Cultured rat glomerular epithelial cells show gene expression and production of transforming growth factor-ß: expression is enhanced by thrombin

Satoru Tsunoda, Hideaki Yamabe, Hiroshi Osawa, Mitsuaki Kaizuka, Kenichi Shirato and Ken Okumura

Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Glomerular crescents play an important role in progressive glomerular injury. The lesions consist of epithelial cells, macrophages, and deposits of fibrin and extracellular matrix. Transforming growth factor beta (TGF-ß) contributes to the modulation of cell growth and extracellular matrix synthesis. Thrombin is involved in fibrin formation in crescents. The purpose of this study was to examine whether glomerular epithelial cells (GEC) could produce TGF-ß, and if so, to clarify the role of TGF-ß in GEC proliferation. We also investigated whether thrombin could modulate the production of TGF-ß and extracellular matrix by GEC.

Methods. Bioassay using the TGF-ß-dependent mink pulmonary epithelial cell line (CCL-64), immunoblot analysis, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to demonstrate TGF-ß production by rat GEC. TGF-ß gene expression was examined by RT-PCR in GEC incubated with thrombin, and type IV collagen and fibronectin were quantified by enzyme immunoassay in culture supernatants of GEC incubated with thrombin or TGF-ß.

Results. TGF-ß activity was demonstrated in GEC supernatants by bioassay. Immunoblot analysis of concentrated culture supernatants using anti-TGF-ß antibody revealed a 12.5-kDa protein, which was compatible with TGF-ß. Concentrated GEC supernatants inhibited GEC proliferation as well as porcine TGF-ß. RT-PCR demonstrated TGF-ß gene expression in GEC. Thrombin (0.5–5.0 U/ml) enhanced TGF-ß mRNA expression in a dose-dependent manner. Thrombin (5.0 U/ml) and porcine TGF-ß (5.0 ng/ml) stimulated the production of type IV collagen and fibronectin by GEC.

Conclusions. Rat GEC produce TGF-ß in vitro. Thrombin may participate in the progression of glomerulosclerosis in crescentic glomerulonephritis through the stimulation of TGF-ß production by GEC.

Keywords: fibronectin; glomerular epithelial cells; rat; TGF-ß; thrombin; type IV collagen



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
It is generally accepted that glomerular crescents play an important role in progressive glomerular injury, the lesions consisting of epithelial cells, macrophages, and deposited fibrin and extracellular matrix. It has been reported that transforming growth factor beta (TGF-ß) is involved in the progression of glomerulosclerosis in glomerulonephritis and diabetic glomerulosclerosis [1,2]. TGF-ß is a multifunctional regulatory protein, and it is known to stimulate the synthesis of matrix components such as type IV collagen, fibronectin, and proteoglycans, which are present in large amounts in normal glomeruli [3,4]. TGF-ß has been shown to participate in the glomerular accumulation of extracellular matrix in glomerulonephritis [1]. It is well known that many kinds of cells can produce TGF-ß. It has been demonstrated that cultured mesangial cells produce TGF-ß when stimulated with fetal calf serum, phorbol esters [5], and thrombin [6]. Cultured human glomerular epithelial cells (GEC) produce TGF-ß, gene expression of which is enhanced with high glucose [7] or low-density lipoprotein (LDL) [8].

Fibrin formation within glomerular crescent has been observed in crescentic glomerulonephritis and is known to be involved in crescent formation. Intraglomerular generation of thrombin may result from the glomerular activation of tissue factor, which has been reported to occur in human proliferative glomerulonephritis [9] and in animal experimental glomerulonephritis [1012]. Thrombin has various biological effects besides its role in haemostasis. For example, it has been reported that thrombin stimulates the mitogenesis of cultured fibroblasts [13,14], lymphocytes [15], endothelial cells [16], vascular smooth-muscle cells [17,18], mesangial cells [19,20], and glomerular epithelial cells [21]. Thrombin stimulates the production of some cytokines by lymphocytes [22] as well as the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor 1 by mesangial cells and epithelial cells [23,24], vascular smooth-muscle cells [18], and mesangial cells [20].

In the present study, we investigated TGF-ß production by cultured rat GEC, and its role in GEC proliferation. We also examined whether thrombin could modulate TGF-ß gene expression in GEC and the production of type IV collagen and fibronectin by GEC.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
GEC culture
The culture of rat GEC was established as described previously [25]. GEC were cultured with K-1 media, 1:1 mixture of Dulbecco's modified eagle's minimum essential medium (DMEM, Gibco Laboratories, Grand Island, NY, USA) and Ham's nutrient mixture F-12 (Gibco Laboratories), supplemented with 2% Nu Serum (Collaborative Research, Bedford, MA, USA) and ITS premix (I, insulin; T, transferrin; S, selenium, Collaborative Research). The cells were used between the 18th and 24th passages.

Characterization of cultured GEC
The cells we used were identified as GEC by standard criteria [26]. They showed cobblestone appearance under phase-contrast microscopy. In immunofluorescence study, they were positive for cytokeratin and FX1A. Neither markers for mesangial cells (Thy-1) nor endothelial cells (Factor VIII-related antigen) were detected in these cells. They were also susceptible to the cytotoxicity of low-dose (10–100 µg/ml) of aminonucleoside puromycin (Sigma, St Louis, MO, USA). It is not currently possible to determine specifically whether GEC in culture originate from visceral or parietal epithelium.

Bioassay for TGF-ß activity
The bioassay for TGF-ß activity was performed using a mink pulmonary epithelial cell line, namely CCL-64 cells (American Type Culture Collection, Rockville, MD, USA), whose growth inhibition is TGF-ß dependent. CCL-64 cells were maintained in Eagle's minimum essential medium (MEM, Gibco Laboratories) with 5% fetal calf serum (FCS, Bioserum, Canterbury, Australia). The cells were placed in 96-well plates (Sumitomo Bakelite Co. Ltd, Tokyo, Japan.) at a density of 4000 cells/well in a volume of 50 µl of MEM with 10% FCS. After 3 h incubation at 37°C in 5% CO2, 50 µl of various concentrations of porcine TGF-ß (3.5 pg/ml–35 ng/ml), which was kindly provided by Dr H. Ohhashi, Kirin Brewer, Maebashi, Japan, and 50 µl of samples were added into each well in a final volume of 100 µl, and the cells were incubated for 48 h. Thereafter proliferation of CCL-64 cells was evaluated by a colorimetric assay using MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide) dye (Promega, Madison, WI, USA). Briefly, cells were incubated for 4 h after addition of 20 µl MTT solution into each well, and solubilization solution (Promega) was added to dissolve the MTT dye incorporated by viable cells. Then the plates were left overnight at room temperature and absorbance at 550 nm with a reference of 650 nm was measured by an ELISA reader.

TGF-ß activity in GEC supernatants
GEC were cultured until confluent in 75-cm2 culture flasks. These cells were washed twice with Hank's balanced salt solution (HBSS, Gibco Laboratories) and incubated with DMEM containing 0.2% lactalbumin (Sigma) for 48 h. The culture supernatants were collected and were concentrated 50–80 times using the ultrafiltration device (Amicon, Inc. Beverly, MA, USA). The concentrated supernatants were stored at -80°C until use. The latent TGF-ß was activated by acidifying GEC supernatants with 1/20 amount of 1 N HCl for 60 min at 4°C, followed by neutralizing with equimolar NaOH. Then TGF-ß activity in the GEC supernatants was measured by bioassay.

Effects of anti-TGF-ß neutralizing antibody on inhibition of CCL-64 cell proliferation by GEC supernatants were examined to confirm the existence of TGF-ß in GEC supernatants. TGF-ß activity in GEC supernatants was evaluated in the presence of 50 µg/ml polyclonal rabbit anti-TGF-ß antibody (King Brewing Co., Kakogawa, Japan) or 50 µg/ml of normal rabbit IgG as control.

Immunoblot analysis of TGF-ß protein
Concentrated GEC supernatants were reduced by heating at 95°C for 4 min in the presence of 2-mercaptoethanol. The reduced samples were electrophoresed on 12% SDS–polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA, USA). Immunoblot analysis was performed using Anti-TGF-ß Polyclonal Antibody Kit (R&D Systems, Minneapolis, MN, USA). This kit uses polyclonal rabbit anti-porcine TGF-ß antibody as a first antibody, biotin-conjugated anti-rabbit IgG as second antibody, and alkaline phosphatase-conjugated streptavidin.

Effect of TGF-ß and GEC supernatants on GEC proliferation
GEC were placed in 96-well plate at a density of 5000 cells/well in a final volume of 50 µl. After 3 h incubation at 37°C in 5% CO2, 50 µl of various concentrations of porcine TGF-ß (3.5 pg/ml–35 ng/ml) and 50 µl of GEC supernatants were added to the wells in a total volume of 100 µl/well. The GEC were incubated for 72 h at 37°C in 5% CO2 and then GEC proliferation was evaluated by an MTT assay as described before. We also examined the effect of anti-TGF-ß antibody on the change of GEC proliferation induced by GEC supernatants. We evaluated GEC proliferation incubated with K1 media only, concentrated GEC supernatants, concentrated GEC supernatants plus anti-TGF-ß antibody (50 µg/ml), and concentrated GEC supernatants plus normal rabbit IgG (50 µg/ml).

Effect of thrombin on TGF-ß gene expression in GEC
We examined the effect of thrombin on TGF-ß mRNA expression in GEC by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Confluent GEC grown in 75-cm2 flasks (Falcon, Becton Dickinson, Franklin Lakes, NJ. USA) were incubated with 0.5–5.0 U/ml of a thrombin (Sigma) for 6 h. Total RNA was extracted from GEC by one-step method using RNA zol (Cinna Biotex, Houston, TX, USA). Five micrograms of total RNA were reverse-transcribed using a RT-PCR kit (Stratagene, La Jolla, CA, USA) to obtain first-strand cDNA according to the manufacturer's instruction. TGF-ß specific primers were purchased from Clontech (Palo Alto, CA, USA). As an internal control, primers for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were also purchased from Clontech. Sequence of these primers were as follows: TGF-ß sense; 5'-CTTCAGCTCCACAGAGAAGAACTGC-3' (bases 1267–1291), TGF-ß antisense; 5'-CACGATCATGTTGGACAACTGCTCC-3' (bases 1540–1564), GAPDH sense; 5'-ACCACAGTCCATGCCATCAC-3' (bases 586–605), GAPDH antisense; 5'-TCCACCACCCTGTTGCTGTA-3' (bases 734–763). The predicted sizes of the amplified product were 298 bp in TGF-ß and 452 bp in GAPDH respectively. Three microlitres of first-strand cDNA solution was mixed with 5 µl reaction buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl pH 8.3, 15 mmol/l MgCl2 and 0.001% gelatine), 4 µl of 2.5 mmol/l dNTP mix (final concentration was 200 µmol/l of dATP, dCTP,dGTP, and dTTP), 1 µl of sense and antisense primer (final concentration, 0.4 µmol/l), and 37.6 µl sterile water and 0.4 µl Taq DNA polymerase (2.0 units). PCR amplification was carried out in the DNA thermal cycler (Parkin/Elmer Cetus, Norwak, CT, USA) with denaturing for 45 s at 94°C, annealing for 45 s at 60°C and extension for 120 s at 72°C. Amplification was performed at 31 cycles for TGF-ß and at 29 cycles for GAPDH as an internal control. Our preliminary experiments have confirmed that these cycles are at exponential phases of amplification. Positive control cDNA for TGF-ß, provided by the manufacturer, was also amplified simultaneously. After PCR amplification, 5 µl of the PCR product was electrophoresed on a 1.3% agarose gel with size marker ({phi}x174/Hinc II digest, Toyobo, Tokyo, Japan), and visualized by ethidium bromide staining.

Effect of thrombin and TGF-ß on the production of type IV collagen and fibronectin by GEC
GEC were incubated with DMEM containing 0.2% lactalbumin (Sigma) and 5 U/ml of {alpha} thrombin or 5 ng/ml of porcine TGF-ß for 48 h in 12-well plate, and then type IV collagen and fibronectin were measured in culture supernatants. We also examined the effect of anti-TGF-ß antibody on the production of type IV collagen and fibronectin stimulated with thrombin. GEC were incubated for 48 h with 5.0 U/ml thrombin, 5.0 U/ml thrombin plus 50 µg/ml anti-TGF-ß antibody, or 5.0 U/ml thrombin plus 50 µg/ml normal rabbit IgG, and the levels of type IV collagen and fibronectin in the cell supernatants were measured. After removing culture supernatants, the cells in each well were lysed in 1 N NaOH and the protein content was measured by the method of Lowry et al. [27] using BSA as a standard. The concentrations of type IV collagen and fibronectin in culture supernatants were expressed as nanograms per microgram of lysed GEC protein. Type IV collagen and fibronectin in culture supernatants were quantified by enzyme-linked immunoassay [28]. Each well in 96-well plate was coated with 10 µg/ml of mouse type IV collagen (Collaborative Research) and 5 µg/ml of rat fibronectin (Sigma) in 100 µl coating buffer during 16 h at 4°C. The culture supernatants of the samples and various concentrations of mouse type IV collagen and rat fibronectin for the standard curve were incubated with equal volumes of 4 µg/ml of polyclonal rabbit antibody against mouse type IV collagen (Collaborative Research) and rat fibronectin (Chemicon, Temecula, CA, USA) for 16 h at 4°C. After the coating, the wells were washed three times with PBS-Tween and 100 µl sample antigen-antibody mixture or standard antigen-antibody mixture were added. The plate was left at room temperature for 30 min. Then all wells were washed three times, followed by addition of 100 µl alkaline phosphatase conjugated anti-rabbit IgG (Dako, Glostrup, Denmark). After 60 min of incubation at room temperature, substrate solution was added and the plate was incubated for another 30 min. The reaction was stopped with 3 N NaOH. The optical density at 405 nm was measured by an ELISA reader. The concentrations of type IV collagen and fibronectin were calculated from standard curves.

Statistical analysis
All data are expressed as the means±1 SD. Results were compared using one-way factorial ANOVA and multiple comparison tests. P<0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Porcine TGF-ß inhibited the proliferation of CCL-64 cells in a dose-dependent manner. TGF-ß activity was observed in GEC supernatants and its amount was equivalent to 72 pg/ml of porcine TGF-ß. This activity was inhibited by anti-TGF-ß neutralizing antibody but not by normal rabbit IgG (Figure 1Go). Immunoblot analysis revealed a single 12.5-kDa protein reacted with anti-TGF-ß antibody in the reduced samples and its molecular size was compatible with that of TGF-ß previously reported (Figure 2Go). RT-PCR showed a single band of 298 bp in total RNA from GEC, which is identical to the predicted size of TGF-ß (Figure 3Go).



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Fig. 1. TGF-ß activity was observed in GEC supernatants by a bioassay using CCL64 and its amount was equivalent to 72.3±13.8 pg/ml of porcine TGF-ß. This activity was significantly blocked by anti-TGF-ß neutralizing antibody, while normal rabbit IgG did not change the TGF-ß activity. Values are means±SD for six wells, and representative data from one of two experiments are shown.

 


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Fig. 2. Immunoblot analysis of GEC culture supernatants revealed a single 12.5-kDa band that reacted with anti-TGF-ß antibody in the reduced sample.

 


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Fig. 3. RT-PCR amplification of both positive control cDNA and cDNA synthesized from GEC total RNA yielded a single band of 298 bp, which is identical to the predicted size of TGF-ß mRNA.

 
Exogenously added porcine TGF-ß inhibited GEC proliferation in a dose-dependent manner. This effect of TGF-ß was observed at concentrations higher than 35 pg/ml (Figure 4Go). We also examined the effect of GEC supernatants on GEC proliferation to determine whether growth of cultured GEC was under the influence of TGF-ß endogenously secreted by GEC. GEC supernatants inhibited GEC proliferation, and addition of anti-TGF-ß neutralizing antibody to GEC supernatants significantly augmented GEC proliferation, though addition of normal rabbit IgG did not influence GEC growth (Figure 5Go).



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Fig. 4. Exogenously added porcine TGF-ß inhibited GEC proliferation in a dose-dependent manner. The effect of TGF-ß was observed at concentrations higher than 35 pg/ml. Values are means±SD for six wells, and representative data from one of two experiments are shown.

 


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Fig. 5. GEC culture supernatants inhibited GEC proliferation. Addition of anti-TGF-ß neutralizing antibody to GEC supernatants significantly augmented the GEC proliferation, although normal rabbit IgG did not influence the GEC growth. Values are means±SD for six wells, and representative data from one of two experiments are shown.

 
In semi-quantitative RT-PCR using GAPDH as an internal control, thrombin (0.5–5.0 U/ml) increased TGF-ß mRNA expression in a dose-dependent fashion though GAPDH gene expression was not changed (Figure 6Go).



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Fig. 6. Thrombin (0.5–5.0 U/ml) augmented TGF-ß mRNA expressions in a dose-dependent manner in semi-quantitative RT-PCR, although GAPDH gene expressions were not changed.

 
Thrombin (5 U/ml) and TGF-ß (5 ng/ml) significantly increased the production of type IV collagen and fibronectin by GEC (Figure 7Go). Anti-TGF-ß neutralizing antibody inhibited the stimulatory effect of thrombin on the production of type IV collagen and fibronectin by GEC, while normal rabbit IgG did not (Figure 8Go).



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Fig. 7. Thrombin (5 U/ml) and TGF-ß (5 ng/ml) significantly increased the production of (A) type IV collagen, and (B) fibronectin by GEC. Values are means±SD for three wells, and representative data from one of two experiments are shown.

 


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Fig. 8. Anti-TGF-ß antibody (50 µg/ml) significantly inhibited the increase of (A) type IV collagen, and (B) fibronectin stimulated with thrombin, while normal rabbit IgG (50 µg/ml) did not. Values are means±SD for three wells and representative data from one of two experiments are shown.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We examined TGF-ß production by rat GEC and the modulatory effect of thrombin on TGF-ß gene expression in GEC and the production of type IV collagen and fibronectin by GEC. TGF-ß production by cultured rat GEC was confirmed by the presence of TGF-ß protein in the culture supernatants, and by the expression of TGF-ß mRNA as demonstrated by RT-PCR.

TGF-ß has various effects on different cell types and can regulate cell proliferation, cell differentiation, the synthesis of extracellular matrix, and the production of various cytokines [26,29,30]. Glomerulosclerosis is characterized by the excessive accumulation of extracellular matrix in the glomeruli, and it has been reported that TGF-ß has an important role in the glomerular accumulation of extracellular matrix in anti-Thy1.1 nephritis and diabetic nephropathy [1,2].

Many kinds of cells can produce TGF-ß, and Kaname et al. [5] reported that TGF-ß produced by mesangial cells regulates mesangial cell growth and differentiation in an autocrine fashion. It has also been demonstrated that cultured human GEC produce TGF-ß and that its gene expression is enhanced with high glucose [7] or low-density lipoprotein (LDL) [8]. However, the interaction between TGF-ß produced by GEC and GEC proliferation is still not fully understood.

In our study, GEC culture supernatants inhibited GEC proliferation, and this inhibitory effect was blocked by anti-TGF-ß antibody. Therefore it was thought that TGF-ß secreted by GEC inhibited GEC proliferation. It is possible that TGF-ß produced by GEC modulates the GEC proliferation and the synthesis of extracellular matrix by GEC as well as mesangial cells, although the regulatory mechanism responsible for GEC-derived TGF-ß production is unknown.

In cultured mesangial cells, TGF-ß production was enhanced with fetal calf serum, phorbol esters [5], and thrombin [6]. In cultured human GEC, Van Det et al. [7] reported that high-glucose medium enhanced TGF-ß protein production and its gene expression as well as fibronectin. Ding et al. [8] reported that oxidized LDL stimulated the expression of TGF-ß as well as fibronectin. Enhanced gene expression of fibronectin was inhibited by anti-TGF-ß neutralizing antibody in both reports. They were of the opinion that these factors are involved in glomerulosclerosis through TGF-ß-mediated mechanisms in arteriosclerosis and diabetic nephropathy.

In our study, thrombin stimulated TGF-ß gene expression in GEC. Thrombin also stimulated the production of type IV collagen and fibronectin by GEC as well as TGF-ß did. Thrombin has various biological effects besides its important role in haemostasis. Thrombin is mitogenic for lymphocytes, endothelial cells, and other types of cells [1318], and it can regulate the production of components of the fibrinolytic system in a variety of cultured cells [22,23]. It has been reported that thrombin stimulates the proliferation of cultured mesangial cells and glomerular epithelial cells [19,20]; it was also reported that thrombin enhances the production of some cytokines by lymphocytes [24], vascular smooth-muscle cells [18], and mesangial cells [20]. Naldini et al. [24] found that thrombin stimulates T-cell proliferation and increases IL-2 and IL-6 production. It has been reported that thrombin induces the proliferation of human vascular smooth muscle cells and expression of the platelet-derived growth factor (PDGF)-A chain gene [18]. Thrombin also upregulates PDGF-B chain gene expression in human mesangial cells [20]. We have reported that thrombin stimulated TGF-ß production and type IV collagen production through TGF-ß-dependent mechanisms in human mesangial cells [6,31]. These findings and our present study imply that thrombin stimulates cytokine production including TGF-ß, in addition to having a mitogenic effect in GEC. It is generally accepted that glomerular crescent plays an important role in progressive glomerular injury. This pathological process consists of proliferating cells including parietal GEC, possibly visceral GEC, and macrophages, and deposits of fibrin and extracellular matrix. Thrombin is generated at sites of glomerular injury by activation of the intrinsic or extrinsic coagulation pathway. Then thrombin stimulates cell proliferation and accelerates TGF-ß production by GEC and mesangial cells. TGF-ß subsequently upregulates the extracellular matrix production by GEC. This mechanism may explain how the hypercellular proliferative lesion evolves to a hypocellular sclerotic lesion in crescentic glomerulonephritis.

In summary, we showed that GEC produce TGF-ß, which in trun inhibits GEC proliferation, and that thrombin stimulates TGF-ß gene expression in GEC, which in turn stimulates the production of type IV collagen and fibronectin by GEC. Thrombin may play an important role in the progression of glomerulosclerosis in crescentic glomerulonephritis through the upregulation of TGF-ß production by GEC, in addition to its mitogenic effect.



   Notes
 
Correspondence and offprint requests to: Hideaki Yamabe MD, Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8216, Japan. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 19. 7.00
Revision received 3. 4.01.