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Experimental
PRIMARY RAT
Cell Research
EPITHELIAL
KIDNEY,
CELL
ENHANCEMENT BY AMMONIUM
JULES BERMAN,
121 (1979) 47-54
CULTURE
OF ADULT
OF CELL
GROWTH
ACETATE
ALAN PERANTONI, HESTER and ELIZABETH KINGSBURY’
MARIE
JACKSON
Experimental Pathology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014 and ‘Litton Bionetics, Kensington, MD 20795, USA
SUMMARY Primary monolayer cultures of epithelial-like cells from adult Lewis rat kidney demonstrated enhanced growth and survival over a 3-week period when cultured in medium supplemented with 10 mM ammonium acetate. In addition to enhancing the growth and survival of epithelial-like cells, 10 mM ammonium acetate also suppressed the growth of a subpopulation of cells having a flbroblast-like morphology. Both epithelial-like and tibroblast-like cells reacted positively in histochemical reactions for gamma glutamyl transpeptidase (GGT) and 5’-nucleotidase, enzymes found in high amounts in kidney tubular epithelium. Despite the enhanced survival of cells in the presence of ammonium acetate, most cells in primary culture eventually died out. Electron microscopic examination of dying epithelial-like cells revealed the presence of numerous large lysosomes filled with amorphous electron-dense material. These findings substantiate the prediction of Seglen (Exp cell res 107 (1977) 207) that ammonia might cause increased growth of primary cells in culture, presumably through its nitrogen-preserving action.
In a recent report, Seglen demonstrated that 10 mM ammonium chloride signiticantly inhibited protein degradation in suspension culture of isolated adult rat hepatocytes [l]. As rat hepatocytes in primary cell culture do not divide, Seglen could not, in his system, observe the growth effects of ammonia. We have developed a technique for establishing primary monolayer cultures of adult rat kidney. These cells have an epithelial morphology as judged by light and electron microscopic examination and divide rapidly for a period of about one week, at which time growth of epithelial-like cells ceases. Concurrently a variant population of cells with fibroblast-like morphology grows and can be subcultured for at least 6 months. We have examined the effect of ammonium 4-791808
acetate on primary monolayer cultures of kidney to determine whether ammonia has a growth enhancing effect on a dividing population of epithelial cells. MATERIALS
AND
METHODS
Kidneys from adult male Lewis rats (LEW/mai, Microbiological Associates, Bethesda, average age 12 weeks) were minced, then shaken for 10 min in Dulbecco’s salts solution containing 0.02% EDTA and no calcium or maanesium. Tissue was then washed and incubated with a solution of 2.25 % trypsin (Flow Laboratories, Rockville. MD) in salts solution containing 0.02% EDTA in a 37 degree shaker for 40 min. At this time, minced tissue was visibly blanched but not dissociated. Trypsin solution was then aspirated leaving behind most tissue fragments. Fragments were then pipetted repeatedly (about a dozen times) with a plastic 10 ml pipet (pipet tip snapped off for wider bore) until tissue fragments dissociated into a cell suspension. The suspension was then transferred to 250 ml of Williams Medium E (WE. Flow Laboratories) supplemented with 2 mM glutamine and 10% fetal bovine serum (Flow Laboratories), and 50 pg/ml Exp Cell
Res 121 (1979)
48
Berman et al.
gentamycin (Microbiological Associates). This suspension was divided into 25 dishes (0 100 mm, Corning). Three hours after plating, dishes were washed twice and refed with 10 ml supplemented WE. Dishes were incubated overnight at which time treatments were begun and cell counts taken. Ammonium acetate, ammonium chloride, and sodium acetate (Fisher Scientific) were dissolved in water at concentrations such that no more than 0.1 ml of solution was ever added to a dish. Media pH (7.4) was not altered by these additions. Solutions were passed through Nalge filters and stored as refrigerated stock solutions. All dishes were refed every 2 days with supplemented WE and varying concentrations of test solution. Cell counts were done by averaging the number of cells in five chosen microscopic fields delineated by a reticle housed in the eyepiece of a phase contrast microscope. Every experimental cell count was determined from the average cell counts of triplicate or quadruplicate dishes. In one experiment, cells were classified cytologically as epithelial-like (having a polygonal structure and growing in well-defined nests) and tibroblast-like (spindled and stellate shapes varying greatly in cell size and having little or no contact with other cells in areas of low cell density, but aligning in parallel arrays in areas of high cell density).
0. 0
Enzyme histochemistry Gamma glutamyl transpeptidase (GGT) activity was demonstrated by the method of Rutenberg [2], without counterstainina. Dishes of cells were fixed by quick rinse with 10 ml of methylbutane (Fisher) ai -2O’C and then stored at -70°C. For assay, 10 ml of reaction mixture [2] were put into each dish and incubated 20 min at 37°C. A 10 pm cryostat-cut section of adult rat kidney was included as a positive control and as a means of comparing the intensity of reaction of those parenchymal kidney cells which do react positively for GGT (proximal tubules). Negative controls consisted of cells incubated in reaction mixture minus gammaglutamyl-4-methoxy-2-naphthylamide (Vega-Fox, Tucson\. ----I5’-Nucleotidase activity was demonstrated histochemically by the technique of Wachstein & Meisel [3], without counterstaining. Cells were fired and stored as described for GGT assay. For histochemical study, reaction mixture [3] was filtered, 10 ml put into each dish and incubated at 37°C for lg h. A 10 pm cryostat-cut section of adult rat kidney was included as a positive control. Negative controls consisted of cells incubated in reaction mixture minus adenosine5’-monophosphate (Sigma, St Louis).
Electron
microscopy
Cells grown in 60 mm tissue culture dishes (Lux) were washed once in 0.1 M cacodylate buffer, pH 7.5, and fixed in cacodylate-buffered 3 % glutaraldehyde. Dishes were post-fixed and stained by 2% osmium tetroxide for 2 h. The cell monolayers were embedded in Epon 812. After embedding, the monolayers were separated from the plastic dishes, and sections were Exp Cell Res 121 (1979)
. I
, 2
, 3
. 0
. 5
, 6
, 7
. 0
. 9
, lo
, II
* 12
Fig. 1. Abscissa: time in primary culture (days); ordinare: no. of cells per dish. ‘I, 10 mM Ammonium 10 mMacetate ammonium chloride; l , water; oacetate. 1. mMn iodium 9 cut, placed on grids, and counterstained with uranyl acetate and lead citrate. The cells were viewed in a Hitachi HUl2A electron microscope.
RESULTS The plated suspension of cells dissociated from the adult kidney represented a mixture of cell clumps of varying sizes, blood cells, a&
debris.
HoWeVer,
one
day
following
plating and washing (3 washes 3 h after plating,
2 WaghOg
the following
morning),
the
dishes ah contained a randomly distributed monolayer of rather uniform-looking epithelial-like cells in small clusters, usually less than ten cells in a cluster. Each dish contained an average of 105 cells (data pooled from three plating experiments), indicating a total yield of 2.5 million cells distributed in 25 dishes the morning following initial plating. Population growth was rapid
Ammonia
eflects on primary
kidney cultures
49
for several days followed by a sharp reduction in cell number (fig. 1). Initially, only epithelial-like cells were seen, but as the epithelial-like population declined, a population of cells with tibroblast-like morphology began to grown and eventually repopulated the dish. The tibroblast-like population could be subcultured for at least 6 months. Addition of 10 mM ammonium acetate to the culture medium reduced the decline in total cell population seen in the first 10 days of culture (fig. 1). Ammonium chloride at 10 mM concentration produced little quantitative change in monolayer growth compared with control dishes. Sodium acetate at 10 mM concentration had no effect compared Fig. 2. Abscissa: time in primary culture (days); ordiwith control dishes (fig. 1). In another set of nate: no. of epithelial-like or fibroblast-like cells per experiments we compared the growth effect dish. V, Epithelial-like cells, 10 mM ammonium acetate; 0, epithefial-like cells, control; V, tibroblast-like of several concentrations of ammonium cells, 10 mM ammonium acetate; 0, fibroblast-like acetate (2.5 mM, 10 mM, 25 mM and 50 cells, control. mM). Ten mM ammonium acetate was optimal for epithelial-like cell growth, and 50 mM ammonium acetate was highly toxic. overgrowth of fibroblast-like cells. Dishes When counted cells were classified as supplemented with 10 mM ammonium aceeither epithelial-like or fibroblast-like, it tate showed very little growth of fibroblastwas observed that 10 mM ammonium ace- like cells compared with control dishes (fig. tate greatly reduced the first week’s de- 2). The ability of ammonium acetate to supcrease in epithelial-like cell population compress tibroblast-like cell growth while enpared with control primary cultures not hancing the survival of epithelial-like cells supplemented with ammonium acetate (fig, was not dependent on the presence of se2). A more or less stable population of epi- rum. Ten mM ammonium acetate added to thelial-like cells emerged in the ammonium dishes supplemented with either O%, 1 %, acetate-treated dishes which persisted for or 10% fetal bovine serum produced enabout 3 weeks and then gradually died out. hanced epithelial-like cell survival and supIn the control dishes not supplemented with pressed tibroblast-like cell growth in all ammonium acetate, a rapidly growing sub- cases. population of cells with a fibroblast-like Epithelial-like cells grew in well-defined morphology emerged after 1 or 2 weeks and nests in tight arrangements of polygonal replaced the epithelial-like cells (fig. 2). It cells (fig. 3). Electron microscopic examishould be noted that the population of epi- nation of one week primary cultures demthelial-like cells died out prior to a large in- onstrated cells with prominent brush borcrease in fibroblast-like cells. Hence, the der, delicate microvilli and numerous miloss of epithelial cells was not the result of tochondria (fig. 4). Addition of ammonium Exp Cell
Res 121 (1979)
50
Berman et al.
Fig. 3. Phase contrast photomicrograph of 2-week-old primary epithelial-like cells in a IO mM ammonium acetate supplemented culture. x 130. Fig. 4. Electron micrograph. Primary culture (1 week) of 10 mM ammonium acetate supplemented kidney cells. Numerous mitochondria, microvilli, and prominent basal lamina are evident. x6200.
Figs 5, 6. Electron micrographs. Kidney cells (3 weeks) supplemented with 10 mM ammonium acetate. (5) Cytoplasm full of autophagocytic structures. x2850. (6) Extracellular structures. x6500.
acetate to early primary cultures produced small cytoplasmic vacuoles visible by phase microscope. Unsupplemented cultures
filled increasingly with dark granules over a period of one week, then died. Ammonium acetate supplemented cultures be-
Exp Cd
Res I21 (1979)
Ammonia
effects on primary
kidney cultures
51
came filled with dark granules more gradually and died at the end of three weeks in culture. Granules could also be observed floating in the medium. Electron microscopic examination of cells at this stage demonstrated that autophagic structures filled the cell cytoplasm (fig. 5). Identical structures were observed extracellularly (fig. 6). After 1 week of 10 mM ammonium acetate supplementation nearly all cells were epithelial-like and demonstrated strong reactivity for GGT (fig. 7) and S-nucleotidase activities (fig. 8), with enzyme activities concentrated along the cell membranes (figs 7, 8). In adult rat kidney, GGT and 5’nucleotidase activities are greatest in the proximal tubules [2,3]. After 1 week cultures not supplemented with ammonium acetate, consisted almost exclusively of fibroblast-like cells. Fibroblast-like cells were positive for 5’-nucleotidase and GGT (fig. 9). However, there was no apparent concentration of GGT or 5’-nucleotidase activities to the plasma membranes.
knowledge, determine the yield of cells in primary culture, we cannot compare our technique (which produced approx. 2.5 million attached epithelial-like cells the morning after initial plating) with other reported techniques. The appearance of a growing population of epithelial-like cells which died out and was .replaced by a fibroblast-like population has been reported by others [7, 171. Seglen has reported that primary suspension cultures of rat hepatocytes are in negative nitrogen balance and exhibit a continuous net release of amino acids and/or urea. Ammonia exerted an inhibitory influence on protein degradation. Recent evidence has confirmed that 20 mM ammonium chloride was non-toxic and inhibited autophagyassociated proteolysis in cultured fibroblasts [18]. Seglen suggested that his observations might be of importance for the growth of cells in tissue culture [ 11. However, as hepatocytes in tissue culture do not divide, he could not study growth effects of ammonia. We have shown that ammonium acetate greatly enhances growth and survival of kidney epithelial-like cells in culture DISCUSSION and dramatically reduces the growth and Techniques for preparing primary cell cul- survival of kidney fibroblast-like cells in culture. In our system, ammonium acetate tures of adult kidney have been described by a number of investigators [4-173. For had a more pronounced effect on epithelialthe most part, kidney cells in culture are like cell growth than did ammonium chloused to propagate viruses, although other ride, for reasons we cannot explain. Howfields of study, including toxicity testing [9, ever, as sodium acetate had no effect, we 11, 121, kidney carcinogenesis [5-7, 14, 151, can infer that the ammonium ion was indeed and kidney metabolism [4], have employed necessary for the effect. It has been sugprimary kidney cell cultures. The technique gested that ammonia is a lysosomotropic here described for preparing primary kid- agent which accumulates in lysosomes and reduces their proteolytic activity [19, 201. ney cell cultures differs from previously aprotinin (trasylol), an inhibitor published techniques primarily in our use of Similarly, enzymes, ina high concentration of trypsin (2.25 %) for of a number of proteolytic a short time (40 min) to prepare a cell sus- creases the survival time of rat kidney in organ culture [21]. This fits nicely with our pension consisting essentially of the entire kidney mass. As no other reports, to our observation that the primary kidney epiExp Cell
Res I21 (1979)
52
Berman
et al.
7. 8. Enzyme histochemistrv of adult rat kidney primary cultures grown in 10 mti ammonium acetate demonstrating epithelial morphology of cells and high enzyme reactivities apparently concentrated on the cell membranes. (7) Gamma glutamy transpeptidase, 14-day primary. x 100. (8) 5’-Nucleotidase, 9-day primary. X240. Fig. 9. Enzyme histochemistry of adult rat kidney primary culture grown without ammonium acetate supplementation demonstrating tibroblast motphology of ceils and high enzyme reactivity distributed throughout the cells. Gamma glutamyl knspeptidase, M-day primary. X 240.
Fins
thelial cells, when near death, were filled with autophagosomes. Ammonium chloride added to primary liver celi cultures is also Exp Cell Res IZI(1979J
known to increase a number of urea cycle enzymes [22]; it is therefore possible that part of the effect of ammonia on primary
Ammonia
kidney cell culture may be mediated through enzyme induction. It is extremely difficult to determine the tissue cell-type from which cells in culture arise. The epithelial-like cells we have observed in primary cell culture are strongly positive for GGT and 5’nucleotidase activities. Among the epithelial cells in kidney, the proximal tubule cells react most strongly for these two enzyme activities, suggesting a tubular origin for the epitheliallike cells observed in culture. In normal kidney 5’nucleotidase is found almost exclusively on the plasma membrane, and GGT activity is greatest on the plasma membranes. A similar distribution of enzyme activities was observed in the epithelial-like cells in culture. Fibroblast-like cells in primary kidney culture also react positively for GGT and 5’-nucleotidase activities, but with enzyme activities not concentrated on the plasma membranes. GGT activity and 5’-nucleotidase activities are low in connective tissue cells of the kidney. The absence of cells with fibroblast-like morphology in the initial population of plated kidney cells and the occasional observation of transition forms between epithelial-like and fibroblast-like cells all suggest that the fibroblast-like cell is a variant of the epithelial-like cells, with growth suppressible by ammonium acetate. The fibroblast-like cells have been described by Hard and coworkers [7]. It is important to remember that kidney epithelium derives from embryonic mesoderm and that under certain conditions epithelial tissue of kidney can revert to a mesenchymal morphology, as seen in the fibrosarcomatoid form of renal adenocarcinoma [23]. The enhancement by ammonium acetate of growth and survival of kidney epitheliallike cells and the suppression of growth of fibroblast-like cells in primary cultures of
effects on primary
kidney cultures
53
kidney is important for several reasons. First, it provides a system wherein the mechanism of action of ammonia on growing cells can be analyzed. Secondly, it provides long-lasting primary cultures of kidney epithelial-like tissue for use in virus propagation, and toxicitylcarcinogenicity studies on epithelial-like kidney cells. Thirdly, since the epithelial-like cells react strongly for GGT, an enzyme involved in amino acid transport in kidney [24], it is now possible to study amino acid transport in a population of morphologically epithelial cells derived from kidney. Finally, the epithelial-like cells in culture eventually die with their cytoplasm tilled with autophagic lysosomes, a process retarded but not prevented by the addition of ammonium acetate to the culture media. Death of cells under conditions of increasing accumulation of autophagic lysosomes is referred to as apoptosis [25], a form of cell death distinguishable from the coagulative type of necrosis encountered most frequently when cells are destroyed in an overtly lethal environment. Apoptosis and coagulative necrosis as they occur in vivo have been discussed [26]. Interestingly, adult rat hepatocytes in primary monolayer culture, after 34 days incubation, become tilled with autophagosomes and shortly thereafter die [27]. The present primary kidney culture system affords the study of apoptosis in a nearly homogeneous population of epithelial-like kidney cells. We are grateful to Dr Jerry Rice for helpful suggestions and for critical reading of the manuscrid, to Mr Ron Newkirk for excellen? preparation of thi electron microscopic material and to Mrs Maxine Bellman for typing the manuscript.
REFERENCES 1. Seglen, P 0, Exp cell res 107 (1977) 207. 2. Rutenberg, A M, Kim, H, Fischbein, J W, Hanker, J C, Wasserkrug, H L & Seligman, A M, J histochem cytochem 17 (1%9) 517. Exp Cell
Res I21 (1979)
54
Berman et al.
3. Wachstein, M & Meisel, E, Am -i clin .path01 27 (1957) 13. 4. Gonzalez, R, Dempsey, M E, Elliott, A Y & Fralev. E E. EXD cell res 87 (1974) 152. 5. Williams, R’ D, &Elliott, A Y; Stein, N & Fraley, E E, In vitro 12 (1976) 623. 6. Stewart, B W & Hard, G C, J natl cancer inst 58 (1977) 1615. 7. Hard, G C, Borland, R&Butler, W H, Experientia 27 (1971) 1208. 8. Sigel, M M, Scotti, T M, Wryk, M A & Dorsey, M, Nature 182 (1958) 1034. 9. Hutton, J C, Schofield, P J, Williams, J F & Hollows, F C, Australian j biol sci 28 (1975) 109. 10. Louis, C J & White, J, Lab invest 9 (1960) 273. 11. Inamoto, H, Ino, Y, Inamoto, M, Wada, T, Kihara, H, Watanabe, I & Asano, S, Lab invest 34 (1976) 489. 12. Walton, J R & Buckley, I K, Agents and actions 5 ( 1975) 69. 13. bye, ‘F J, Oncology 24 (1970) 137. 14. Ochiai, M, Japanese j urol assoc 62 (1971) 1. 15. Mason, D H & Takemoto, K K, Int j cancer 19 (1977) 391. 16. Kozuch, 0 & Mayer, V, Acta virol19 (1975) 498. 17. Malinin, T I, Clatlin, A J&Block, N L, In vitro 13 (1977) 144.
Exp Ceil
Res 121 (1979)
18. Amenta, J S, Hlivko, T J, McBee, A G, Shinozuka, H & Brochner, S, Exp cell res 115 (1978) 357. 19. Seglen, P 0 & Reith, A, Exp cell res 100 (1976) 276. 20. Glimelius, B, Westermark, B & Wasteson, A, Exp cell res 108 (1977) 23. 21. Davis, H, Gascho, C & Kiernan, J A, In vitro 12 (1976) 192. 22. Gebhardt, R, Bellemann, P & Mecke, D, Exp cell res 112 (1978) 431. 23. Bennington, J L & Beckwith, J B, Tumors of the kidneyrrenal pelvis and ureter, fascicle 12, p. 145. Armed Forces Institute of Pathology, -. Washington (1975). 24. Meister, A, Science 180 (1973) 33. 25. Kerr, J F R, Wyllie, A H & Currie, A R, Brit j cancer 26 (1972) 239. 26. Wyllie, A H, J clin path01 27 suppl. (1974) 35. 27. Bemaert, D, Wanson, J, Drochmans, P & Popowski, A, J cell biol74 (1977) 878.
Received June 30, 1978 Revised version received January 2, 1979 Accepted January 4, 1979
Experimental
PRIMARY RAT
Cell Research
EPITHELIAL
KIDNEY,
CELL
ENHANCEMENT BY AMMONIUM
JULES BERMAN,
121 (1979) 47-54
CULTURE
OF ADULT
OF CELL
GROWTH
ACETATE
ALAN PERANTONI, HESTER and ELIZABETH KINGSBURY’
MARIE
JACKSON
Experimental Pathology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014 and ‘Litton Bionetics, Kensington, MD 20795, USA
SUMMARY Primary monolayer cultures of epithelial-like cells from adult Lewis rat kidney demonstrated enhanced growth and survival over a 3-week period when cultured in medium supplemented with 10 mM ammonium acetate. In addition to enhancing the growth and survival of epithelial-like cells, 10 mM ammonium acetate also suppressed the growth of a subpopulation of cells having a flbroblast-like morphology. Both epithelial-like and tibroblast-like cells reacted positively in histochemical reactions for gamma glutamyl transpeptidase (GGT) and 5’-nucleotidase, enzymes found in high amounts in kidney tubular epithelium. Despite the enhanced survival of cells in the presence of ammonium acetate, most cells in primary culture eventually died out. Electron microscopic examination of dying epithelial-like cells revealed the presence of numerous large lysosomes filled with amorphous electron-dense material. These findings substantiate the prediction of Seglen (Exp cell res 107 (1977) 207) that ammonia might cause increased growth of primary cells in culture, presumably through its nitrogen-preserving action.
In a recent report, Seglen demonstrated that 10 mM ammonium chloride signiticantly inhibited protein degradation in suspension culture of isolated adult rat hepatocytes [l]. As rat hepatocytes in primary cell culture do not divide, Seglen could not, in his system, observe the growth effects of ammonia. We have developed a technique for establishing primary monolayer cultures of adult rat kidney. These cells have an epithelial morphology as judged by light and electron microscopic examination and divide rapidly for a period of about one week, at which time growth of epithelial-like cells ceases. Concurrently a variant population of cells with fibroblast-like morphology grows and can be subcultured for at least 6 months. We have examined the effect of ammonium 4-791808
acetate on primary monolayer cultures of kidney to determine whether ammonia has a growth enhancing effect on a dividing population of epithelial cells. MATERIALS
AND
METHODS
Kidneys from adult male Lewis rats (LEW/mai, Microbiological Associates, Bethesda, average age 12 weeks) were minced, then shaken for 10 min in Dulbecco’s salts solution containing 0.02% EDTA and no calcium or maanesium. Tissue was then washed and incubated with a solution of 2.25 % trypsin (Flow Laboratories, Rockville. MD) in salts solution containing 0.02% EDTA in a 37 degree shaker for 40 min. At this time, minced tissue was visibly blanched but not dissociated. Trypsin solution was then aspirated leaving behind most tissue fragments. Fragments were then pipetted repeatedly (about a dozen times) with a plastic 10 ml pipet (pipet tip snapped off for wider bore) until tissue fragments dissociated into a cell suspension. The suspension was then transferred to 250 ml of Williams Medium E (WE. Flow Laboratories) supplemented with 2 mM glutamine and 10% fetal bovine serum (Flow Laboratories), and 50 pg/ml Exp Cell
Res 121 (1979)
48
Berman et al.
gentamycin (Microbiological Associates). This suspension was divided into 25 dishes (0 100 mm, Corning). Three hours after plating, dishes were washed twice and refed with 10 ml supplemented WE. Dishes were incubated overnight at which time treatments were begun and cell counts taken. Ammonium acetate, ammonium chloride, and sodium acetate (Fisher Scientific) were dissolved in water at concentrations such that no more than 0.1 ml of solution was ever added to a dish. Media pH (7.4) was not altered by these additions. Solutions were passed through Nalge filters and stored as refrigerated stock solutions. All dishes were refed every 2 days with supplemented WE and varying concentrations of test solution. Cell counts were done by averaging the number of cells in five chosen microscopic fields delineated by a reticle housed in the eyepiece of a phase contrast microscope. Every experimental cell count was determined from the average cell counts of triplicate or quadruplicate dishes. In one experiment, cells were classified cytologically as epithelial-like (having a polygonal structure and growing in well-defined nests) and tibroblast-like (spindled and stellate shapes varying greatly in cell size and having little or no contact with other cells in areas of low cell density, but aligning in parallel arrays in areas of high cell density).
0. 0
Enzyme histochemistry Gamma glutamyl transpeptidase (GGT) activity was demonstrated by the method of Rutenberg [2], without counterstainina. Dishes of cells were fixed by quick rinse with 10 ml of methylbutane (Fisher) ai -2O’C and then stored at -70°C. For assay, 10 ml of reaction mixture [2] were put into each dish and incubated 20 min at 37°C. A 10 pm cryostat-cut section of adult rat kidney was included as a positive control and as a means of comparing the intensity of reaction of those parenchymal kidney cells which do react positively for GGT (proximal tubules). Negative controls consisted of cells incubated in reaction mixture minus gammaglutamyl-4-methoxy-2-naphthylamide (Vega-Fox, Tucson\. ----I5’-Nucleotidase activity was demonstrated histochemically by the technique of Wachstein & Meisel [3], without counterstaining. Cells were fired and stored as described for GGT assay. For histochemical study, reaction mixture [3] was filtered, 10 ml put into each dish and incubated at 37°C for lg h. A 10 pm cryostat-cut section of adult rat kidney was included as a positive control. Negative controls consisted of cells incubated in reaction mixture minus adenosine5’-monophosphate (Sigma, St Louis).
Electron
microscopy
Cells grown in 60 mm tissue culture dishes (Lux) were washed once in 0.1 M cacodylate buffer, pH 7.5, and fixed in cacodylate-buffered 3 % glutaraldehyde. Dishes were post-fixed and stained by 2% osmium tetroxide for 2 h. The cell monolayers were embedded in Epon 812. After embedding, the monolayers were separated from the plastic dishes, and sections were Exp Cell Res 121 (1979)
. I
, 2
, 3
. 0
. 5
, 6
, 7
. 0
. 9
, lo
, II
* 12
Fig. 1. Abscissa: time in primary culture (days); ordinare: no. of cells per dish. ‘I, 10 mM Ammonium 10 mMacetate ammonium chloride; l , water; oacetate. 1. mMn iodium 9 cut, placed on grids, and counterstained with uranyl acetate and lead citrate. The cells were viewed in a Hitachi HUl2A electron microscope.
RESULTS The plated suspension of cells dissociated from the adult kidney represented a mixture of cell clumps of varying sizes, blood cells, a&
debris.
HoWeVer,
one
day
following
plating and washing (3 washes 3 h after plating,
2 WaghOg
the following
morning),
the
dishes ah contained a randomly distributed monolayer of rather uniform-looking epithelial-like cells in small clusters, usually less than ten cells in a cluster. Each dish contained an average of 105 cells (data pooled from three plating experiments), indicating a total yield of 2.5 million cells distributed in 25 dishes the morning following initial plating. Population growth was rapid
Ammonia
eflects on primary
kidney cultures
49
for several days followed by a sharp reduction in cell number (fig. 1). Initially, only epithelial-like cells were seen, but as the epithelial-like population declined, a population of cells with tibroblast-like morphology began to grown and eventually repopulated the dish. The tibroblast-like population could be subcultured for at least 6 months. Addition of 10 mM ammonium acetate to the culture medium reduced the decline in total cell population seen in the first 10 days of culture (fig. 1). Ammonium chloride at 10 mM concentration produced little quantitative change in monolayer growth compared with control dishes. Sodium acetate at 10 mM concentration had no effect compared Fig. 2. Abscissa: time in primary culture (days); ordiwith control dishes (fig. 1). In another set of nate: no. of epithelial-like or fibroblast-like cells per experiments we compared the growth effect dish. V, Epithelial-like cells, 10 mM ammonium acetate; 0, epithefial-like cells, control; V, tibroblast-like of several concentrations of ammonium cells, 10 mM ammonium acetate; 0, fibroblast-like acetate (2.5 mM, 10 mM, 25 mM and 50 cells, control. mM). Ten mM ammonium acetate was optimal for epithelial-like cell growth, and 50 mM ammonium acetate was highly toxic. overgrowth of fibroblast-like cells. Dishes When counted cells were classified as supplemented with 10 mM ammonium aceeither epithelial-like or fibroblast-like, it tate showed very little growth of fibroblastwas observed that 10 mM ammonium ace- like cells compared with control dishes (fig. tate greatly reduced the first week’s de- 2). The ability of ammonium acetate to supcrease in epithelial-like cell population compress tibroblast-like cell growth while enpared with control primary cultures not hancing the survival of epithelial-like cells supplemented with ammonium acetate (fig, was not dependent on the presence of se2). A more or less stable population of epi- rum. Ten mM ammonium acetate added to thelial-like cells emerged in the ammonium dishes supplemented with either O%, 1 %, acetate-treated dishes which persisted for or 10% fetal bovine serum produced enabout 3 weeks and then gradually died out. hanced epithelial-like cell survival and supIn the control dishes not supplemented with pressed tibroblast-like cell growth in all ammonium acetate, a rapidly growing sub- cases. population of cells with a fibroblast-like Epithelial-like cells grew in well-defined morphology emerged after 1 or 2 weeks and nests in tight arrangements of polygonal replaced the epithelial-like cells (fig. 2). It cells (fig. 3). Electron microscopic examishould be noted that the population of epi- nation of one week primary cultures demthelial-like cells died out prior to a large in- onstrated cells with prominent brush borcrease in fibroblast-like cells. Hence, the der, delicate microvilli and numerous miloss of epithelial cells was not the result of tochondria (fig. 4). Addition of ammonium Exp Cell
Res 121 (1979)
50
Berman et al.
Fig. 3. Phase contrast photomicrograph of 2-week-old primary epithelial-like cells in a IO mM ammonium acetate supplemented culture. x 130. Fig. 4. Electron micrograph. Primary culture (1 week) of 10 mM ammonium acetate supplemented kidney cells. Numerous mitochondria, microvilli, and prominent basal lamina are evident. x6200.
Figs 5, 6. Electron micrographs. Kidney cells (3 weeks) supplemented with 10 mM ammonium acetate. (5) Cytoplasm full of autophagocytic structures. x2850. (6) Extracellular structures. x6500.
acetate to early primary cultures produced small cytoplasmic vacuoles visible by phase microscope. Unsupplemented cultures
filled increasingly with dark granules over a period of one week, then died. Ammonium acetate supplemented cultures be-
Exp Cd
Res I21 (1979)
Ammonia
effects on primary
kidney cultures
51
came filled with dark granules more gradually and died at the end of three weeks in culture. Granules could also be observed floating in the medium. Electron microscopic examination of cells at this stage demonstrated that autophagic structures filled the cell cytoplasm (fig. 5). Identical structures were observed extracellularly (fig. 6). After 1 week of 10 mM ammonium acetate supplementation nearly all cells were epithelial-like and demonstrated strong reactivity for GGT (fig. 7) and S-nucleotidase activities (fig. 8), with enzyme activities concentrated along the cell membranes (figs 7, 8). In adult rat kidney, GGT and 5’nucleotidase activities are greatest in the proximal tubules [2,3]. After 1 week cultures not supplemented with ammonium acetate, consisted almost exclusively of fibroblast-like cells. Fibroblast-like cells were positive for 5’-nucleotidase and GGT (fig. 9). However, there was no apparent concentration of GGT or 5’-nucleotidase activities to the plasma membranes.
knowledge, determine the yield of cells in primary culture, we cannot compare our technique (which produced approx. 2.5 million attached epithelial-like cells the morning after initial plating) with other reported techniques. The appearance of a growing population of epithelial-like cells which died out and was .replaced by a fibroblast-like population has been reported by others [7, 171. Seglen has reported that primary suspension cultures of rat hepatocytes are in negative nitrogen balance and exhibit a continuous net release of amino acids and/or urea. Ammonia exerted an inhibitory influence on protein degradation. Recent evidence has confirmed that 20 mM ammonium chloride was non-toxic and inhibited autophagyassociated proteolysis in cultured fibroblasts [18]. Seglen suggested that his observations might be of importance for the growth of cells in tissue culture [ 11. However, as hepatocytes in tissue culture do not divide, he could not study growth effects of ammonia. We have shown that ammonium acetate greatly enhances growth and survival of kidney epithelial-like cells in culture DISCUSSION and dramatically reduces the growth and Techniques for preparing primary cell cul- survival of kidney fibroblast-like cells in culture. In our system, ammonium acetate tures of adult kidney have been described by a number of investigators [4-173. For had a more pronounced effect on epithelialthe most part, kidney cells in culture are like cell growth than did ammonium chloused to propagate viruses, although other ride, for reasons we cannot explain. Howfields of study, including toxicity testing [9, ever, as sodium acetate had no effect, we 11, 121, kidney carcinogenesis [5-7, 14, 151, can infer that the ammonium ion was indeed and kidney metabolism [4], have employed necessary for the effect. It has been sugprimary kidney cell cultures. The technique gested that ammonia is a lysosomotropic here described for preparing primary kid- agent which accumulates in lysosomes and reduces their proteolytic activity [19, 201. ney cell cultures differs from previously aprotinin (trasylol), an inhibitor published techniques primarily in our use of Similarly, enzymes, ina high concentration of trypsin (2.25 %) for of a number of proteolytic a short time (40 min) to prepare a cell sus- creases the survival time of rat kidney in organ culture [21]. This fits nicely with our pension consisting essentially of the entire kidney mass. As no other reports, to our observation that the primary kidney epiExp Cell
Res I21 (1979)
52
Berman
et al.
7. 8. Enzyme histochemistrv of adult rat kidney primary cultures grown in 10 mti ammonium acetate demonstrating epithelial morphology of cells and high enzyme reactivities apparently concentrated on the cell membranes. (7) Gamma glutamy transpeptidase, 14-day primary. x 100. (8) 5’-Nucleotidase, 9-day primary. X240. Fig. 9. Enzyme histochemistry of adult rat kidney primary culture grown without ammonium acetate supplementation demonstrating tibroblast motphology of ceils and high enzyme reactivity distributed throughout the cells. Gamma glutamyl knspeptidase, M-day primary. X 240.
Fins
thelial cells, when near death, were filled with autophagosomes. Ammonium chloride added to primary liver celi cultures is also Exp Cell Res IZI(1979J
known to increase a number of urea cycle enzymes [22]; it is therefore possible that part of the effect of ammonia on primary
Ammonia
kidney cell culture may be mediated through enzyme induction. It is extremely difficult to determine the tissue cell-type from which cells in culture arise. The epithelial-like cells we have observed in primary cell culture are strongly positive for GGT and 5’nucleotidase activities. Among the epithelial cells in kidney, the proximal tubule cells react most strongly for these two enzyme activities, suggesting a tubular origin for the epitheliallike cells observed in culture. In normal kidney 5’nucleotidase is found almost exclusively on the plasma membrane, and GGT activity is greatest on the plasma membranes. A similar distribution of enzyme activities was observed in the epithelial-like cells in culture. Fibroblast-like cells in primary kidney culture also react positively for GGT and 5’-nucleotidase activities, but with enzyme activities not concentrated on the plasma membranes. GGT activity and 5’-nucleotidase activities are low in connective tissue cells of the kidney. The absence of cells with fibroblast-like morphology in the initial population of plated kidney cells and the occasional observation of transition forms between epithelial-like and fibroblast-like cells all suggest that the fibroblast-like cell is a variant of the epithelial-like cells, with growth suppressible by ammonium acetate. The fibroblast-like cells have been described by Hard and coworkers [7]. It is important to remember that kidney epithelium derives from embryonic mesoderm and that under certain conditions epithelial tissue of kidney can revert to a mesenchymal morphology, as seen in the fibrosarcomatoid form of renal adenocarcinoma [23]. The enhancement by ammonium acetate of growth and survival of kidney epitheliallike cells and the suppression of growth of fibroblast-like cells in primary cultures of
effects on primary
kidney cultures
53
kidney is important for several reasons. First, it provides a system wherein the mechanism of action of ammonia on growing cells can be analyzed. Secondly, it provides long-lasting primary cultures of kidney epithelial-like tissue for use in virus propagation, and toxicitylcarcinogenicity studies on epithelial-like kidney cells. Thirdly, since the epithelial-like cells react strongly for GGT, an enzyme involved in amino acid transport in kidney [24], it is now possible to study amino acid transport in a population of morphologically epithelial cells derived from kidney. Finally, the epithelial-like cells in culture eventually die with their cytoplasm tilled with autophagic lysosomes, a process retarded but not prevented by the addition of ammonium acetate to the culture media. Death of cells under conditions of increasing accumulation of autophagic lysosomes is referred to as apoptosis [25], a form of cell death distinguishable from the coagulative type of necrosis encountered most frequently when cells are destroyed in an overtly lethal environment. Apoptosis and coagulative necrosis as they occur in vivo have been discussed [26]. Interestingly, adult rat hepatocytes in primary monolayer culture, after 34 days incubation, become tilled with autophagosomes and shortly thereafter die [27]. The present primary kidney culture system affords the study of apoptosis in a nearly homogeneous population of epithelial-like kidney cells. We are grateful to Dr Jerry Rice for helpful suggestions and for critical reading of the manuscrid, to Mr Ron Newkirk for excellen? preparation of thi electron microscopic material and to Mrs Maxine Bellman for typing the manuscript.
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3. Wachstein, M & Meisel, E, Am -i clin .path01 27 (1957) 13. 4. Gonzalez, R, Dempsey, M E, Elliott, A Y & Fralev. E E. EXD cell res 87 (1974) 152. 5. Williams, R’ D, &Elliott, A Y; Stein, N & Fraley, E E, In vitro 12 (1976) 623. 6. Stewart, B W & Hard, G C, J natl cancer inst 58 (1977) 1615. 7. Hard, G C, Borland, R&Butler, W H, Experientia 27 (1971) 1208. 8. Sigel, M M, Scotti, T M, Wryk, M A & Dorsey, M, Nature 182 (1958) 1034. 9. Hutton, J C, Schofield, P J, Williams, J F & Hollows, F C, Australian j biol sci 28 (1975) 109. 10. Louis, C J & White, J, Lab invest 9 (1960) 273. 11. Inamoto, H, Ino, Y, Inamoto, M, Wada, T, Kihara, H, Watanabe, I & Asano, S, Lab invest 34 (1976) 489. 12. Walton, J R & Buckley, I K, Agents and actions 5 ( 1975) 69. 13. bye, ‘F J, Oncology 24 (1970) 137. 14. Ochiai, M, Japanese j urol assoc 62 (1971) 1. 15. Mason, D H & Takemoto, K K, Int j cancer 19 (1977) 391. 16. Kozuch, 0 & Mayer, V, Acta virol19 (1975) 498. 17. Malinin, T I, Clatlin, A J&Block, N L, In vitro 13 (1977) 144.
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18. Amenta, J S, Hlivko, T J, McBee, A G, Shinozuka, H & Brochner, S, Exp cell res 115 (1978) 357. 19. Seglen, P 0 & Reith, A, Exp cell res 100 (1976) 276. 20. Glimelius, B, Westermark, B & Wasteson, A, Exp cell res 108 (1977) 23. 21. Davis, H, Gascho, C & Kiernan, J A, In vitro 12 (1976) 192. 22. Gebhardt, R, Bellemann, P & Mecke, D, Exp cell res 112 (1978) 431. 23. Bennington, J L & Beckwith, J B, Tumors of the kidneyrrenal pelvis and ureter, fascicle 12, p. 145. Armed Forces Institute of Pathology, -. Washington (1975). 24. Meister, A, Science 180 (1973) 33. 25. Kerr, J F R, Wyllie, A H & Currie, A R, Brit j cancer 26 (1972) 239. 26. Wyllie, A H, J clin path01 27 suppl. (1974) 35. 27. Bemaert, D, Wanson, J, Drochmans, P & Popowski, A, J cell biol74 (1977) 878.
Received June 30, 1978 Revised version received January 2, 1979 Accepted January 4, 1979
