Virchows Arch [Pathol Anat] (1986) 51:161-176

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9 Springer-Verlag 1986

Kidney glomerular explants in serum-free media: role of individual medium components in cell outgrowth* Terry D. Oberley 1'2, Bruce W. Steinert 2, and Paul J. Anderson 1 1 Pathology Service, William S. Middleton Memorial Veterans Hospital 2500 Overlook Terrace, Madison, WI 53705, USA 2 Department of Pathology, University of Wisconsin Medical School 600 Highland Avenue, Madison, WI 53792, USA

Summary. Guinea pig glomeruli were grown for 22 days in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, and antibiotics. To this basic medium was added insulin, transferrin, selenium (Se), tri-iodothyronine, or fibronectin (FN) - either singly, or in various combinations - and sequential quantitative studies of the glomerular outgrowths were performed. Total cells in glomerular outgrowths, mitotic index, and glomerular attachment rate were determined and compared with values for glomerular outgrowths in media containing either no additions or all of the above components. FN was required for whole glomerular attachment, while transferrin plus FN was required for mitosis in glomerular cell outgrowths. Insulin and tri-iodothyronine slightly increased glomerular cell outgrowth by slightly increasing whole glomerular attachment, but had little effect on mitosis in glomerular outgrowths. The effect of Se was complex. Se did not affect whole glomerular attachment or mitosis in the presence of transferrin plus FN. However, in a medium containing transferrin, FN, and 3-amino-l,2,4-triazole (AT) (an inhibitor of catalase and glutathione peroxidase), Se increased total cell number but had little effect on the glomerular attachment rate or the mitotic index. Morphologic analysis of glomeruli early in culture suggested that Se may act by decreasing the amount of or delaying the time of cell death. In all of the media tested, total DNA was relatively constant over the course of 22 days, suggesting the possibility that glomerular cells cultured in a serum-free medium are part of a cell renewal system. Offprint requests to: T.D. Oberley, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, WI 53705, USA * Supported by grants to T.D.O. from the University of Wisconsin Graduate School and the Veterans Administration. B.W.S. was a predoctoral fellow supported by NIH training grant 5-T32-ESO715

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Key words: Glomeruli - Transferrin - Fibronectin - Selenium - Culture

Introduction

Isolated kidney glomeruli from many species, placed under standard in vitro culture conditions in the presence of serum (Norgaard 1983; Kreisberg and Karnovsky 1983) or in certain chemically defined serum-free media containing fibronectin (FN) (Oberley et al. 1982, 1983; Oberley and Steinert 1983), will attach to a culture substrate, and an outgrowth of glomerular cells then appears. The mechanism by the which this outgrowth occurs is poorly understood. Our laboratory has begun a series of studies on glomerular cell outgrowth in serum-free media, since the outgrowth of cells under these conditions can be carefully controlled. Previous studies demonstrated the usefulness of this system by proving that FN was required for whole glomerular attachment (Oberley 1985). The present study examines the role of other media components in cell outgrowth. Such studies are difficult to perform in cells grown in serum because the serum contains unknown quantities of growth factors. Glomerular cell outgrowth in culture is an extremely complex process. Previous morphologic evidence suggested that the predominant cell type observed in glomerular cell outgrowths in serum-free media were visceral epithelial cells (Oberley et al. 1982, 1983). However, glomerular cells in vitro cannot be identified unequivocally until biochemical or histochemical cell markers become available. Further, the outgrowth of cells from glomeruli involves several processes, including whole glomerular adhesion, cell migration, mitosis, and cell death (Oberley 1985). Despite the complex nature of this process, the mechanism by which cells leave the explant and spread on the culture substrate is an interesting phenomenon which may provide intriguing insights into such processes as cell migration and the regulation of mitosis. The present study demonstrates that transferrin is required for mitosis in glomerular outgrowths and our results suggest that selenium (Se) may have a protective effect against cell death in vitro. Materials and methods Animals. Five-week-old randomly bred Hartley guinea pigs (300-400 g) of both sexes (O'Brien,

Oregon, WI) were maintained on laboratory chow and drinking water supplemented with ascorbic acid ad libitum. Glomerular isolation. Glomeruli were isolated as previously described, using a nylon screening technique (Oberley et al. 1979). Glomerular culture. Glomeruli were cultured in a basic medium of Waymouth's MB 752/1, sodium pyruvate (1%), nonessential amino acids (i %), and penicillin and streptomycin (100 units/ml) (all GIBCO). This basic medium was then supplemented with various combinations of FN (7.7 gg/ml, a gift from Dr. Deane Mosher, University of Wisconsin), insulin (5 ~tg/ml, Collaborative Research), transferrin (5 gg/ml, Collaborative Research), tri-iodothyronine (7 ng/ ml, Sigma Chemical), Se (5 ng/ml, Collaborative Research), and 3-amino-l,2,4-triazole (AT) (10-6 M, Sigma Chemical). The medium containing insulin, transferrin, selenium, tri-iodothyr-

Kidney glomerular explants: role of media components

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onine and fibronectin was designated as "complete" medium although this medium was not the optimal medium for glomerular outgrowth. Recent studies in our laboratory demonstrated that outgrowth is better in medium containing serum rather than in this "complete" medium. The present studies were performed in two-well tissue culture chamber dishes (Tissue-Tek, Miles) (4 cm2/well). Initially each well contained 2 ml of medium. Each day, 0.75 ml of the appropriate medium without FN was added. In a standard experiment, cells were fed supplements plus FN on day 0 and then fed supplements only from days 1 to 22. This protocol was adopted because FN can, under certain conditions, inhibit growth (Yamada and Kennedy 1984; Oberley, unpublished observations).

Quantitation ofglomerular cell outgrowth. Glomerular cell outgrowth, mitosis, and glomerular attachment were measured after Giemsa staining, as previously described (Oberley 1985; Oberley et al. 1985). Cell outgrowths were analyzed 7, 10, 14, 18 and 22 days after initiation of culture. Since it was impossible to pipet the same number of glomeruli into each tissue culture chamber, cell outgrowth is expressed as number of cells per initial glomerulus. For the same reason, whole glomerular attachment is expressed as the glomerular attachment rate (%), defined as the number of attached glomeruli divided by the number of initial glomeruli times 100. The mitotic index (%) is defined as the total number of mitoses divided by the total cells times 100. Mitoses were not counted in the parent explant, since its three-dimensional nature precluded visualization of the glomerular interior. Each experiment was performed in duplicate, and several experiments were performed in different media as described in the text. Means and standard errors were calculated for each medium and analyzed as described in the section on statistical analysis. It should be emphasized that glomeruli placed in serum-free media grow as colonies (Oberley et al. 1982). Further, only approximately 5% of attached glomeruli actually produce colonies (Oberley et al. 1983). Therefore, although the ratio of the number of cells in the outgrowths per initial glomerulus was quite low even at the time of maximal outgrowth (four cells per glomerulus on day 10 in "complete" medium), this number actually represents a substantial number of cells: a typical experiment resulted in 5,000 cells in glomerular outgrowths per Tissue-Tek chamber on day 14. Therefore, although the ratio of cells per initial glomerulus was quite low, individual colonies contained up to approximately 500 cells.

Morphologic analysis of unattached glomeruli in early culture. No significant glomerular attachment occurred before day 7. Unattached glomeruli from days 0 to 7 of culture were fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS), centrifuged to form a pellet, secondarily fixed in 1% osmium tetroxide in veronal buffer, dehydrated in an ethanol series, and embedded in Epon. Semithin sections (1 p.m thick) were cut and stained with toluidine blue. Glomerular cells were identified by their position with respect to the glomerular basement membrane: visceral epithelial cells were present external to the basement membrane, while endocapillary cells were either internal to the basement membrane (endothelial cells) or surrounded by mesangial matrix (mesangial cells). Cells were considered viable if their nuclei displayed dispersed heterochromatin and the cells were in their correct anatomic positions. Cells were considered necrotic if their nuclei were pyknotic (clumped heterochromatin) or if the cell had separated from its basement membrane so that it was no longer in the correct anatomic configuration (epithelial or endothelial cells). Cells from ten glomeruli from each of the media were counted using a Zeiss bright field microscope with a 100 • objective lens (oil immersion). Means and standard deviations were calculated and analyzed as described in the section on statistical analysis. Glomerular cells were scored by a pathologist (T.D.O) without knowledge of the medium or the age of the culture. Quantitation of total DNA. DNA was assayed fluorometrically (Downs and Wilfinger 1983) after sonication of tissue for 15 s on ice at 75% maximum power with a Biosonik sonicator (Bronwill Scientific, Rochester, NY) using a half-inch probe. We measured the binding of the fluorescent dye Hoechst 33258 to DNA. Total DNA (from attached and unattached glomeruli and cells) was measured. Each experiment was performed in triplicate and several experiments were performed in each medium as described in the text. Means and standard errors were calculated for each medium and analyzed statistically.

T.D. Oberley et al.

164

Statistical analys&. Significance of the differences was determined by analysis of variance (ANOVA) and Duncan's post hoc test at the p<0.05 Level (Duncan 1955). Most values were compared statistically to values for control tissue (day 0 at time of isolation) and various days of culture, and values from different media on the same day of culture were compared to each other. However, since we did not know the mitotic index on day 0, this variable was analyzed by comparing later days with day 7, the first day for which the mitotic index was determined. Only the most important statistically significant results are reported here.

Results

Quantitative studies on the role of transferrin in glomerular cell outgrowth Previous studies demonstrated that FN is required for whole glomerular attachment (Oberley 1985). Therefore, the present study tested individual components of the medium in combination with FN. This study and a preliminary study (Oberley 1985) indicated that either insulin or tri-iodothyronine in the presence of FN increased glomerular cell outgrowth only minimally by slightly increasing the glomerular adhesion rate without affecting the mitotic index. In contrast, transferrin in the presence of FN increased glomerular cell outgrowth. To verify this result, quantitative analyses were performed on seven experiments in which glomeruli were grown in the basic medium plus transferrin and FN. The results were compared to those from five experiments in which glomeruli were grown in basic medium plus FN alone. For further comparison we included two experiments in which glomeruli were grown in the basic medium and two in which insulin, transferrin, Se, tri-iodothyronine, and FN (designated as "complete" medium) were all present. In the following discussion, media will be named by their chemiFig. 1. Cells per initial glomerulus: role of transferrin. Results are expressed as mean___SEM. Seven experiments were performed in medium containing transferrin and FN, five in medium containing FN, two in basic medium, and two in complete medium. Each experiment consisted of duplicate measurements (two separate tissue culture wells were seeded with glomeruli and counted per experiment). Asterisks indicate a statistical value of p < 0.05 compared to day 0, when no cell outgrowths were present. Comparisons between media are described in the text for this and subsequent figs. t~--t~, complete medium; zx---~, transferrin and fibronectin; o---o, basic medium and fibronectin; x---x, basic medium alone

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cal additives (e.g., transferrin plus FN), but in all cases the components of the basic medium were also present. The results are shown in Figs. 1-3. The number of cells per initial glomerulus increased from days 7 to 14 in all media containing F N and then slowly declined during days 18 to 22. Figure 1 demonstrates that the number of cells per initial glomerulus was greatest in complete medium. However, there were more cells per initial glomerulus in medium containing transferrin and F N than in medium containing only FN. Very little cell outgrowth occurred when glomeruli were grown in basic medium without any supplements. In comparing results with different media on various days, the basic medium was found to have significantly fewer cells than the other three media on days 7, 10, 14, and 22. Most importantly, on day 14, medium containing F N had significantly fewer cells than medium containing transferrin plus F N or complete medium (which contained transferrin). The glomerular attachment rate increased from days 7 to 14 in all media containing F N and then slowly declined from days 18 to 22. Results for glomerular attachment rate are shown in Fig. 2. All media containing F N supported similar attachment rates, which were much greater than in the basic medium, in which very little whole glomerular attachment occurred. In comparison to day 0, when no glomerular attachment had yet occurred, statistical analysis showed significant attachment of whole glomeruli in all media except basic medium. We did not know the initial mitotic rate in the parent explant. Mitotic index was first measured on day 7, by which time enough glomerular attachment had occurred for meaningful data to be obtained, and statistical analyses compared subsequent days to day 7. In all media, the mitotic index was greatest on day 7 and then declined rapidly (Fig. 3). The mitotic index

T.D. Oberley et al.

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Fig. 6. Mitotic index: role of selenium. Results are expressed as mean 4-SEM. The experiments were the same as those in Fig. 1. Asterisks indicate a statistical value of p<0.05 compared to day 7. Comparison with day 0 was not possible because we could not determine the mitotic index of the parent explants

was significantly greater in medium containing transferrin plus F N than in basic medium or medium supplemented only with FN.

Quantitative studies on the role of selenium in glomerular cell outgrowth In preliminary experiments we could not demonstrate an effect o f Se on glomerular cell o u t g r o w t h in medium containing transferrin plus F N . Because Se is a cofactor for the enzyme glutathione peroxidase (Flohe et al. 1973), we decided to test the effect o f Se in the presence o f an inhibitor (AT) of the antioxidant enzymes catalase (Heim et al. 1955) and peroxidase ( D o n i and Piro 1983). T w o experiments were performed to compare cell outgrowths in basic medium containing (i) transferrin plus F N , (ii) transfer-

Fig. 7. Light microscopy of glomerulus early in culture. Glomeruli were embedded in Epon on various days after being placed in culture and then examined by light microscopy. Cells were classified as viable (V) or necrotic (N) and as epithelial (EP), mesangial (ME), or endothelial (EN); viable endothelial (V-EN), necrotic endothelial (N-EN), viable epithelial (V-EPI), and viable mesangial (V-ME) cells, x 1715. Nuclear morphology (dispersed vs clumped heterochromatin) was the chief distinguishing feature in separating viable from necrotic epithelial cells. Cells were examined with a 100 x objective lens; the present micrograph was photographed with a 25 x objective lens, x 1715

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Kidney glomerular explants: role of media components

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rin, FN, and AT, and (iii) transferrin, FN, AT, and Se. The results are shown in Figs. 4-6. Again, the number of cells per initial glomerulus increaed on days 7 to 14, then decreased slightly (Fig. 4). The number of cells per initial glomerulus was significantly greater in medium containing transferrin, FN, AT, and Se than in medium containing transferrin plus FN on days 10 and 14, while on day 14 the number of cells in medium containing transferrin, FN, AT, and Se was significantly greater than in media containing transferrin, AT, and FN. The glomerular attachment rate increased in these experiments on days 7 to 14, then decreased slightly (Fig. 5). There were no significant differences between media on any given day. Mitotic indices were similar in all three media (Fig. 6). Compared to day 7, the mitotic index was significantly lower on days 10, 14, 18, and 22 in all media, and there were no differences between media on any given day.

Morphologic analysis of unattached glomeruli in early culture Several studies have indicated that Se may protect against oxidant stress (Chance et al. 1969). To determine whether Se has a protective effect on

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Fig. t0. Morphologic analysis of unattached glomeruli early in culture: endothelial cells. Results are expressed as mean + SEM. The experiment is the same as that described in Fig. 8 and the glomeruli were processed similarly. Asterisks indicate a statistically significant value of p < 0.05 compared to day 0 (may be either higher or lower)

glomeruli in culture, we examined semithin sections of glomeruli cultured in various media over a period of several days. The media tested were basic medium supplemented with (i) FN, (ii) transferrin and FN, (iii) transferrin and FN plus AT, and (iv) transferrin, FN, AT, and Se. Viable and necrotic cells were counted and classified as visceral epithelial, mesangial, or endothelial (Fig. 7). Light microscopic examination of glomeruli in early culture often showed viable and necrotic cells adjacent to each other. Results of these studies are shown in Figs. 8-10. The number of viable visceral epithelial cells decreased with time in culture, while the number of necrotic cells remained relatively constant (Fig. 8). The number of viable mesangial cells decreased dramatically with time in culture, and the number of necrotic cells increased correspondingly (Fig. 9). The nurhber of viable endothelial cells actually increased with time, while the number:of necroic cells remained relatively constant (Fig. 10). There were several important significant differences between various media. However, we shall focus on the differences between media containing Se and the other media. On day 3, media containing FN, transferrin plus FN, or transferrin plus FN and AT all showed significantly fewer viable visceral epithelial cells than medium containing transferrin, FN, AT, and Se. On days 2 and 4 there were significantly more viable mesangial cells in medium containing transferrin, FN, AT, and Se than in the same medium without Se. Finally, on day 3 there were significantly more viable endothelial

Kidney glomerular explants: role of media components 22201816[q-

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Quantitation of total DNA Total D N A was constant in all media tested. Results for basic medium, basic medium plus FN, or basic medium plus transferrin and FN are shown in Fig. 11. There were no significant differences over time or between media. Similar results were obtained with media containing transferrin, FN, and AT or transferrin, FN, AT, and Se. Results for total D N A in complete medium have been previously published; total D N A slightly increased with time in culture (Oberley et al. 1985). Discussion This study provides preliminary results on the role of individual media components in glomerular cell outgrowth. While no previous studies have

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T.D. Oberleyet al.

been performed on the specific molecular requirements for cell migration from glomerular explants to the culture substrate, interesting studies have been performed with renal cortical explants. Minuth (1983) has demonstrated that rabbit renal cortical explants will migrate to a culture substrate in the presence of serum and form globular bodies surrounded by an epithelium of differentiated collecting duct cells. This study demonstrated that outgrowth was arrested by cytoskeletal blocking agents such as vinblastine, colchicine, and cytochalasin B. In contrast, inhibitors of DNA synthesis such as cytosine arabinoside, mitomycin, and hydroxyurea had no effect on cell outgrowth. These results suggest only a minor role for mitosis in cell outgrowth in this system. Consistent with this hypothesis is the lack of mitotic figures in the outgrowths (Minuth and Kriz 1982). In the glomerular system described here, mitotic activity plays a prominent role in cell outgrowth. We have conducted studies on glomerular explants in the presence of serum (Oberley et al. 1979, 1981) and have recently found quantitative differences between cell outgrowths from glomeruli grown in serum-free and serum-containing media, with serum-containing media showing few mitoses in cell outgrowths (unpublished observations). A future paper will deal with the mechanistic differences in glomerular cell outgrowth in these two media. A previous study (Oberley 1985) and this one document that FN is required for whole glomerular attachment. FN is a large glycoprotein found in loose mesenchyme, basement membranes, and blood (Ruoslahti et al. 1981). The effect of FN is quite complex, however, since it promotes both whole glomerular attachment and outgrowth of glomerular cells. Fibronectin has well known cell adhesive properties (Yamada and Olden 1978). That is, it promotes the attachment of cells to a substrate. However, it also promotes cell growth, including the growth of glial cells (Baron-Van Evercooren et al. 1982) and fibroblasts (Bitterman et al. 1983). Further, under certain conditions, FN can inhibit cell growth (Yamada and Kennedy 1984). The results of the present study with cultured adult kidney glomerular explants are in contrast to those of Thesleff et al. (1984), who demonstrated that FN was not required for the differentiation and proliferation that occured when embryonic mouse kidney mesenchyme was induced to form kidney tubules in vitro. However, these systems are not strictly comparable for two reasons: (i) we are using adult tissue, and (ii) our system requires attachment before outgrowth can occur. Recently Sariola et al. (1984) provided evidence that in vivo glomerular visceral epithelial cells do not stain immunohistochemically when reacted with anti-FN antibodies. However, again, these investigators used embryonic cells; and further, they demonstrated that in in vitro outgrowths from embryonic glomeruli there was a subpopulation of cells that expressed FN in strong fibrillar and granular patterns with immunofluorescence techniques. Hence, there is the possibility that a subpopulation of glomerular cells can adhere to FN, and it may be these ceils that attach to the culture substrate and produce colonies in our studies. Future experiments will be designed to test this possibility. Transferrin increased cell outgrowth in our glomerular explant system.

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Transferrin is an iron carrier required for proliferation of many cell types in chemically defined media (Barnes and Sato 1972; Vogt et al. 1969; Tormey et al. 1972) and has also been shown to stimulate embryonic development (Ekblom et al. 1983). Transferrin has been shown to bind to a cellsurface receptor. This transferrin-receptor complex is then internalized via coated pits, the transferrin loses its iron in acidic endosomes, and finally apotransferrin is returned to the extracellular space (Dautry-Varsat et al. 1983; Klausner et al. 1983; Martinez-Medeilin and Schulman 1967; Sibille et al. 1982; Takahashi and Tavossoli 1982; van Renshonde et al. 1982). The growth-stimulating effects of transferrin apparently require the presence of its receptor, since interference with the metabolism of transferrin by antibodies to the receptor can affect the cell cycle and growth (Trowbridge and Lopez 1982), and the expression of the transferrin receptor at the cell surface is related to cell proliferation (Shindelman et al. 1981). The important role of transferrin in branching morphogenesis, growth, and differentiation of the embryonic kidney has been demonstrated by Thesleff and Ekblom (1984), who showed that primitive kidney mesenchyme differentiates into kidney tubules in vitro after an inductive stimulus only if transferrin is present. Further, the proliferation that occurs during this differentiation process also requires transferrin. Although the present study demonstrates that transferrin is required for optimal mitosis in cell outgrowths from kidney glomeruli, it is not certain whether it is actually iron that mediates mitosis. Numerous attempts in our laboratory to resolve this question with iron chelators have always resulted in cellular toxicity (unpublished observations). However, since Landschulz et al. (1984) have demonstrated that a lipophilic iron chelator can replace transferrin as a stimulator of embryonic kidney cell proliferation and differentiation, future studies in our laboratory will attempt to clarify the role of transferrin and iron. Morphologic studies described in this paper suggest that (i) cell death and possible cell renewal were occurring within glomeruli during early culture; (ii) the mesangial cell appears to rapidly die in serum-free media, suggesting that either epithelial or endothelial cells are the cells appearing in culture (however, a previous morphologic study [Oberley et al. 1985] suggests that only visceral epithelial cells attach to the culture substrate and participate in cell outgrowth); and (iii) Se may have a protective effect against cell death in cultured glomeruli. Selenium is a cofactor for the enzyme glutathione peroxidase (Flohe et al. 1973). This enzyme functions synergistically with vitamin E in protecting cellular membranes from the process of lipid peroxidation, the oxidative deterioration of polyunsaturated lipids leading to the formation of highly unstable free radical intermediates (Hoekstra 1975). The present study demonstrated that there are more viable cells (epithelial, mesangial, and endothelial) in glomeruli grown in medium containing transferrin, FN, and AT and Se than in the same medium without Se. Further, there were more cell outgrowths in medium containing transferrin, FN, AT and Se than in the same medium without Se. Since neither mitotic index nor glomerular

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attachment rate was significantly different in these two media, it is possible that the increase in cell number in cell outgrowths was due to a decrease in the amount of cell death or possibly to a delay in the time of cell death. During the same experiments, we also measured hydrogen peroxide levels and demonstrated that AT increased intracellular hydrogen peroxide when included in medium with transferrin and FN, while Se dramatically reduced intracellular hydrogen peroxide in medium containing transferrin, FN, and AT (unpublished observation). As hydrogen peroxide is known to kill cells in culture (Simon et al. 1981), it seems likely that Se causes an apparent increase in cell outgrowth by preventing the cell killing caused by inhibition of the enzymes responsible for hydrogen peroxide production. In future studies we will measure cell death in culture using biochemical assays (e.g., lactic dehydrogenase release). Hornsby et al. (1985) recently demonstrated that Se is not necessary for the growth of cultured bovine adrenocortical cells, but also found that Se deficiency can increase the susceptibility to peroxide-mediated toxicity in these same cells. Thus, their results and the results of this present study are in close agreement. Thesleff and Ekblom (1984) have demonstrated that transferrin dramatically increases total DNA during the in vitro differentiation of embryonic kidney mesenchyme into kidney tubules. In contrast, in the system described here, total DNA was constant in the presence of transferrin and FN. The most likely explanation for our results is that our adult glomerular culture system is a cell renewal system. That is, whenever a cell dies [which does occur, as demonstrated by morphology (Oberley et al. 1985 and Figs. 7-10)], a precursor cell is stimulated to divide and replace the lost cell. Pabst and Sterzel (1983) have used autoradiography after [3H]thymidine labeling to demonstrate a cell renewal system in normal rat glomeruli in vivo. Future studies will test the hypothesis that glomerular cells in serum-free medium are part of a cell renewal system. In summary, both FN and transferrin have been shown to be required for glomerular cell outgrowth. Future studies will be attempt to establish the molecular mechanisms by which outgrowth occurs. References Barnes D, Sato G (1980) Serum-free culture: a unifying approach. Exp Cell Res 74:113-169 Baron-Van Evercooren A, Kleinman HK, Seppa HEJ, Rentier B, Dubois-Daleq M (1982) Fibronectin promotes rat Schwann cell growth and motility. J Cell Biol 93:211-216 Bitterman PB, Rennard SI, Adelberg S, Crystal RG (1983) Role of fibronectin as a growth factor for fibroblasts. J Cell Biol 97:1925-1932 Chance B, Sies H, Boveris A (1969) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527-605 Dautry-Varsat A, Crechanouer A, Lodish HF (1983) pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci (USA) 80:2258-2262 Doni MG, Piro E (1983) Glutathione peroxidase blockage inhibits prostaglandin biosynthesis in rat platelets and aorta. Haemostasis 13:240-243 Downs TR, Wilfinger WW (1983) Fluorometric quantitation of DNA in cells and tissue. Anal Biochem 131:538-549 Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1-42 Ekblom P, Thesleff I, Saxen L, Miettinen A, Timpl R (1983) Transferrin as a fetal growth

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factor: acquisition of responsiveness related to embryonic induction. Proc Natl Acad Sci (USA) 80: 2651-2655 Flohe L, Gunzler WA, Schock HH (1973) Glutathione peroxidase: a selenoenzyrne. FEBS Lett 32:132-134 Heim WB, Appleman D, Pyfrom HT (1955) Production of catalase changes in animals with 3-amino-l,2,4-triazole. Science 122:693-694 Hoekstra WG (1975) Biochemical function of selenium and its relation to vitamin E. Fed Proc 43 : 2083-2089 Hornsby PJ, Pearson DW, Autor AP, Aldren KA, Harris SE (1985) Selenium deficiency in cultured adrenocortical cells: Restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium. J Cell Physiol 123:33-38 Klausner RD, Ashwell G, Van Rensuonde J, Harford JB, Bridges KR (1983) Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci (USA) 80: 2264-2266 Kreisberg JI, Karnovsky MJ (1983) Glomerular cells in culture. Kidney Int 23:438-447 Martinez-Medeilin J, Schulman HM (1967) The kinetics of iron and transferrin incorporation into erythroid cells and the nature of stromal-bound iron. Biochem Biophys Acta 264: 272-284 Landschulz W, Thesleff I, Ekblom P (1984) A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation. J Cell Biol 98: 596-601 Minuth WW (1983) Induction and inhibition of outgrowth and development of renal collecting duct epithelium. Lab Invest 48: 543-548 Minuth WW, Kriz W (1982) Culturing of renal collecting duct epithelium as globular bodies. Cell Tissue Res 224:335-348 Norgaard JOR (1983) Cellular outgrowth from isolated glomeruli: origin and characterization. Lab Invest 48: 526-542 Oberley TD (1986) The possible role of reactive oxygen metabolites in glomerular cell outgrowth. In: Oberley LW (ed) Superoxide dismutase III: Pathologic states. CRC Press, Boca Raton (in press) Oberley TD, Steinert BW (1983) Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells. Virchows Arch [Cell Pathol] 44:337-354 Oberley TD, Burkholder PM, Barber TA, Hwang CC (1979) Cytochemical characterization of cultures adult guinea pig glomerular cells. Invest Cell Pathol 2:27-44 Oberley TD, Murphy-Ullrich JE, Muth JV (1981) The effect of fetal calf serum on the biology of cultured glomerular cells. Diag Histopathol 4:117-128 Oberley TD, Murphy PJ, Steinert BW, Albrecht RM (1982) A morphologic and immunofluorescent analysis of primary guinea pig glomerular cell types grown in chemically defined media: evidence for clonal growth and cell differentiation. Virchows Arch [Cell Pathol] 41 : 145-170 Oberley TD, Murphy-Ullrich JE, Albrecht RM, Mosher DF (1983) The effect of the dimeric and multimeric forms of fibronectin on the adhesion and growth of primary glomerular cells. Exp Cell Res 145:265-276 Oberley TD, Steinert BW, Yang AH, Anderson PJ (1986) Kidney glomerular explants in serum-free media: sequential morphologic and quantitative analysis of cell outgrowths. Virchows Arch [Cell Pathol] 50: 209-235 Pabst R, Sterzel RB (1983) Cell renewal of glomerular cell types in normal rats: an autoradiographic analysis. Kidney Int 24:626-631 Ruoslahti E, Engvall E, Hayman EG (1981) Fibronectin: current concepts of its structure and function. Coll Relat Res 1:95-128 Sariola H, Kuusela P, Ekblom P (1984) Cellular origin of fibronectin in interspecies hybrid kidneys. J Cell Biol 99:2099-2107 Shindelman JE, Ortmeyer AE, Sussman HH (1981) Demonstration of the transferrin receptor in human breast cancer tissue. Potential marker for identifying dividing cells. Int J Cancer 27: 329-334 Sibille JC, Octave Y, Schneider J, Trouet A, Crichton RR (1982) Transferrin protein and iron uptake by cultured hepatocytes. FEBS Lett 150:365-369

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Simon RH, Scoggin CH, Patterson D (1981) Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals. J Biol Chem 256:7181-7186 Takahashi K, Tavossoli M (1982) Biphasic uptake of iron transferrin complex by L210 murine leukemia cells and rat reticulocytes. Biochim Biophys Acta 685:6-12 Thesleff 1, Ekblom P (1984) Role of transferrin in branching morphogenesis, growth, and differentiation of the embryonic kidney. J Embryol Exp Morphol 82:147-161 Thesleff I, Ekblom P, Kuusela P, Lehtonen E, Ruoslahti E (1983) Exogenous fibronectin is not required for organogenesis in vitro. In Vitro 19:903-910 Tormey DC, Imrie RC, Mueller GG (1972) Identification of transferrin as a lymphocyte growth promotor in human serum. Exp Cell Res 74:113-169 Trowbridge IS, Lopez F (1982) Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits tumor cell growth in vitro. Proc Natl Acad Sci (USA) 79:1175-1179 van Renshonde J, Bridges KR, Harford JH, Klausner RD (1982) Receptor-mediated endocystosis of transferrin and the uptake of Fe in K562 cells. Identification of non-lysosomal acidic compartment. Proc Natl Acad Sci (USA) 79:6186-6190 Vogt AR, Mishell I, Dutton RW (1969) Stimulation of DNA synthesis in cultures of mouse spleen suspensions by bovine transferrin. Exp Cell Res 54:195-200 Yamada KM, Kennedy DW (1984) Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J Cell Biol 98 : 29-36 Yamada KM, Olden K (1978) Fibronectin. Adhesive glycoproteins of cell surface and blood. Nature 275:179-184 Received September 25 / Accepted December 21, 1985