of the monolayers is illustrated in Fig. 1, which shows an HMC image of an 8-day-old monolayer. It can be seen that the majority of cells display a polygonal 'cobblestone' appearance, but there were also some cells in the monolayer with a circular profile. This is in keeping with the normal appearance of the mature CCD epithelium (O'Neil & Hayhurst, 1985), and also with CCD in culture (Bello-Reuss & Weber, 1987). The appearance of a round cell type may be consistent with expression of intercalated cells in these cultures which is also suggested by our observations of PNA binding to some cells in the monolayers (see below). At the ultrastructural level (Figs 2 and 3) the cells showed a morphology which was very similar to that reported for CCD principal cells (Kaissling & Kriz, 1979; Ridderstrale, Kashgarian, Koeppen, Giebisch, Stetson, Ardito & Stanton, 1988) with small apical projections and well-developed tight junctions (Fig. 3 B and C). Cells with 'dark' and with 'light' cytoplasmic staining were apparent (Fig. 2 B). The scanning electron micrographs
Fig. 1. HMC image of anl 8-day-old monolayer (mtagnification x 400; scale bar 10 /im). Most of the cells have a polygonall profile, but somiie cells (arrow-heads) have a round profile.
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A
Fig. 2. Transmission electron micrographs of 9-day-old monolayers. A, magnification x 5000; scale bar 1-0 /im. B, cell on the left shows 'dark' cytoplasm contrasting with 'light' cytoplasm of the cell on the right (magnification x 3000; scale bar 1 /im).
(Fig. 4) showed that the cells were of generally uniform appearance with short, apical projections, and that some cells possessed a single cilium. These results demonstrate that when the epithelial cells are allowed to grow out from the cortical explants onto a permeable growth support, they develop typical epithelial organization. These observations are in marked contrast to cells grown on plastic, which did not develop differentiated characteristics (Minuth, 1987). PNA binding The intercalated cell of the CCD can be distinguished by its distinctive morphology (O'Neil & Hayhurst, 1985) and by binding of PNA to the apical membrane (Le Hir, Kaissling, Koeppen & Wade, 1982). It appears that PNA is bound selectively by the fisubtype of intercalated cells, which are the cell type effecting HC03- secretion (Schwartz et al. 1985). Figure 5 shows a fluorescence micrograph of FITC-conjugated PNA binding to a 7-day monolayer. Two patterns of binding are apparent. Some cells show strong PNA binding. These cells constitute 23 ± 3 00 (n = 6) of cells in the monolayer. Other cells (unquantifled) exhibited a lower level of binding of the lectin. The reasons for the differential density of staining are unknown at present. Binding of PNA could be prevented by incubation in HEPES-solution containing 20 mm-galactose (data not shown) suggesting the presence of galactosyl (/1-1,3) N-acetyl galactosamine residues on these cells. These results are consistent with expression of /3-type intercalated cells in these cultures. This is
NEoINATE COLLECTING DUCT IN CULTURE
1 33
B*N ffP~~
Flia. 3. TraImMISSIOn electroni microgra,phs of 7-dav old moniol ayecs shoW~ing tight junictions. A magnificationi x 5)0(0 sclale hatr omii B. hoxed dItcd fromi A (nidgnificdtion x 15000: sc aie hit- ami). C boxecd area fromi B (maignification x )00)) scale bar 02 urn). in contra,-Ist to an earlier study in which neonate CCD cells were maintained on their own kidnev-specific SuIpport (MinuLth., Gilbert, LaLter. Akteries & Gross, 1986) and in whilch
PNA
bindin_g was
absent.
Electrical properties By 7 davs monola-yers did niot achieve fuIll conflulence (a-ssessed visually) aLnd this was r-eflected by the laIck of a- signiflicanit 1, and low! R,,, (22-5 -V 1 Q cillui1= 24). However, we tested wheitheri cells expr-essed an amilloride-sensitixve conductanice. a pr-operty consistent withi pr-incipal cell funtCtionl. Figurec 6A shiows 'an examiple of a whole-cell current-voltage relaLtioniship recor-ded fr-omi anii isolated cell. The miean wxhole-cell current wxas 1-35 -1 0-1 nS (n 8; Fig. 6 B). Additioni of 100 pNi-amniloride to the bath caused a signiificanit inhibition of the mnw ard Cuirr-ent (Fig. 6 A anid B) to 0-9 + 0-2 niS (P < 0-05 paired ( test; n 7), and hiyperpolarized the cell memnbra--ne potential ; the mieani memiibrane potenitial uinder conitrol
134
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WHITF AND H. RFF.V
Fig. 4. Scanning electroin micrograph of a ninle day-old monolaver (magnification x 3500; scale balr 5 /m) showxing xiew of apical membrane of monolaver cells. Note short apical projections atnd occasionall central ciliumii.
conditions was -36+ 2 mV. aid -50+ 3 mV in the presence of amiloride (P < 0 01 1 = 7) as would be expected for inhibition of a catioin-selective conductaince. The eflects of amiloride were reversible (Fig. 6B). More thain half of the cells tested displayed low seal resistance ( < 0 5 GQ) rand were therefore not included in the analysis (see DiscussioIn). In summary. in spite of lack of trainsepithelial measui-emilenits, these experimenits show that these cells express armiloride-sensitiV e compoIneInts of the wrhole-cell conduIctaInce which is a characteristic of principal cells of the CCD. DlISCUSSI () N
The use of cultul-ed models of defined renal epithelia is cUrrently of great interest for a v ariety of reasons. Firstly. the 'flat sheet coInfigluatioin of cells grow n in imlonolayer is more convenient for electrophiysiological inxestigation than the isolated perfused tubule. Secondly, isolated tuibule peerfusion1 studies generally do not gener-ate suflicienit amounIts of imaterial to be able to corr-elate biochemical ecvents with electrophysiological responises. Fin -ally, the comparatively short time of xviability of most tubule SeKJIllellts in itio mlakes microperfusion ill-suited to study phenoimiena with a prolonged time COUrIse. Several different methlods hlaxe beein described ftor the cultuir-e of' cells which display propeities consistent with the CCD (Bello-Reuss & Weber, 1987: Fejes-Toth & Naray-Fejes-Toth, 1987: Minuth, 1987). An explaiit culture derixed fi-omil neonatal collecting duct (Minuth. 1987) can be grown13 on plistic ring supports aind used for transepitheliial electrophysiology. The properties of such cuIltuir-es are consisteint with ain homogeinous population of principal cells (Gross, MinutIl. Kri/ & Fromter, 1986). Paitch clalIlIp stuidies haxe also beeni conduc-
NEONATE COLt,ECTING DUCT IN
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CU'LTURF1
Fig. 5. Fluorescence image (see Methods) of FITC PNA binding to a 7-dcav-old monolaver (maignification sc,ale bar 10/om).
x
400:
ted (Gitter, Beyenbach, Christine, Gross, Minuth & Fromter, 1987), which describe K+ channels, similar to those described in CCD principal cells. However, a major disadvantage of the explant preparation, is that the epithelial cells grow on top of a thick (> 100 /im) layer of fibroblasts and other nephrogenic material, rendering visualization of the epithelial cells in an undistorted monolayer impossible. The opacity of the preparation makes the use of fluorescence techniques impracticable and microelectrode impalements or patch-clamp study extremely difficult (Laskowski, Christine, Gitter, Beyenbach, Gross & Fromter, 1990). Attempts to culture the epithelial cells away from their kidney-specific support on plastic culture dishes resulted in rapid loss of epithelial polarization and de-differentiation (Minuth, 1987). It is well known that a significant factor in maintaining cultured epithelial characteristics is the provision of growth factors from the basolateral aspect of the cells. This can only be provided by growing the cells on a permeable support (Handler, Perkins & Johnson, 1980; Handler, 1986; Steel, Preston, Johnson & Handler, 1986). It was with this in mind that the current study was undertaken. Groit/i th11(1 morphology, The growth of these cells is rather slow, so that even after I week in culture, monolayers were still not fully confluent over the 1 13 cm! area of the millicells. However, this slow rate of growth is probably related to the composition of the media used, and we are currently experimenting with different types of media to ascertain the optimum growth conditions for the epithelial cells. The slow growth rate of the monolayers notwithstanding, the morphology of the cells was highly similar to CCD cells of native rabbit kidney and in cultured monolayers
S. WHITE AND H. REEVE
136 A
200
I (pA)
100
-150
-100 --
I
I
50
.P
V (mV) -100 K
-200 L
B
Control 16r
100
pM-amiloride
-
c)
1-4
Q ct _
0
*~~~
1.2
-
Q
3 10
0-8L
I
I
Fig. 6. A, whole-cell current-voltage relationship under control conditions (0) and in the presence of 100 /iMamiloride (0). Recordings were made at room temperature with a solution containing 140 mM-NaCl; 4-5 mMKCI in the bath and 140 mM-potassium gluconate (pH 7 1 with KOH and 10 mM-HEPES) in the pipette. B, mean (± S.E.M.) data for whole-cell conductance for control and in the presence of amiloride. Right-hand box is post-amiloride. * indicates P < 0 05 (two-tailed) for a Student's t test on paired data.
(Kaissling & Kriz, 1979; Bello-Reuss & Weber, 1987). In complete contrast to an earlier study (Minuth, 1987) where epithelial organization and morphology was lost when the epithelial cells were grown on plastic culture dishes, the cells grown on the collagen-coated permeable supports maintained their distinctive morphology and epithelial organization (Figs 1-3). The factors that determine differentiation of cells in culture are for the most part unknown. Primary cultures of CCD obtained from adult kidney grow to form an epithelial organization, with tight junctions and exhibit a polygonal cellular morphology (BelloReuss & Weber, 1987; S. White, unpublished). The reasons why cells derived from neonate kidney are not able to behave in a similar fashion (Minuth, 1987) are undetermined. However, our results illustrate very clearly the importance of polarized supply of growth factors to growing epithelial cells for development of the differentiated phenotype (Handler, 1986).
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binding Our finding that FITC-conjugated PNA bound to a proportion of the cells in monolayer is consistent with the presence of fl-type intercalated cells in the monolayers. The proportion of PNA-binding cells was found to be very similar to that reported for CCDs perfused in vitro and in other culture models (Le Hir et al. 1982; O'Neil & Hayhurst, 1985; Schwartz et al. 1985; Schwartz, Satlin & Bergmann, 1988; Emmons, Matsuzaki, Stokes & Schuster, 1991). In an earlier study (Minuth et al. 1986), binding of PNA could not be demonstrated in cells grown on explants. This difference in lectin binding may be due to the loss of some inhibitory factor(s) released by the nephrogenic material of the explant when the epithelial cells migrate from the floating explant, or may be related to differences in the age of the cultures in the two studies. The earlier study used cultures which were only 24 h, rather than the 7-day-old cultures used in the present work. In the neonate CCD functional intercalated cells are very sparse and increase in number with age (Satlin & Schwartz, 1987). Although we observed round-profile cells within the monolayers (Fig. 1), which is consistent with intercalated cell morphology, we could find no correlation of cell morphology with binding of PNA (Fig. 5). The factors controlling the development of PNA
intercalated cells are for the most part unknown. Indeed it is not clear whether or not
principal and intercalated cells may be derived from a common germ cell or arise from different cell types (Minuth, Gilbert, Rudolph & Spielman, 1989). It may be that the expression of cell surface binding sites for PNA precedes the development of mature cell morphology in these cultures, resulting in staining of cells which are at different stages of development. We have not yet determined whether the proportion of PNA-positive cells varies with time in culture. However, in preliminary experiments we have shown that the number of intercalated cells that are expressed can be influenced by the presence of aldosterone (White & Reeve, 1991). Since it is possible to visualize living cells in these monolayers, it seems likely that this monolayer preparation may be of great use as a model for investigating the factors involved in controlling cell expression in the developing collecting duct.
Electrophysiological properties The lack of confluence of these cultures was the reason for a lack of transepithelial electrical potential and the low resistance of the monolayers. Clearly, more experiments are required to determine the optimum growth conditions required to obtain areas of confluent cells which will be suitable for transepithelial electrophysiology. However, the presence of amiloride-sensitive whole-cell currents is a good indicator that some cells within the monolayer express properties consistent with those of principal cells. We found that not all cells isolated displayed amiloride-sensitive currents. All of these cells were rejected on the grounds of low seal resistance (see Methods). It is likely that this group included the 'intercalated-type' cells. Intercalated cells are characterized by low membrane potential and microelectrode impalements are often 'leaky' or unstable (Muto, Sansom & Giebisch, 1987). The absolute magnitude of the recorded currents was low in comparison to those recorded from principal cells of the rat CCD (Frindt, Sackin & Palmer, 1990). The difference may be due to the fact we used isolated single cells or, more likely, that the culture conditions play a major role in defining transport properties. In preliminary studies (White & Reeve, 1991), we have found that both the amiloride-sensitive and insensitive components of whole-cell conductance are increased after incubation of the monolayers for 24 h in media containing aldosterone.
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From the whole-cell clamp experiments, we cannot determine in which membrane the amiloride-sensitive conductance is situated. Patch-clamp studies of the apical membrane of the explant cultures (Laskowski et al. 1990) have failed to demonstrate amiloride-sensitive sodium channels of the type observed in the rat CCD (Palmer & Frindt, 1986). Likewise, we have not yet identified whether these monolayer cells contain amiloride-sensitive sodium channels in the apical membrane. In summary, we have found that when neonatal CCD explants are grown in culture on permeable supports, the cells form monolayers which exhibit morphological, histochemical and some electrophysiological properties consistent with the CCD. The ease with which these cultures can be propagated, and the excellent optical properties of these monolayers make them a significantly improved model over explant preparations for certain types of experiment. This work was supported by the Wellcome Trust, the Nuffield Foundation and the University of Leeds Research Fund. We wish to express our gratitude to Professor W. Minuth (University of Regensburg) for showing us the neonatal preparation and for continued helpful advice during the course of this study, and to Dr Malcolm Hunter for comments on an earlier version of the manuscript. REFERENCES
BELLO-REUSS, E. & WEBER, M. R. (1987). Electrophysiological studies of primary cultures of rabbit distal tubule cells. American Journal of Physiology 252, F899-909. EMMONS, C. L., MATSUZAKI, K., STOKES, J. B. & SCHUSTER, V. L. (1991). Axial heterogeneity of rabbit cortical collecting duct. American Journal of Ph/vsiologj' 260, F498-505. FEJES-TOTH, G & NARAY-FEJES-TOTH, A. (1987). Differentiated transport functions in primary cultures of rabbit collecting ducts. American Journal of Physiology 253, F1302-1307. FRINDT, G., SACKIN, H. & PALMER, L. G. (1990). Whole-cell currents in rat cortical collecting tubule: low-Na diet increases amiloride-sensitive conductance. American Journal of Physiology 258, F562-567. GITTER, A. H., BEYENBACH, K. W., CHRISTINE, C. W., GROSS, P., MINUTH, W. W. & FROMTER, E. (1987). High conductance K+ channel cultured from rabbit renal cortical collecting duct anlagen. Pflugers Archir 408, 282-290. GROSS, P., MINUTH, W. W., KRIZ, W. & FROMTER, E. (1986). Electrical properties of renal collecting duct principal cell epithelium in tissue culture. Pfluigers Archiv 406, 380-386. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluigers Archiv 391, 85-100. HANDLER, J. S. (1986). Studies of kidney cells in culture. Kidney International 30, 208-215. HANDLER, J. S., PERKINS, F. M. & JOHNSON, J. P. (1980). Studies of renal cell function using cell culture techniques. American Journal of Physiology 238, Fl-9. HORSTER, M. F., FABRITIUS, J. & SCHMOLKE, M. (1985). The study of epithelial function by in vitro culture of nephron cells. Pfluigers Archiv 405, suppI. 1, S158-162. KAISSLING, B. & KRIZ, W. (1979). Structural Analysis of the Rabbit Kidney, pp. 1-121. SpringerVerlag, Heidelberg. LASKOWSKI, F. H., CHRISTINE, C. W., GITTER, A. H., BEYENBACH, K. W., GROSS, P. & FROMTER, E. (1990). Cation channels in the apical membrane of collecting duct principal cell epithelium in culture. Renal Physiology and Biochemistry 13, 70-81. LE HIR, M., KAISSLING, B., KOEPPEN, B. M. & WADE, J. B. (1982). Binding of peanut lectin to specific epithelial cell types in kidney. American Journal of Physiology 242, 117-120. MEROT, J., BIDET, M., GACHOT, B., LE MAOUT, S., KOECHLIN, N., TAUC, M. & POUJEOL, P. (1989).
Electrical properties of rabbit early distal convoluted tubule in primary culture. American Journal of Physiology 257, F288-299. MINUTH, W. W. (1983). Induction and inhibition of outgrowth and development of renal collecting duct epithelium. Laboratory Investigation 48 (5), 543-548.
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MINUTH, W. W., (1987). Neonatal rabbit kidney cortex in culture as tool for the study of collecting duct formation and nephron differentiation. Differentiation 36, 12-22. MINUTH, W. W., GILBERT, P., LAUER, G., AKTERIES, K. & GROSS, P. (1986). Differentiation properties of renal collecting duct cells in culture. Differentiation 33, 156-167. MINUTH, W. W., GILBERT, P., RUDOLPH, U. & SPIELMAN, W. S. (1989). Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney. Difterentiation 93, 19-25. MUTO, S., GIEBISCH, G. & SANSOM, S. (1987). Effects of adrenalectomy on CCD: evidence for differential response of two cell types. American Journal of Physiology 253, F742-752. O'NEIL, R. G. & HAYHURST, R. A. (1985). Functional differentiation of cell types of cortical collecting duct. American Journal of Physiology 248, F449-453. PALMER, L. G. & FRINDT, G. (1986). Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proceedings of the National Academy of Sciences 83, 2767-2770. RIDDERSTRALE, Y., KASHGARIAN, M., KOEPPEN, B. M., GIEBISCH, G., STETSON, D., ARDITO, T. & STANTON, B. A. (1988). Morphological heterogeneity of the rabbit collecting duct. Kidney International 34, 655-670. SATLIN, L. M. & SCHWARTZ, G. J. (1987). Postnatal maturation of rabbit renal collecting duct: intercalated cell function. American Journal of Physiology 253, F622-635. SCHWARTZ, G. J., BARASCH, J. & AL-AWQATI, Q. (1985). Plasticity of epithelial polarity. Nature 318, 368-371. SCHWARTZ, G. J., SATLIN, L. M. & BERGMANN, J. E. (1988). Fluorescent characterization of collecting duct cells: a second H+-secreting type. American Journal of Physiology 255, F1003-1014. STEELE, R. E., PRESTON, A. S., JOHNSON, J. P. & HANDLER, J. S. (1986). Porous-bottom dishes for culture of polarized cells. American Journal of Physiology 251, C136-139. WHITE, S. J. & REEVE, H. (1991). Effects of aldosterone on cultured collecting duct cells from neonate rabbit kidney. In Aldosterone: Fundamental Aspects, ed. BONVALET, J.-P., FARMAN, N., LOMBES, M. & RAFESTIN-OBLIN, M.-E. p. 330. John Libbey, Paris.
