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Identification of Cells Responsible for Urokinase-Type Plasminogen Activator Synthesis and Secretion in Human Diploid Kidney Cell Cultures

Posted on: Wednesday, 25 August 2004, 06:00 CDT

SUMMARY

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.

Key words: urokinase plasminogen activator.

INTRODUCTION

Urokinase-type plasminogen activator (uPA) is a specific neutral serine protease that converts plasminogen to plasmin, a broad- spectrum trypsin-like protease (Vassalli et al., 1991; Hirsh and Weitz, 1999). Il is produced and secreted by a variety of human cells and cultured cell lines (Lewis, 1979; Rondeau el al., 1989; Sappino et al., 1991).

The kidney is a recognized site of uPA synthesis and secretion in vivo (Brown et al., 1994). The presence of uPA is essential for the prevention of fibrin accumulation in the kidney and urine (Brown et al., 1994). Impairment in the ability of the kidney to secrete uPA can cause extensive fibrin deposition and subsequent obstruction of vessels and tubules. Studies using normal mouse tissues have demonstrated uPA immunoreaclivity and the presence of uPA messenger RNA within the medullary rays and portions of the proximal and distal tubules of the kidney (Tomooka et al., 1992; Brown et al., 1994). A similar distribution of uPA immunoreactivity has been noted within the normal human kidney (Angles-Cano et al, 1985; Wagner et al, 1996).

The kidney is composed of at least 15 different cell types (Ardaillou el al., 2000). The synthesis of uPA has been attributed to several renal cell types (Ferraiuolo et al., 1984; Angles-Cano et al., 1985). The synthesis of uPA is a sequential process in which inactive pro-uPA is initially produced and converted to active highmolecular weight uPA (Kasai el al., 1985a, 1985b). High- molecular weighl uPA can be further cleaved by metalloprotease 7 to give low-molecular weight uPA (LMW-uPA) (Marcotte et al., 1992).

We have identified two kidney cell types in purified and heterogeneous cell populations that synthesize and secrete uPA in vitro. Our results demonstrated that human glomerular visceral epithelial (GVE) and kidney tubular epithelial (KTE) cells synthesize and secrete uPA into the culture medium in cultures established from kidney tissues and purified populations of cells isolated from these tissues.

MATlCItIALS AND MlCTHODS

Preparation and cultivation of human kidney cell cultures. Normal human diploid cell cultures prepared from neonatal kidneys were used for these studies. Parental permission was obtained for the use of all kidney tissues used in this study. Blood samples from the mother and neonate were tested for the presence of CMV, HlV, HTLV I/II, and hepatitis B viruses and were negative for the presence of these viruses.

The kidney capsule, ureter, renal vein and arlery, and kidney calyx were removed before mincing the tissue for cell dissociation. The kidneys were minced into small pieces and digested in Medium 199 containing 10% fetal bovine serum (FBS), 200 units/ml penicillin, 200 g/ml streptomycin, and 200 units/ml of type 1 collagenase at 355 C for approximately 1 h. After cell dissociation, the cell suspension was filtered through a sterile gauze to remove undigested tissue fragments and was centrifuged at approximately 260 g for 10 min. The cells were rinsed with Medium 199 containing 20% FBS, centrifuged, and suspended in Medium 199 with 20% FBS and 7.5% dimethyl sulfoxide. The cell suspension was inoculated into ampules, frozen, and stored in liquid nitrogen.

Cells were thawed from liquid nitrogen, and 3.2 10^sup 6^ cells were inoculated into 490-cm^sup 2^ roller bottles containing Medium 199 with 10% FBS at a medium volume to surface area ratio of 0.53 ml/ cm^sup 2^ (Ryan et al., 1975). The cell viability was approximately 35% after the thaw of primary cells from liquid nitrogen storage and inoculation of cells into roller bottles. Cultures were gassed with 5% CO2-95% air and incubated for 7 d at 37 C on a roller rack rotating at 0.16-0.25 rpm. Cells were harvested by removing the growth medium, rinsing the cultures with calcium- and magnesium- free phosphatebuffered saline (PBS), and adding 300^00 units/ml trypsin in PBS containing 0.1% human serum albumin and 1% glucose. The cell viability of all harvests was > or =90%. Cells were inoculated into roller bottles containing growth medium and incubated as described above. Cells lines used in the studies described below had completed less than 15 population doublings or less than 60% of their in-vitro life span. Cells were tested for the presence of mycoplasma and viruses toward the end of the cell line life span. all cell lines were negative for the presence of mycoplasma and viruses. Antibiotics were not added to any media used in these studies.

Human renal cortical, proximal, mesangial, and mesenchymal cells. Populations of purified normal adult human renal cortical epithelial (HRCE), renal proximal tubular epithelial (RPTE), mesangial (NHM), and mesenchymal (MSCH) cells were obtained from Cambrex, Inc. (Walkersville, MD). The HRCE cell cultures were identified by staining positive for the presence of pan-cytokeratin by the vendor. The RPTE cells were identified by staining positive for the presence of [gamma]-glutamyl transpeptidase, a marker enzyme associated with cultured proximal tubule cells (Chung et al., 1982). The NHM cells tested positive for the presence of fibronectin and negative for cylokeralin 19 and von Willebrand factor according to the vendor. The absence of cytokeratin and von Willebrand factor has been identified with cultured NHM cells (Ardaillou et al., 2000). The MSCH cells were identified by the vendor by their abilily to differentiate into osteogenic, chondrogenic, and adipogenic lineages. The MSCH cells were positive for CD105, CD166, CD29, and CD44. These tested negative for CD14, CD34, and CD45. Adult bone marrow MSCH cells do not produce uPA and were used as a negative control in the experiments described below. all cultures received from the vendor had completed no more than two passages before receipt and were subcultured no more than three times before conducting the experiments described below. We estimate that these cultures had completed less than 15 population doublings when they were used for these experiments. Each cell type was inoculated into T75 flasks at 1 10^sup 5^ cells per flask and grown in the medium and at the temperature described above.

Production of uPA using heterogeneous and purified cell populations. Cells were grown in Medium 199 containing 8.3% newborn calf serum and 0.5% beef embryo extract before uPA production (Dalton et al., 1996). Confluent cultures were rinsed with Medium 199 without FBS, and a serum-free, uPA production medium consisting of basal salts, BME vitamins, 3 g/L glucose, 10 ng/ml epidermal growth factor, 1 M sodium orthovanadate, 2 mM glutamine, 0.1% human serum albumin, and 10% hydrolyzed bovine blood fibrin was added to the cultures. Sodium chloride was added to the medium to achieve a medium osmolality between 490 and 539 mOsm/kg. Cells were incubated in this medium for 42-63 d at approximately 35 C during uPA production. The cultures were not refed during the 42- to 63-d incubation period. The presence of uPA activity in the culture medium was determined using an amidolytic assay (Wang et al., 2000).

Cell identification. Indirect immunofluorescent (IF) staining of cell cultures and Western blot analysis of cell lysates were used to identify specific kidney cell types and to detect the presence of uPA antigen. Antibody against Wilrn's tumor antigen (WT-I) was used to identify GVE cells (Hosono et al., 9 1999; Scharnhorst et al., 2001). Antibody against pan-cytokeratin antigen was used to identify KTE cells (Helbert et al., 2001; van Kooten et al, 2001). The NHM and MSCH cells were identified using monoclonal antibodies against Thy-1 and smooth muscle [alpha]-actin, respectively (Floege el al., 1994; Oite et al., 1996).

Antibodies. Rabbit polyclonal antibody against WT-I was obtained from Genetka (Montreal, Canada). Mouse monoclonal antibody against pan-cytokeratin, rat monoclonal antibody against Thy-1 antigen, and fluorescein isothiocyanate (FITC)-labeled anti-mouse and anti- rabbit antibodies were obtained from Santa Cruz Bio (Santa Cruz, CA). Monoclonal anti-smooth muscle [alpha]-actin antibody, anti- goat IgG, and rabbit anti-goat FITC-labeled antibodies were obtained from Sigma Chemical Co. (St. Louis, MO). Goal polyclonal anti-uPA antibody was used to stain for uPA (Abbotl Laboratories, North Chicago, IL). Horseradish peroxidase-conjugated anti-mouse and anti- rabbit IgG antibodies \were obtained from BioRad (Richmond, CA). An enhanced chemiluminescence (ECL) detection kil was purchased from Amersham Biosciences Corp. (Piscalaway, NJ).

immunohistochemistry, Cells grown on Lab Tek chamber slides were rinsed with PBS (pH 7.2), fixed in cold 100% ethanol for 10 min, and washed three times with PBS. The slides were then incubated with blocking solution (3% bovine serum albumin in PBS) for 30 min at room temperature. Cells were incubated with primary antibody for l h at room temperature and then were exposed to three 10-min washes in PBS containing 0.1% Tween 20. The cells were incubated with FITC- conjugated secondary antibody for l h at room temperature in the dark. The slides were washed three times for 5 min eaeh and mounted with mounting solution. Stained cells were observed using a Leica DMIKH inverted fluorescence microscope (Lcica Microsystems, WeI7.1ar, Germany).

Western blot- analysis. Kidney cells were lysed in I sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 62 mM Tris, 10% glycerol, pH 7.4), and the protein content was determined using the bicinchoninic acid protein assay (Pierce Inc., Rockford, IL). Thirty micrograms of total protein was eleclrophoresed on a 4-20% SDS- polyacrylamide gel (Invitrogen, Carlsbad, CA) and transferred onto a nitrocellulose membrane (BioRad). The membrane was blocked with 5% nonfat dry milk and probed with primary antibody. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody for l h and detected by ECL.

RESULTS

Cell cultures prepared from human neonatal kidney tissue, grown in Medium 9 199 containing 10% FBS, and maintained in uPA production medium for 42 d produced pro-uPA and LMW-uPA. The intracellular presence of uPA antigen during cell growth and incubation of cells in uPA production medium was detected using IF staining of cultured cells (Fig. 1A) and by Western blot analysis of cell lysates (Fig. 1B). The secretion of uPA into growth and production media was demonstrated by detection of uPA amidolytic activity and Western blot analysis of samples from growth and production media (Fig. 1C and D). The amidolytic activity of uPA in the growth medium at day 7 was approximately 100 IU/ml. The uPA activity in production medium increased from approximately 400 units/ml at day 14 to almost 1000 units/ml at 42 d incubation (Fig. 1C).

The human diploid kidney cell (HKC) cultures were stained with antibodies for the presence of uPA, WT-] (GVF cells), pan- eytokeratin (KTE cells), and Thy-1 (NHM cells) antigens. The results demonstrated that the presence of uPA in HKC cultures (Fig. 2A) was associated with the presence of KTF and CVF cell populations (Fig. 2B and C, respectively) but not with the presence of HNM cells (Fig. 20). The presence of uPA antigen was associated with cells that contained WT-I and pan-cytokeratin antigens during the 42-d incubation period in uPA production medium (Fig. 2E). The amount of LMW-uPA in the culture medium increased, whereas the amount of pro- uPA decreased during the 42-d incubation period (Fig. 2E). Thy-1 antigen (NHM cells) was detected only on days 7 and 14 using Western blot analysis (Fig. 2E).

To further investigate these findings, HKC cultures and purified cell cultures of HRCE, RPTE, NHM, and MSCH cells wen; stained for the presence of uPA, WT-1, pan-cytokeratin, Thy-1, and smooth muscle [alpha]-actin antigens. The results (Fig. 3) demonstrated that HKC, HRCE, and RPTE cell cultures stained positive for the presence of uPA antigen and contained primarily GVE and KTE cells, as indicated by the presence of WT-I and pan-cytokeratin antigens. Thy-1 and [alpha]-actin antigens associated with NHM and MSCH cells were not detected in HKC or purified HHCE and RPTE cell populations. The NHM and MSCH cell cultures stained positive for the presence of Thy-1 and [alpha]-actin, respectively, but failed to stain positive for the presence of WT-I, pan-cytokeratin, or uPA antigens. These results were confirmed using Western blot analysis of whole-cell lysates (Fig. 4). Although WT-I antigen was detected in NHM and MSCH cell populations when assayed using Western blot analysis, both cell populations failed to stain positive for the presence of uPA antigen (Fig. 4).

The amidolytic assay of the culture medium lor uPA activity (Table 1) confirmed our results using IF staining of cell cultures and Western blot analysis of cell lysates. The HKC, HRCE, and RPTE cultures secreted uPA into the culture medium, whereas NHM and MSCH cultures failed to secrete significant amounts of uPA into the culture medium. The amidolytic activity of uPA in the culture medium from HKC, HRCE, and RPTE cell cultures increased from 701-792 lU/ml at 21 d in culture to 11707 -1570 IU/ral at 63 d in culture. The amidolytic activity in the culture medium from NHM or MSCH was 18 ITJ/ml or less throughout the 63-d incubation period.

FIG. 1. Synthesis and secretion of urokinase-type plasminogen activator (uPA) by cultured human diploid kidney cell (HKC). Indirect immunofluorescent staining of HKC cultures for the presence of uPA during cell growth (A, left panel} and during the maintenance of cells in uPA production medium (A, right panel). (B) Western blot analysis of cell lysates from cells at 5 and 7 d in growth medium (Gr Medium) and at 7, 14, 21, 28, 35, and 42 d of incubation in uPA production medium. (C) UPA amidolytic activity at 7 d growth (GrM) and at 7-42 d incubation in uPA production medium (ProdM). (D) Western blot detection of pro-uPA and low-molecular weight uPA at 5 and 7 d growth and at 7 through 42 d in uPA production medium. Figure is published in color online at http://inva.allenpress.com/ invaonline/?request=index-html.

FlG. 2. Indirect immunofluorescenl slaining of human diploid kidney cell (HKC) cultures maintained in urokinase-type plasminogen activator (uPA) production medium for the presence of uPA (A), pan- cytokeratin antigen (kidney tubular epithelial cells) (B), Wilm's lumor antigen (WT-I) (g)omerular visceral epithelial cells) (C), and Thy-1 antigen (mesangial cells) (O). (E) Western blot analysis of HKC culture lysates for the presence of pro-uPA, low-molecular weight uPA, WT-I, pan-cytokeratin (Pan-Cyto), and Thy-1 antigen at 1- 42 d in uPA production medium. Figure is published in color online at http://inva.allenpress.com/invaonline/?request=index-html.

FiG. 3. Human diploid kidney cell (HNK), human renal cortical epithelial (HRCE), renal proximal tubular epithelial (RPTE), mesangial (NHM), and mesenchymal (MSCH) cell cultures were stained for the presence of urokinase-type plasminogen activator (uPA), Wilm's tumor antigen (WT-I), pan-cytokeratin (pan-Cyto), Thy-1, and ct-actin antigens. The HNK, HRCE, and RPTE cultures stained positive for the presence of uPA and contained primarily glomerular visceral epithelial and kidney tubular epithelial cells, as shown by positive staining using WT-I and pan-Cyto. The NHM and MSCH cell cultures failed to stain for the presence of uPA, WT-I, and pan-Cyto but stained positive for the presence of Thy-1 and a-actin, respectively. Figure is published in color online at http:// inva.allenprcsH.com/ invaonline/?request-index-html.

DISCUSSION

The presence of uPA activity in human urine, kidney tissue, and kidney cell cultures has been documented (Wagner et al., 1996). The kidney is a significant source of uPA expression in all mammalian species (Sappino el al., 1991). The precise cellular site of uPA synthesis in the kidney has not been determined, though previous work using murine and human tissues has shown immunoreactivity in glomerular and tubular cells (Angles-Cano et al., 1985). Brown et al. (1994) have reported the presence of uPA in the medium from pure cultures of human glomerular epithelial cells, cocultures of human glomerular and NHM cells, and whole-glomeruli cultures. UPA was not detected in pure cultures of NHM cells. They also demonstrated localization of uPA messenger RNA in the cytoplasm ol human glomerular cells but not in NHM cells. However, Reid et al. (1992) have reported uPA synthesis by NHM cells. Lacave et al. (1989) have reported the presence of tissue plasminogen activator and of an inhibitor of plasminogen activator (PAI-I) in the supernatant from HNM cell cultures.

Our results confirm the results reported by Brown et al. (1994) and suggest that KTE cells as well as GVE cells produce uPA in the kidney. The basis for the fact that both KTE and CVE produce uPA is that the expression of WT-I antigen is observed only in the podocyte (GVE) layer of the mature glomerulus (Hosono et al., 1999) and that pan-cytokeralin is expressed only in KTK cells (HeIhert et al., 2001). The results of our studies demonstrated that both WT-I and pan-cytokeratin were expressed in HNK cell cultures that produced uPA and suggests lhat both eell types are present. It is possible that the patterns of antigen expression changed from the original tissue during serial cell propagation and that both antigens are present on one cell type. However, any change in the pattern of antigen expression would have had to occur within 15 population doublings from the tissue origin.

FlG. 4. The expression of pro-urokinase-lype plasminogen activator (uPA), low-molecular weight uPA (LMW-uPA), Wilm's tumor antigen (WT-I), pan-cytokeratin (pan-Cylo), Thy-1, and smooth muscle [alpha]-aclin ([alpha]-actin) in human diploid kidney cell (HNK), human renal cortical epithelial (HRCE), renal proximal tubular epithelial (RPTE), mesangial (NHM), and mesenchymal (MSCH) cells by Western blot analysis of cell lysates. The presence of pro-uPA and LMW-uPA was associated with HNK, HRCE, and RPTE cells but not with NHM or MSCH cell populations.

Studies in which purified cultures of RPTE and HCRE cells were used to study uPA production showed the presence of both WT-I and pan-cytokeratin in both cell populations. We believe the expression of both anti\gens in purified populations of HCRE and RPTE cultures to be the result oi the presence of other cell types in these populations. This is supported by the vendor's claim that the purity of these was 90% or more. However, it cannot be exeluded that both antigens could be present on a single cell type. More studies using additional characteristics that are associated with each cell type should be conducted.

Purified cultures of NHM cells or mixed cell populations containing NHM cells did not express or secrete detectable amounts of uPA. These results appear to contradict results by Lacave et al. (1989) and Reid et al. (1992), who reported uPA production by NHM cells. This discrepancy may result from several possible explanations. The uPA expression by NHM cells could be due to permissive or instructive interactions with other kidney epithelial cells (Master, 1976; Barasch et al., 1999), or the production of uPA may reflect the presence of other cell types in the NHM cell preparation used by these investigators. The lack of uPA production by NHM cells in our studies may he caused by exposure of these cells to uPA production medium with a high osmolality.

TABLE 1

UPA AMIDOLYTIC ACTIVITY (IU/ml) OF HKC, HRCE, RPTE, NHM, AND MSCH CELL CULTURES IN PRODUCTION MEDIUM AT 21, 35, 49, AND 63 D(a)

Kidney tubular cells line the proximal and distal tubules of the nephron. Consequently, they are exposed to conditions in which the osmolality can vary significantly. The normal response of these cells to hypertonic conditions may be to increase production of uPA to provide an additional safeguard against fibrin formation in the kidney and to enhance the overall function of the kidney in allowing urine clearance.

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SAFEDTN BKQAJ, ANUJA M. SHAH, AND JON M. RYAN1

Biologies Technical Operations, Department 456, Abbott Laboratories, 1400 Sheridan Road, North Chicago, Illinois 60064

(Received 17 December 2003; accepted 25 February 2004)

1 To whom correspondence should be addressed at E-mail: jon.ryan@ abbott.coma

Copyright Society for In Vitro Biology Mar/Apr 2004

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