1 Department of Clinical Sciences, Division of Nephrology, University La Sapienza, Rome, 2 Institute of Histology, and 3 Department of Medical and Surgical Sciences, Division of Nephrology, University of Padua, Padua, Italy
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Abstract |
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Methods. HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcriptionpolymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor ß1 (TGF-ß1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG.
Results. U937 adhesion at 13 h was increased in HG (from 54.9±6.6 to 87.1±5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993±505 µ2 with 1.2±0.1 hillocks/high-power field, which increased to 13 651± 1114 µ2 with 0.5±0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9±1.7 vs 4.2±0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-ß1 (+590%) were observed in co-cultures in HG. COL IV and TGF-ß1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1±0.6 vs 4.0±0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response.
Conclusions. High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes.
Keywords: collagen; diabetic nephropathy; matrix; mesangium; metalloproteinase; monocytes
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Introduction |
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The pathogenesis of diabetic nephropathy (DN) is unclear; a number of mechanisms have been suggested, and genetic susceptibility is strongly suspected. Whatever the cause(s), after a variable length of time glomerular sclerosis ensues, resulting from the abnormal synthesis of fibrous matrix by glomerular cells, which eventually occupies the entire capillary space [1,2].
Many experimental studies have shown that renal cells grown in high glucose (HG) media, mimicking the diabetic microenvironment, exhibit impaired sensitivity to vasoconstrictors, reduced proliferation rates, and a tendency towards differentiation in a secretory phenotype [35]. This results into deposition of extracellular matrix, reminiscent of nodular proliferation in vivo. Altered phospholipase C signalling and particularly increased protein kinase C translocation to the plasma membrane are early biochemical events possibly encoding for such phenotypic changes [36]. Increased biosynthesis of transforming growth factor-ß1 (TGF-ß1) is an additional hallmark of in vitro diabetes models, probably responsible for enhanced matrix deposition [5,7,8].
While many morphological and biological features of the mesangial cell (MC) in DM have been thoroughly investigated, a still-neglected issue of both experimental and human DN [9,10] is mesangial infiltration by lymphomonocytes/macrophages. Evidence from the experimental streptozotocin model suggests that the infiltration, which occurs very early, is specifically due to metabolic intermediates of insulin deficiency, being prevented by its administration [9,10].
The interaction between leukocytes and resident glomerular cells has been the subject of extensive investigation in recent years in spontaneous and experimental inflammatory nephropathies, placing emphasis on the cross-talk between infiltrating and intrinsic cells with respect to cellular events such as apoptosis, proliferation, and matrix synthesis [11,12]. In view of the potential importance of the monocytic infiltration of diabetic kidneys as well, we sought to combine a co-culture approach employing undifferentiated monocytic cells of the U937 line, which spontaneously adhere to cultured human MC (HMC) [11,12], with a high glucose culture environment, in order to answer the following questions: (i) does HG potentiate leukocyte chemoattraction onto HMC? (ii) is such interaction responsible for altered survival of the latter? (iii) does co-culture enhance matrix accumulation by increasing protein synthesis or by inhibiting degradation through metalloproteinases, suggesting that monocytes may initiate glomerulosclerosis?
Our results indicate that monocytes indeed modify the proliferative behaviour, survival and matrix accumulation of cultured HMC in HG, with HG acting to promote their influx in the diabetic kidney. Therefore, infiltration of leukocytes should be indeed considered a factor in the progression of DN.
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Subjects and methods |
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Monocyte adhesion assay
Undifferentiated myelomonocytic leukaemia cells of the U937 line (ATCC code CRL 1593) were grown in suspension cultures employing the same FBS-supplemented RPMI 1640 medium. Cultures were expanded by seeding 2x106 cells into 25 ml in 75 cm2 flasks (Costar, Cambridge, MA) every 72 h. Fresh medium was added every 24 h. Prior to an experiment, the suspension was centrifuged at 1000 g for 10 min, the cells rinsed once in serum-free RPMI 1640, counted and spun again before final resuspension at the density of 2x106 cells/ml. The U937 cells were then layered onto confluent monolayers of HMC in 24-well plates (total volume 500 µl/well). After incubation at 37°C for 3 h, chosen on the basis of preliminary time-course experiments, the suspension was aspirated, and non-adherent U937 cells were dislodged by rinsing the monolayers three times with sterile phosphate-buffered saline (PBS, pH 7.3). The co-cultures were then fixed with 70% ethyl alcohol (v/v) followed by staining with the conventional MayGrünwaldGiemsa method.
Image analysis microscopy
Co-cultured U937 cells and HMC grown on 8-well glass Lab-Tek chamber slides (Nunc, Naperville, IL) were stained by MayGrünwaldGiemsa after fixation in 70% ethyl alcohol. The slides were then examined on a Zeiss Axioplan inverted-stage microscope equipped with an image analysis set-up based on ImageMeasure 5200 (Microscience Inc.) software in an ASEM 386 personal computer. Outlining of randomly chosen cells for cross-sectional area was performed with a hand-held device in a blinded fashion by an operator viewing the enlarged optical field directly on the computer screen. The following parameters were examined: (i) U937 and HMC cell number per surface area units; (ii) number of hillocks of HMC, defined as nodular, stellate proliferation areas around a central core of amorphous matrix [13]; (iii) cross-sectional area of HMC, easily distinguished from U937 monocytes due to their spread, >20-fold larger surface; (iv) number of apoptotic HMC, as evidenced by chromatin condensation indicative of DNA fragmentation (see following paragraph).
Detection of apoptotic cells
Cells undergoing DNA fragmentation as a result of apoptosisor programmed cell deathwere detected by in situ nick-end labelling according to the TUNEL technique. Following fixation of cells on 8-well Lab-Tek chamber-slides with 4% paraformaldehyde in 0.1 mol NaH2PO4, pH 7.4 for 60 min at room temperature, the slides were treated with 20 µg/ml proteinase K for 30 min at 37°C. After rinsing with 2 d H2O, DNA nick-end labelling was carried out with terminal transferase (TdT) for 60 min at 37°C, followed by incubation with avidinFITC and counterstaining with 0.5 µg/ml propidium iodide for 15 min at 4°C. Tween 20-permeabilized cells (0.5% for 30 min) treated with 1 µg/ml DNase I for 1 h at 37°C served as positive control. All reagents were supplied by Biomolecular Co., Torrance, CA; (kit DN-001). Fluorescence microphotographs of randomly chosen fields were later analysed for percentage of TUNEL+ cells out of the total number of cells counted.
Reverse transcriptionpolymerase chain reaction (RTPCR)
RTPCR was used for comparing the expression of mRNA for the following: 1 chain of type IV collagen, matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP), tissue inhibitor 1 of MMP (TIMP-1), TIMP-2 and urokinase plasminogen activator (uPA), and TGF-ß1, according to specific methods previously described [14] and after normalization of each value against the corresponding GAPDH expression (Table 1
). Cultured cells were suspended in Ultraspec solution, and total RNA was extracted using the RNAzol method (Ultraspec RNA, Biotecx). RNA yield and purity were checked by spectrophotometric determinations at 260 and 280 nm. Reverse transcription of 5 mg of total RNA was carried out as already described [14]. The cDNA solution was then diluted 1:10, divided into aliquots in amplification tubes and stored at -20°C until PCR. A typical PCR-reaction mixture was prepared as follows: 1.5 U of Taq DNA polymerase (5000 units/ml, Pharmacia) was added to 5 ml of reaction buffer 10x (Pharmacia), 1 ml of dNTP (10 mmol/l each), 2 ml of primers (5 mmol/l each) as described elsewhere [14], 20 ml of cDNA dilution (see below) and water to 50 ml final volume. Amplification was performed in sequential cycles including 1 min denaturation at 94°C, annealing conditions as shown in Table 1, and 1 min extension at 72°C; after the last cycle, all samples were incubated for an additional 10 min at 72°C. For each target molecule, preliminary linear range-finding experiments were performed in all specimens by PCR-amplifying increasing amount of cDNA (six dilutions from 60 pg to 24 ng of total RNA) for 25 cycles using fixed reaction conditions (see above), and the appropriate first-third calibration curve dilution was used for PCR amplification. Following amplification, triplicate 20 ml aliquots were electrophoresed in 7.5% polyacrylamide gel (19:1 acrylamide:bis-acrylamide) and PCR products highlighted by silver staining (Silver Staining Plus Kit, Bio-Rad, Hercules CA). Densitometric evaluation was performed in a GS300 Scanning Densitometer (Hoefer, San Francisco CA). The linear range of amplification was again verified for each target molecule in highly scoring samples. The means of triplicate amplicons were used for comparisons; when values of triplicates differed more than 15%, the amplification was repeated.
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Zymography
Gelatinolytic activity in cell-conditioned media was assayed as described [15]; 13 µg of cell pellet was analysed by 0.1% gelatin zymography. The gels were stained with Coomassie Brilliant Blue R-250, and clear bands represented areas of gelatinolysis. Culture medium conditioned by HT1080 melanoma cells was co-electrophoresed as control to identify pro- and active MMP-2 and MMP-9 [15]. Digestion bands were analysed by a GS300 Hoefer Scanning Densitometer. The pixel intensity for each band was analysed with imaging software.
ELISA for type IV collagen
Type IV collagen (COL IV) was measured by a sandwich ELISA commercial kit using two monoclonal antibodies to the 7S domain and to the non-7S and non-NC1 domains (Fuji Chemical Industries, Ltd, Toyama, Japan). In brief, each well of a microtitre plate was coated with 100 µl of monoclonal antibody solution (100 µg IgG/ml) at 4°C overnight. This antibody recognizes the NH2-terminal 7S domain of the chain of COL IV. In another non-coated plate 50 µl culture medium or standard antigen solution was incubated with 200 µl peroxidase-labelled Fab' (80 ng/100 µl). This antibody recognizes the central triple-helical domain near the COOH terminus of COL IV. Subsequently, 100 µl of the sample mixture was transferred to the washed antibody-coated plate and incubated for 1 h at room temperature. After washing the plate, 100 µl of the enzyme substrate containing 4 mg/ml O-phenylene-diamine and 0.02% H2O2 was added and incubated at room temperature for 15 min. The reaction was stopped by adding 100 µl 1.33 N H2SO4 and absorbance at 500 nm was measured to calculate the medium concentration of COL IV.
Statistics
Data are expressed as mean±SE unless otherwise stated. In all morphological experiments, comparisons were carried out by one-way analysis of variance (ANOVA), with significance set at P<0.05 or greater. RTPCR and ELISA data were evaluated by unpaired Student's t-test following normalization for the 3:1 HMC/U937 ratio in the co-cultures.
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Results |
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As inferred from Table 2, HMCs co-cultured with monocytes in NG did not show modification of their COL IV expression rate; its apparent decrease is indeed explained by the dilution effect by U937 cells (nd). On the contrary, this gene was over-expressed up to 137% in HG (P=0.036) (Figure 4
). Upregulation of COL IV was confirmed by ELISA (+78% in HG vs NG, P=0.0033, Table 3
). In co-culture conditions, compared to the sum of separate contributions, a striking increase of MMP-9 mRNA levels was observed, in both NG and HG (three- and sixfold respectively, P=0.001 for both conditions); MMP-9 upregulation was confirmed by zymography (Table 3
). No apparent modification of MMP-2 and TIMP-2 mRNA and MMP-2 protein expression, considering the dilution effect by U937 cells, was noted, as also confirmed by zymography (Table 3
). On the contrary, expression of MMP-1, uPA, and TGF-ß1 was remarkably different between NG and HG: it was less than 20 and 50% (all P<0.005) in NG respectively, and more than twofold higher in HG for all markers (P=0.002; P=0.002, P=0.004 respectively), compared to the sum of the individual contributions. TIMP-1 was significantly downregulated in NG (P=0.02), while in HG no modification was apparent. Also the MMP-1/TIMP-1 and MMP-1/TIMP-2 mRNA ratios were modulated in a fashion similar to MMP-1 alone, being higher in HG (P<0.001 for both ratios vs NG). The MMP-2/TIMP-2 mRNA ratio was not substantially modified (Figure 5
).
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Discussion |
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Our co-culture model was designed to reproduce some aspects of the early glomerular changes occurring in DM. U937 cells were previously shown by us and other laboratories to duplicate most functional features of peripheral blood monocytes, including adhesion to cultured mesangial cells through ligation of specific adhesion molecules [11,12,16]. One important advantage of using the U937 line is the unlimited supply of viable cells with a reproducible phenotype, quite different from freshly isolated cells from healthy donors, suffering from intra- and inter-individual variability. Early infiltration in vivo is most probably driven by the glomerular expression of the adhesion molecules VCAM-1 and ICAM-1, along with the chemotactic factor MCP-1 [11]. This is in keeping with previous observations by our group and others, demonstrating that the in vitro interaction between U937 cells and HMCs occurs through ICAM-1/ VCAM-1 and the corresponding leukocyte counter-receptors LFA-1/VLA-4 [11,12,16]. That the diabetic milieu, and particularly in HG conditions, induced mesangial cell hypertrophy is a well-known phenomenon [5,7,8]. Our data suggest that the hypertrophic effect is mostly due to HG, while the interaction with monocytes does not seem to further contribute. Co-culture in HG is rather associated with reduced proliferation in response to an initial decrease in the number of HMC. This proliferative pattern, consistent with earlier findings in NG media, possibly reflects a rapid cytotoxic effect of U937 cells on HMC, followed by a reparative response at longer intervals. We have previously provided evidence for a direct, cell-mediated type of injury, involving the release of reactive oxygen species [12], although other mechanisms such as toxic cytokines or pore-forming membrane ligands could also be implicated. Obviously, these effect of U937 cells require their functional integrity; indeed, they are largely vital in the continued presence of FBS, with evidence of apoptosis by TUNEL assay in only 10.9±1.2% of U937 cells in the final suspension.
The co-culture model permits a convenient separation of the two components of this cellular interaction, that is monocytes and HMC, along with the products arising from their interplay. Only when total RNA is extracted from co-cultures it is not possible to identify the precise source, as adhesion of monocytes is irreversible and physical isolation of the two populations cannot be accomplished without disrupting the cells. On the other hand, U937 cells do not express collagen, which therefore represents an example of a gene upregulated by co-culture in a selected population, that is, HMC. The opposite situation applies to MMP-9, which is only expressed in U937 cells. Other genes reflect the contribution of both cell lines, and transcription in co-culture should be evaluated against each individual line at the corresponding NG/HG condition.
As often seen, hypertrophic cell changes generally involve differentiation towards a synthetic phenotype and reduced proliferation rates. This is the case for co-cultured HMC in HG, as demonstrated by the increased expression of COL IV (+137% increase above NG) and TGF-ß1 genes (sevenfold); the parallel antimitogenic effect of co-culture in a diabetic milieu is demonstrated by the reduced number of HMC at 48 h and hillocks in HG vs NG. The dramatic increase of TGF-ß1 gene expression most probably drives these phenotypic changes [7,8].
Low apoptotic rates observed in the HMC co-cultured at 48 h probably contribute to the parallel increase in cell number observed in NG; to the contrary, stable rates of apoptosis may account for the reduced cellularity in response to co-culture in HG. TGF-ß can also mediate apoptosis in a number of cells, among which MC [5,7,8]. The present results on the lack of effect of HG on apoptosis of pure HMC cultures, as well as of HMC/U937 co-cultures, seem to suggest that apoptosis is not a pivotal phenomenon in the pathogenesis of diabetic glomerulosclerosis.
Contrary to most inflammatory glomerulopathies, diabetic mesangial expansion is due to extracellular matrix deposition rather than hypertrophic and/or hyperplastic changes of MC. This is a dual process, involving both an abnormal synthetic and degradative activity of the MC, possibly related to overexpression of growth factors, particularly TGF-ß1 [7,8]. Such upregulation has for example been reported for COL IV, in what is considered a central event in the process of diabetic glomerulosclerosis [5]. Results from this study confirm the above data, and furthermore disclose a synergism between MC and monocytes in HG, responsible for a rather massive expression of the profibrogenetic growth factor TGF-ß1 (+100% compared to separate HMC cultures in NG).
MMPs are a family of metalloendopeptidases engaged, in conjunction with tissue serine proteinases (i.e. uPA), in the turnover of matrix in the pericellular environment. Acting in concert, the MMPs have the capability to degrade all the components of extracellular matrix. MMPs can be activated by both uPA and MT1-MMP, and inhibited by TIMPs [18]; disruption of the balance between these molecules can lead to either net matrix degradation or accrual. In DN, downregulation of degradative processes has been proposed as a leading phenomenon in mesangial expansion. In this cell co-culture system, while MT1-MMP is not expressed, uPA has the potential to activate gelatinases, although apparently not as a crucial limiting step; despite overexpression of the uPA gene in co-cultures in HG, this is not paralleled by a corresponding increase of activated gelatinases in the zymography. Our data also suggest overzealous matrix degradation in HG co-cultures. Indeed, the mRNA ratios MMP-1/TIMP-2 and MMP-1/TIMP-1, uPA, and MMP-9 mRNA were all consistently increased in these conditions. Accordingly, overexpression of the TGF-ß1 gene and the resulting increased COL IV synthesis observed in our in vitro model of the diabetic glomerulus may constitute one of the two components of a balance leading to renal scarring.
Diabetic conditions do not seem to uniformly affect all MMPs, as shown by the reduced expression of MMP-2 and constancy of the MMP-2/TIMP-2 ratio, confirming previous in vitro results on MC cultures, in animal models, and in diabetic patients [18]. The dramatically increased expression of MMP-9 in HG co-cultures compared with separate U937 cells in HG (+647%) and with NG conditions (+397%), is consistent with the recent report by Ebihara et al. [19], who demonstrated that increased plasma levels of MMP-9 precede the development of microalbuminuria in NIDDM patients; since MMP-9 is prominently produced and secreted by macrophages, it was suggested that macrophages infiltrating the glomerulus are the source of increased levels of MMP-9. Furthermore, it was speculated that this infiltration is causally linked to the onset of microalbuminuria, due to the effect of macrophage MMP-9 on glomerular endothelial cells.
Based on the current findings, the interaction with monocyte/macrophages would seem at first deleterious for the mesangium. However, another reading of the data is also possible. In trying to link our experimental model to the in vivo situation, the normal glomerular mesangium, with generally scanty monocytes/ macrophages, might correspond to separate HMC cultures in NG, while the diabetic mesangium might correspond to co-cultures in HG. The comparison between these two conditions would be therefore more sensible, in pathophysiological terms, than between pure HMC and co-cultures both in NG or HG. Thus, it can be seen from Figures 3a,c
that HMC number and the percentage apoptotic are not significantly different between NG controls and co-cultures in HG at 48 h. It appears that monocytes may indeed normalize cell turnover in HG, a condition in which the number of cells generally decreases. Furthermore, the reduction of the number of hillocks might suggest a beneficial effect of monocytes on HMC growth in diabetic conditions, if one considers hillocks as an in vitro correlate of glomerulosclerosis [13]. Supporting this idea is also the observation that, notwithstanding the elevated synthesis of COL IV, matrix degradation actually increases, as shown by higher expression of MMP-9, uPA, MMP-1/TIMP-1, and MMP-1/ TIMP-2, and the constant level of MMP-2/ TIMP-2. The effect of macrophage supernatants on hillock formation has been investigated by Mattana et al. [20]. They demonstrated that in NG, number and size of hillocks are enhanced in a concentration-related manner by the addition of macrophage supernatants. Our data suggest that U937 cells may have in HG a normalizing effect on HMC function and growth, since they reduce their tendency to form nodular foci of exaggerated growth of cells and matrix deposition.
In conclusion, although the precise contribution of inflammatory monocyte/macrophage infiltration of renal tissues on the pathogenesis of DN is still unclear, the present results support the concept that in a diabetic microenvironment infiltrating leukocytes modulate the synthetic and proliferative phenotype of MC, and that they could play an important role in the progression towards renal failure. Further studies are warranted to gain a better understanding of this generally underexamined pathological feature of DN.
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Acknowledgments |
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Notes |
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References |
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