1 Department of Internal Medicine and Institute of Physiological Chemistry, University of Würzburg, 3 Institute of Anatomy, University of Dresden, 4 Department of Anatomy and Cell Biology, RWTH University of Aachen, Germany, 2 Institute of Preventive and Clinical Medicine, Bratislava, Slovakia and 5 Nagoya University Daiko, Medical Centre, Nagoya, Japan
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Abstract |
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Methods. Arrested cells were exposed to vehicle (control), AGE-BSA (1976 µM) and BSA (38 µM) in the presence or absence of trypsin (0.6255.0 µg/ml) (2.5 µg/ml) for 24 h. We evaluated cell proliferation by cell count and by [3H]thymidine incorporation, TGF-ß1 expression by reverse transcription-polymerase chain reaction (RT-PCR), and TGF-ß1 protein by ELISA. In addition, cell accumulation of AGEs was studied by immunohistochemical staining of the AGE imidazolone.
Results. AGE-BSA inhibited [3H]thymidine incorporation, lowered cell number and increased cell protein content as well as TGF-ß1 mRNA and protein as compared with control and BSA. Immunohistochemical staining revealed a marked intracellular accumulation of the AGE imidazolone. Co-incubation of AGE-BSA with trypsin ameliorated the impaired thymidine incorporation, the decreased cell count and the enhanced cell protein content. TGF-ß1 overexpression was normalized, while TGF-ß1 protein declined insignificantly. Intracellular imidazolone accumulation was strikingly suppressed.
Conclusions. In the tubule cell line LLC-PK1, AGE-BSA exerts an antiproliferative effect, most probably due to TGF-ß1 overproduction. The co-administration of trypsin abrogated this alteration, very likely as a result of an interaction with AGE-binding protein(s), which is supported by the decreased intracellular AGE accumulation. These findings may be the starting point for the development of specific proteolytic enzymes to interfere with the interaction between AGEs and their receptors/binding proteins.
Keywords: AGEs; cell proliferation; imidazolone; TGF-ß1 mRNA; trypsin and tubule cells
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Introduction |
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To counteract the toxic intra-/extracellular effects of AGEs, numerous drugs were developed and studied in in vitro and in vivo experiments. These include blockers of AGE formation (aminoguanidine [12] and the thiazolidine derivative OPB-9195 [13]), the protein cross-link breaker N-phenacylthiazolium bromide [14], anti-oxidants (taurine, vitamin E, alpha-lipoic acid and pyridoxamine [15]), as well as the administration of the soluble receptor of AGEs [16]. Another approach could be the blocking of AGE binding to their cell surface receptors by proteolytic enzymes. Thus, in cultured endothelial and lung cells, the AGERAGE interaction is suppressed by the serine protease trypsin, associated with decreased oxidative stress [9,17]. It is conceivable that the protease-induced lowering of AGE-binding also protects renal cells from maladaptive responses, such as the enhanced cytokine/growth factor formation and their consequences. Therefore, in the current study, we investigated the potential modulatory action of the serine protease trypsin on various AGE effects. Our interest focused on TGF-ß1 formation, which is of fundamental importance in the development of cell hypertrophy and accumulation of ECM [18,19]. Since tubular cells are the main target of renal hypertrophy in diabetes, we examined the porcine tubule cell line LLC-PK1. Surprisingly, nothing is known about AGE effects on TGF-ß1 expression in the tubule cells. The involvement of this growth factor seems to be probable, since exposure to AGE-BSA was followed by alterations characteristic of TGF-ß1 effects, such as cell hypertrophy, impaired protein degradation and lowered cathepsin activity [20]. Additionally, we studied the intracellular accumulation of the AGE imidazolone in the presence and absence of trypsin.
The results obtained show that the exposure of LLC-PK1 cells to AGE-BSA enhanced cell protein content and increased the expression of TGF-ß1 in a dose-dependent manner. Cell proliferation was reduced. Co-administration of trypsin abrogated these alterations. Immunohistochemical staining showed a marked intracellular AGE accumulation, which was lowered noticeably by trypsin, indicating a blockade of cellular AGE uptake.
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Materials and methods |
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AGE-modified BSA was characterized by immunological and spectroscopical methods. The preparation was recognized by two different AGE-specific antibodies using both the ELISA (imidazolone and carboxymethyllysine antibodies) and western blot techniques (data not shown). The AGE-BSA showed fluorescence spectra typical for AGEs (max emission=440 nm at an excitation wave-length of 370 nm). After purification by chromatography on a polymyxin column (Pierce, Germany) no endotoxin could be detected by the Limulus Amebocyte Lysate (LAL) test (E-TOXATE; Sigma).
Cell culture and experimental treatment
LLC-PK1 cells, a porcine cell line that exhibits the properties of proximal tubules, were grown at 37°C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagles medium (DMEM) with 100 mg/dl glucose supplemented with 10% foetal calf serum (FCS; Gibco-BRL), 25 mM Hepes, 100 U/ml penicillin and 100 ng/ml streptomycin. Cells were plated separately on 100 mm dishes for isolation of RNA or six-well plates for the cell proliferation assay.
After reaching 7080% confluence, the subconfluent cells were synchronized by serum-free medium (SFM) for 24 h, followed by treatment with vehicle (control), BSA or AGE-BSA (1976 µM) related to BSA content with or without trypsin (0.6255 µg/ml) and high glucose (450 mg/dl).
Examination of a potential AGE-BSA degradation by trypsin
Two AGE-BSA batches and unmodified BSA (25 µM in PBS) were incubated with a high dose of trypsin (50 µg/ml; MUCOS GmbH, Geretsried, Germany) for 24 h at 37°C. In neither case was any degradation observed after sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE). Prolonged incubation for >72 h under the same conditions caused significant degradation, but some undegraded AGE-BSA still was present after this time (data not shown).
Cytotoxicity was evaluated by both Trypan Blue exclusion and release of lactate dehydrogenase.
Cell number was analysed using the Casy-1 System (Schaerfe, Reutlingen, Germany), based on the Coulter Counter principle.
Cellular proliferation assay
Cellular proliferation was determined using [3H]thymidine incorporation. The subconfluent monolayer was quiescented by being grown in SFM for 24 h, then incubated for 12, 16, 20 and 24 h, respectively, in SFM in the absence (control) or presence of AGE-BSA or non-glycated BSA with or without trypsin at various concentrations, as well as an anti-TGF-ß1 antibody. During the last 4 h, cells were labelled with 0.5 µCi per well of [3H]thymidine (50 µCi/mmol; Amersham, Germany). The cells were rapidly washed three times with ice-cold PBS and solubilized in 2% SDS, followed by precipitation with 1 ml of 20% trichloroacetic acid (TCA). The precipitates were collected with a cell harvester onto a glass microfibre filter (Schleicher Schuel, Dassel, Germany) and washed sequentially with 10 and 5% TCA, and finally with ethanol. Incorporated [3H]thymidine was measured in a liquid scintillation counter.
Cell protein content
Total protein concentration in cell lysate was measured using bicinochoninic acid (BSA protein assay; Pierce, Bonn, Germany [22]).
Measurement of TGF-ß1 expression by reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from subcultured LLC-PK1 cells treated with vehicle, BSA or AGE-BSA in the presence or absence of trypsin by single step method [23]. The purity and concentration was determined by measuring the optical densities at 260 and 280 nm. The A260/A280 ratio ranged from 1.70 to 1.95. The integrity of RNA was confirmed by electrophoresis on 1% agarose gels, and the RT-PCR reaction was performed as described previously [24].
Briefly, cDNA was synthesized by reverse transcription from 1 µg of total RNA in a volume of 25 µl containing 1xFirst-Strand synthesis buffer (10 mM Tris-HCI pH 9.0, 50 mM KCI, 1.5 mM MgCI), 1 mM of dNTPs, 0.5 µl of ribonuclease inhibitor (40 U/µl), 0.5 µl of Maloney murine leukemia virus (MMLV) reverse transcriptase (50 U/µl, Stratagene, La Jolla, CA), and 0.4 ng/µl of random primers for 1 h at 37°C.
PCR amplification was performed by the standard procedure using primers (sense 5'-CTGAGGCTCAAGTTAAAAG-3' and anti-sense 5'-GAACCCGTTAATTTCCAC-3') deduced from the TGF-ß1 sequence of pigs, giving a PCR product size of 246 bp [25]. GAPDH cDNA was co-amplified as an internal control using the following primer sequences (5'3'); the sense 5'CGGAGTCAACGGATTTGGTCG-3' and anti-sense 5'AGCCTTCTCCATGGTGGTGA AGAC-3' with a final product size of 306 bp [23]. Both TGF-ß1 and GAPDH were amplified for 35 cycles using the following conditions: denaturation at 94°C for 30 s, annealing at 55°C for 45 s and extension at 72°C for 1 min. The PCR products were analysed by 2% agarose gel electrophoresis and quantified by densitometry. A sample of RNA not subjected to RT-PCR was used as a negative control in each experiment.
TGF-ß1 protein and bioactivity assay
After incubation with AGE-BSA (38 µM) for 24 h, total TGF-ß1 protein was determined in the supernatant of cultured cells by an ELISA assay according to commercial instruction (Promega, Heidelberg, Germany). Its concentration was related to cell number.
For the evaluation of the bioactivity of TGF-ß1 induced by AGE-BSA, the cell protein concentration as well as the antiproliferative effect was investigated after co-incubation with the polyclonal rabbit antibody of TGF-ß1 (Promega) according to Wolf et al. [26]. The LLC-PK1 cells were made quiescent for 24 h in serum-free DMEM and were then incubated for an additional period of 24 h with AGE-BSA either alone or in the presence of 20 µg/ml of the neutralizing anti-TGF-ß1 antibody. Incorporation of [3H]thymidine into LLC-PK1 cells was used for measurement of cell proliferation as described before.
Immunohistochemical detection of imidazolone
To evaluate the cellular accumulation of AGEs, immunostaining of imidazolone (a common epitope of AGE-modified proteins [27]) was performed using the peroxidaseantiperoxidase method with the monoclonal anti-imidazolone (AG-1) antibody as follows. Cell culture medium was removed and cells washed with PBS. Cultured cells were mixed with 1.5 ml fibrin glue (Tissuecol Duo S; Immuno GmbH, Heidelberg, Germany), scraped off, transferred into reactive tubes and centrifuged at 1000 g for 10 s. The pellet was coagulated with a drop of a thrombin solution (Immuno GmbH). A spoon of cells in a cup were fixed with 0.1 M PBS containing 4% paraformaldehyde for 1 h at 4°C, washed three times for 30 min in PBS, further fixed in 40%, 70%, 96% and absolute ethanol as well as xylol for 2030 min, embedded in paraplast, cut (5 µm sections) and mounted on silane-coated slides. The sections were dewaxed, dried overnight and irridiated with microwaves in 0.01 M sodium citrate buffer (pH 6.0), twice for 5 min at 850 W. After being washed in PBS (pH 7.4) the sections were treated with 0.3% hydrogen peroxide for 30 min, incubated with respective normal sera and then incubated for 1 h at 37°C with primary antibodies. After removing the normal rabbit sera, the sections were incubated with monoclonal anti-imidazolone antibody at 4°C overnight. Thereafter, they were washed with PBS and incubated with biotinylated secondary antibodies followed by a streptavidin/biotin-peroxidase complex (Veceastain Elste, Vector Burlingame, CA, USA) at 37°C for 30 min. They were then washed twice with PBS and the sections were completed by the addition of diaminobenzidine-H2O2 solution for 530 min. After being washed with PBS, the slides were counterstained with haematoxylin.
Analysis of the sections was carried out in an unbiased manner by means of a C.A.S.T. Grid system (Olympus, Albertslund, Denmark) (for more details see [28]). All cells were evaluated on the television screen monitor at a final magnification of x3453 (objective used x100, oil, Numerical Aperture (NA)=1.35) that came into focus with unbiased counting frames systematically, randomly spaced throughout the sections (area of the counting frames 1477 µm2). The distance between counting frames in mutually orthogonal directions x and y was 100 µm. Accordingly, on average, 350 cells were evaluated per section, which were found in
64 unbiased counting frames per section. Degree of imidazolone staining was divided into four rates: negative (-), weak positive (+), intermediate positive (++) and strong positive (+++). Afterwards, negative and weak positive data were rated as negative, and the others were counted as positive.
Statistical analysis
The mean for the replicates of each experiment was determined. Results are presented as mean±SD, with n indicating the number of experiments. A comparison between the two groups was made using paired and unpaired t-test. Group differences (more than three) were evaluated employing analysis of variance (ANOVA). Statistical significance was defined as a P<0.05.
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Results |
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Cell protein content and cell volume
Cell protein content rose from 0.730±0.07 ng/cell (control) to 0.894±0.06 ng/cell after AGE-BSA (P<0.05), and BSA incubation resulted in an insignificant rise to 0.806±0.01 ng/cell. After co-incubation of AGE-BSA with trypsin, the cell protein content was nearly normalized (0.772±0.05 ng/cell). Cell volume showed, after AGE-BSA, a significant rise from 2.828±0.10 to 3.092±0.102 fl (P<0.05), and was nearly normalized by co-incubation with trypsin (2.633±0.098 fl). After BSA incubation, cell volume did not change (2.612±0.097 fl).
Expression of TGF-ß1 mRNA in LLC-PK1 cells
We hypothesized that the AGE-BSA-induced cell effects could be mediated, at least in part, by a rise in endogenous TGF-ß1 production. Therefore, TGF-ß1 gene expression and TGF-ß1 protein in the supernatant of cell culture were investigated. The results revealed induction of TGF-ß1 mRNA by AGE-BSA (38 µM) as well as by high glucose medium (30 mmol/l glucose). AGE-BSA increased TGF-ß1 mRNA levels standardized to GAPDH mRNA levels by 5.3±1.2 (serum-free; n=6) and high glucose by 4.1±0.9 of control (n=6; Figure 2). Elevated expression of TGF-ß1 mRNA transcripts was not significantly changed after prolongation of the incubation period to 72 h (data not shown). AGE-BSA (19, 38 and 76 µM) increased the expression of TGF-ß1 mRNA after 24 h in a concentration-dependent manner (Figure 3
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Effect of AGE-BSA on TGF-ß1 protein
As shown in Figure 4 , the incubation of LLC-PK1 cells with AGE-BSA (38 µM) resulted in a significant rise of total TGF-ß1 protein in the supernatant in relation to cell number after 24 h. Co-incubation with trypsin (2.5 µg/ml) weakened this effect.
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Neutralizing TGF-ß1 antibody against the AGE-BSA-induced antiproliferative effect
To assess whether the AGE-BSA-induced cellular responses are mediated by TGF-ß1, a neutralizing anti-TGF-ß1 antibody was used. Addition of 20 µg/ml to AGE-BSA (38 µM)-containing media increased thymidine incorporation to levels that approximated the basal thymidine incorporation in cells grown in control medium; the antibody itself did not influence cell proliferation (Figure 5) and prevented the increase of cell protein content. Thus, the AGE-BSA-induced rise of cell protein from 0.40±0.04 to 0.49±0.02 ng/cell (P<0.05) was reduced to 0.42±0.01 ng/cell (P<0.01) after co-incubation with the neutralizing anti-TGF-ß1 antibody. These responses suggest that the growth inhibitory action of AGE-BSA was predominantly attributable to endogenous TGF-ß1 bioactivity.
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Immunohistochemical evaluation of AGE accumulation in the cells
Potential AGE accumulation in the cells was evaluated by an immunohistochemical detection method for the AGE imidazolone [31], which demonstrated in the AGE-BSA-treated group a mean positive staining of 65.5% of the cells, which averaged only to 34.4% after co-incubation of AGE-BSA with trypsin (Figure 6).
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Discussion |
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In accordance with our recent studies [20], a rise in cell protein content and cell volume was found after incubation with AGEs. Incubation with BSA, on the other hand, did not alter these parameters significantly.
As a next step, we could show that the incubation of LLC-PK1 cells with AGE-BSA increased expression of TGF-ß1 mRNA in a dose-dependent manner. In addition, a significant rise in TGF-ß1 protein could be demonstrated in the supernatant 24 h after AGE incubation, which indicates that the expression of TGF-ß1 was translated into protein. The physiological relevance of increased TGF-ß1 synthesis was evaluated by co-incubation with a neutralizing anti-TGF-ß1-antibody, which led to a reversal of both the AGE-enhanced cell protein content and the antiproliferative action.
Our data on increased TGF-ß1 formation is consistent with similar in vitro studies in rodent mesangial cells after incubation with AGE-albumin [32], as well as in vivo investigations in the glomeruli of normal rodents after parenteral administration of AGE-albumin [33]. The AGE-BSA-induced TGF-ß1 formation is of particular interest with regard to renal hypertrophy in the early stages of DN and the later development of renal fibrosis [2] and corresponds to our earlier finding of an impaired protease activity (cathepsin B+L activity) after incubation of LLC-PK1 cells with AGE-BSA [20]. Currently, activation of transcription factor by AGEs has been reported for the major inducible transcription factor NF-B via the RAGE receptor [11]. In our own investigations in LLC-PK1 cells, we could demonstrate an activation of p42MAP-kinase (Erk) as well as its downstream target, the AP1-complex, after incubation with AGE-BSA but not with BSA alone [34]. This indicates that AGEs appear to induce specific signal transduction pathways in LLC-PK1 cells.
Co-incubation of AGE-BSA with trypsin ameliorated the anti-proliferative action of AGE-BSA as well as the enhanced cell protein content. Moreover, TGF-ß1 expression was markedly reduced. The elevated TGF-ß1 protein levels showed only a tendency to lower values. This surprising observation can be attributed to various factors. One possible explanation is that the observation period might have been insufficient to detect a significant decline of TGF-ß1 protein.
Concerning the modulatory effect of trypsin on tubule cell function, a partial degradation of AGE-BSA into two smaller fragments, with a consequent decline of cellular uptake, could be involved. This possibility could be excluded by incubation of two AGE-BSA batches with a high concentration of trypsin. Within an observation period of 24 h, no degradation could be detected.
Therefore, we assume that the trypsin-mediated effects result from an impaired interaction between AGEs and their binding proteins/receptors at the tubule cell surface, as formerly demonstrated for endothelial cells by binding studies [9,17]. Our findings of the immunostaining of the AGE imidazolone in the tubule cells are in line with this data. By use of an imidazolone-specific antibody, a marked staining of the tubule cells could be demonstrated after AGE-BSA incubation, which was reduced by >50% in the presence of trypsin. Hence, it can be deduced that the amelioration of the AGE-BSA effects observed in our study resulted in particular from a decreased cellular AGE content. Trypsin is a widely used protease for enzymatic digestion of fixed tissues to enhance immunostaining. In our study, there was a strong fixation and paraffin-embedding step between the biological experiment (e.g. trypsin treatment of living cells) and the immunohistochemical detection of AGEs. Therefore, later interference of trypsin with the immunodetection of AGEs can be excluded.
Currently, we cannot offer data concerning the specificity and selectivity of the trypsin action. It is likely that other proteases, such as certain metalloproteases (MMPs), produce similar effects or even higher specificity and affinity to the AGE-binding proteins. Thus MMPs have been implicated in the cleavage of TNF- and other receptors [35]. Therefore the findings presented here may be the starting point for the development of new specific proteolytic enzymes.
In conclusion, our data shows that AGE-BSA exerts marked effects on LLC-PK1 cells, most likely due to enhanced formation of TGF-ß1. These alterations are substantially reversed by co-administration of trypsin, which lowers the intracellular accumulation of the AGE imidazolone. Thus, an interaction of this serine protease with AGE-binding proteins on the surface of LLC-PK1 cells is assumed.
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Notes |
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References |
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