1 Division of Nephrology, Department of Internal Medicine IV, J. W. Goethe-University, Frankfurt/M., and 2 Division of Nephrology, Department of Medicine II, Hospital München Harlaching, Germany
Correspondence and offprint requests to: Patrick C. Baer, Johann Wolfgang Goethe University, Department of Internal Medicine IV, Division of Nephrology, Theodor Stern-Kai 7, D-60590 Frankfurt/M., Germany.
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Abstract |
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Methods.Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.2550 µM) or MPA plus guanosine (100 µM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1ß+IFN+TNF
=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis.
Results.MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression.
Conclusions.We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.
Keywords: distal tubule; epithelial cells; human; mycophenolic acid; proliferation; proximal tubule
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Introduction |
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Renal tubular epithelial cells play a fundamental role in modulating inflammatory responses during inflammatory kidney diseases, autoimmune disorders and renal allograft rejection. Expression of chemokines, human leukocyte antigens, and adhesion molecules may be involved in renal infiltration by lymphocytes, monocytes and granulocytes, thus amplifying inflammatory diseases [810]. A family of monocyte-specific chemoattractant and activating cytokines (chemokines) has been described to be upregulated during graft rejection, including the chemokine regulated upon activation, normal T cell expressed and secreted (RANTES) [8]. RANTES is a member of the superfamily of proinflammatory cytokines called CC chemokines that is a chemoattractant for memory T cells, monocytes, and eosinophils [11]. Moreover, recognition of foreign antigens by T lymphocytes during these immune responses requires the presence of human leukocyte antigens (HLA) on antigen-presenting cells [12]. The immunoglobulin-like molecule intercellular adhesion molecule-1 (ICAM-1) increases the avidity of T lymphocytes interactions with antigen-presenting cells bearing HLA antigens [12]. Proinflammatory cytokines like -interferon (
IFN), interleukin 1ß (IL1ß), and tumour necrosis factor
(TNF
) were found to be potent inductors of the expression of different cell surface antigens and soluble factors like RANTES, HLA-DR, and ICAM-1 in vitro [1214]. Inhibition of this cross-talk between infiltrating cells and local renal cells by immunosuppressant drugs may result in an improvement in the treatment of transplant rejection.
Therefore the object of this study was to investigate the effect of MPA on proliferation and cytokine-induced antigen expression on highly purified proximal and distal tubular cells from human kidney.
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Subjects and methods |
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Isolation, culture and stimulation of human proximal and distal tubular cells
Human proximal (PTC) and distal tubular cells (DTC) were separated using antibody coated magnetic beads as described previously [15]. Cells were prepared after tumour nephrectomies from those portions of the human kidney not involved in renal-cell carcinoma. The tissue was minced into approximately 1 mm2 pieces and digested with RPMI 1640 containing collagenase/dispase (1 mg/ml) at 37°C for 1 h. The digested tissue fragments were passed through a 106 µm mesh and incubated for 45 min with RPMI 1640 supplemented with collagenase IV (1 mg/ml), DNase (100 µg/ml) and MgCl2 (5 mM). After Percoll density gradient centrifugation, the cell pellet was preincubated in 10 ml ice-cold RPMI 1640 for 20 min with human immunoglobulin G (hIgG; 2.5 mg/ml) added. Cells were incubated for 20 min on ice with the primary antibody (in RPMI plus hIgG; 5 µg/107 cells). To enrich PTC we used a mAb against aminopeptidase M (CD13), specific for the proximal tubule. DTC were isolated through a mAb recognizing Tamm Horsfall glycoprotein (THG), a specific antigen of the thick ascending limb of Henle's loop and the early distal convoluted tubule. Then, cells were washed with PBS containing 5 mM EDTA and 0.5% BSA. Finally, cells were incubated with secondary antibody for 20 min, washed, passed through a fine mesh (pore size 40 µm), and isolated by immunomagnetic separation applying the Mini-MACS system [15].
After immunomagnetic isolation cells were seeded in six well plates precoated with human collagen IV (20 µg/ml). The cells were grown in medium 199 with 10% FCS at 37°C and 5% CO2 in a humidified atmosphere. The culture medium was supplemented with the antibiotic meropenem (100 µg/ml) during the first 23 days after isolation. Culture medium was changed every 34 days.
Proximal primary isolated cells were strongly positive for aminopeptidase M (98.6%); however, cells of the distal portion were strictly negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparatus and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and fewer apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin. Stainings for desmin, smooth-muscle actin, a fibroblast-specific marker, and von Willebrand factor to exclude the possibility of contaminating cells were negative [15]. In vitro differentiation of PTC and DTC was shown by scanning electron microscopy and flow cytometry [16, Baer et al., unpublished data]. Activity of brush border enzyme alkaline phosphatase was detectable in cultured proximal cells only, cultured distal cells revealed no activity [15]. A strong expression of the epidermal growth factor receptor in cultured DTC was shown, whereas the signal in cultured PTC was low [Baer et al., unpublished data]. Cultured cells displayed different adenylate cyclase responsiveness to hormonal stimulation. Parathyroid hormone (10-6 M) increased cAMP production in DTC up to 32.8-fold of the basal level, in PTC up to 3.5-fold only. Calcitonin (10-6 M) stimulated adenylate cyclase in DTC 4.3x, whereas only a low calcitonin response was found in PTC. Arginine-vasopressin (10-6 M) increased the distal cAMP production 1.9-fold of the basal level; the proximal cAMP production was negligible [15].
Assay for cytotoxicity
As an indicator of cytotoxicity, the activity of lactate dehydrogenase in cell culture supernatants of PTC and DTC was determined by a commercial assay, measuring the decrease of absorbance at 340 nm, resulting from conversion of NADH to NAD in the presence of pyruvate. Supernatants from cell cultures treated with MPA were collected and assayed for LDH release.
Fluorometric assay
For the measurement of the total cell number, a fluorometric assay with the DNA-intercalating fluorochrome DAPI (4,6-diamino-2-phenylindole) was used [17]. These measurements represent cell numbers at a specific time point of cell culture in media supplemented with MPA or MPA plus guanosine. Cells (2x103) were seeded in medium 199 with 10% FCS and different concentrations of MPA (0.25 µM, 2.5 µM, 25 µM, 50 µM) in 96-well microtitre plates and cultured for 96 h. Medium 199 with 10% FCS served as a positive control, and medium 199 without FCS served as a negative control. The cells were washed twice with PBS, and lysed with 100 µl buffer (0.02% w/v SDS, 150 mM NaCl, 15 mM sodium citrate) at RT for 15 min. Finally 100 µl DAPI (2 µg/ml) was added to each well, and incubated at RT for 5 min. Fluorescence was measured in relative fluorescence units (FU) in a FluoStar fluorescence reader (BMG, Offenburg, Germany). A 355 nm excitation filter and a 460 nm emission filter were used. All results were expressed as mean values and standard deviations (n=6).
Stimulation of RANTES, HLA-DR, and ICAM-1 and treatment with mycophenolic acid
A combination of proinflammatory cytokines, -interferon (
IFN; 200 U/ml), interleukin 1ß (IL1ß; 25 U/ml) and tumour necrosis factor
(TNF
; 10 ng/ml), was used for stimulation. Because our previous studies showed that a combination of cytokines resulted in a strong RANTES induction, we also used this cytomix in the current study.
Cells were grown in 24-well culture dishes until confluence. For stimulation of RANTES protein expression, cells were washed and stimulated with a mix of all three cytokines (cytomix) for 48 h in medium 199 without FCS. Stimulation of HLA-DR and ICAM-1 expression was done with cytomix for 96 h. To test the influence of MPA on the cytomix-induced expression of RANTES, HLA-DR, and ICAM-1 cells were pre-treated with media containing MPA (0.25 µM, 2.5 µM, 25 µM, 50 µM) at 37°C for 2 h. The cells were washed twice and incubated with the cytomix together with MPA. As a control, cells were incubated with medium 199 alone (basal) or with cytomix plus dexamethasone (10 µM). Supernatants were collected after 48 h for RANTES quantification, or cells were harvested after 96 h for flow cytometric analysis of HLA-DR and ICAM-1 expression.
For determinations of RANTES protein, supernatants were collected and assessed for the chemokine or stored at -20°C for later measurement. Cell number was assessed from trypsinized suspensions and counted in a Neubauer chamber. For flow cytometrical determination of HLA-DR and ICAM-1, cells were detached by trypsin/EDTA treatment for exactly 7 min, followed by immunostaining (see section `Flow cytometry').
Quantification of RANTES expression
The chemokine RANTES was quantified using sandwich enzyme-linked immunosorbent assay. In brief, wells of 96-well microtitre plates were coated with an anti-human RANTES mAb overnight at room temperature. Non-specific binding sites were blocked with PBS/1% BSA/5% sucrose. After 1 h of incubation the plates were washed with PBS/0.05% Tween. Standard and samples were added to the wells and incubated for 2 h at room temperature. The plates were washed and incubated with biotinylated rabbit anti-RANTES for 2 h at room temperature, washed again and incubated with horseradishperoxidasestreptavidin for 20 min. After washing, substrate reaction was followed by measurement of the optical density at 450 nm. Recombinant human RANTES was used as a standard.
Flow cytometry
Confluent monolayers were washed twice with PBS. Suspensions were prepared by incubation with 0.05% trypsin and 0.02% EDTA for 7 min. The cells were washed twice with PBS. Aliquots of single cell suspension (1 to 2x105 cells) were incubated for 15 min at room temperature with directly labelled antibodies. Cells were washed with 2 ml PBS and pelleted by centrifugation at 300 g for 5 min. Cells were fixed in 1% paraformaldehyde. The labelled cells were analysed using a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany). All investigations included negative controls without antibodies and with non-relevant antibodies to evaluate the positive staining profile. The data of the level of fluorescence intensity, forwards and sideways (90°) scatter of 105 cells were processed using WinMDI Flow Cytometry Application Version 2.5 analysis software. Median of fluorescence intensity was calculated. Antigen expression of MPA-treated cells was correlated with the cytomix-stimulated antigen expression (100%). Each cell purification procedure was taken as a single, independent experiment, i.e. the number `n' in statistical analysis refers to the number of separate isolations.
Statistical analysis
The data of RANTES protein release were expressed as means±SD. For statistical analysis, Student's t-test for unpaired samples was used. P values <0.05 were considered significant; P values <0.01 were considered highly significant.
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Results |
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After 4 days of cell culture, a significant decrease in total cell number (compared to normal cell culture medium as a control) was seen when MPA was added in a high concentration (50 µM: PTC -74.6%/DTC -74.8%; 25 µM: -71.8/-71.9; 2.5 µM -62.9/-51.9) (Figure 1A, B). This inhibition of cell growth was abolished by the addition of guanosine, indicating the inhibitory effect of MPA on IMPDH. The lower MPA concentration (0.25 µM) had no effect on tubular cell growth (Figure 1A, B
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Discussion |
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Only limited data about the effects of MPA on renal tubular epithelial cells, a pivotal cell type involved in the rejection process, are available. Major side-effects of MPA therapy, i.e. gastrointestinal disorders with diarrhoea and eosinophilic infiltration indicate the possible effects of MPA on epithelial cells [21]. In this study, we investigated the effects of MPA on human renal proximal and distal tubular epithelial cells in vitro. In renal transplant recipients, therapeutically given MPA reaches the tubular epithelium in the graft at the basolateral cell membranes and therefore may affect tubular cell metabolism. The concentrations of MPA used in this in vitro study are in the range of blood concentrations obtained in vivo (cmax=up to 26 µg/ml) [22].
The effect of MPA on lymphocyte proliferation and the molecular mechanisms underlying this effect are well described [7,23]. In case of the action mechanism of MPA by specific inhibition of purine de novo synthesis, the antiproliferative effect should be lymphocyte-specific. In-vitro studies showed the total inhibition of DNA synthesis in mitogen-stimulated lymphocytes [24]. MPA depletes GTP in human peripheral blood monocytes, but not in neutrophils [7]. Nevertheless, an inhibitory effect on cell proliferation of smooth-muscle, endothelial, and mesangial cells has been described [25,26], whereas Eugui et al. found no inhibition of endothelial and fibroblast cell proliferation [27]. In mice, MPA prevented DNA synthesis in lymph nodes but not in testicular germinal cells and basal epithelial cells of the small intestine [7,28]. In our study, we could demonstrate that the total number of human tubular epithelial cells decreased after MPA treatment due to an inhibition of tubular cell proliferation. The abolition of this effect by guanosine supplementation conforms to the described action mode of MPA.
In renal and cardiac allografts, the chemokine RANTES is expressed at high levels 1 week after transplantation and correlates with the influx of T cells and macrophages during rejection [8,2931]. The expression and secretion of RANTES by cytokine-treated tubular epithelial cells and other cell types, and the inhibition by glucocorticoids has been shown in different studies [14,3234]. Therefore our interest was to evaluate the effect of MPA on this cytokine-induced chemokine expression. Incubation of tubular cells with cytokines and MPA resulted in a synergistic upregulation of RANTES secretion, whereas dexamethasone inhibited the cytomix-induced RANTES production. This synergistic effect of cytokines plus MPA seems not to be restricted to tubular epithelial cells since it could be demonstrated in human endothelial cells (unpublished data). The mechanisms of the synergistic effect of MPA on RANTES expression are not currently known. Recent data revealed that an inhibition of nitric oxide (NO) results in an upregulation of LPS-stimulated RANTES expression [34]. Others described the inhibition of cytokine-induced NO production by MPA, and suggested that the activity of NO synthase is dependent on cellular GTP level [35]. We found a partial abolition of the synergistic effect by additional incubation with guanosine. We postulate that inhibition of tubular cell NO production by MPA is responsible for the synergistic upregulation of RANTES through MPA and the cytomix. The clearest benefit of MPA is in the post-transplantation period, where a combination therapy of MPA with cyclosporin A and steroids reduces biopsy-proven acute rejection [1].
A possible downregulation of RANTES expression during rejection episodes in patients treated with a combination therapy may be due to the action of steroids only. Therefore the synergistic effect of cytokines and MPA may not result in clinical implications during the treatment of rejection episodes. Furthermore, the main effect of MPA is on leukocytes and the inhibition of their proliferation results not only in a reduced infiltration but also in a reduced release of cytokines in the transplant.
Recognition of foreign antigens by T cells requires the presence of human leukocyte antigens (HLA) on antigen-presenting cells. The avidity of this interaction may be increased by the expression of other cell surface proteins such as intercellular adhesion molecule-1 (ICAM-1) [12]. Moreover, antigen-independent interactions between T cells or monocytes and their target cells are essential for generating and affecting the immune response [9]. This cellular cross-talk requires adhesion molecules like ICAM-1 on the membranes of the target cells [9,12,36]. The relationship between ICAM-1 and HLA (class II antigens) in immune responses is synergistic, and in cases of limiting HLA expression, ICAM-1 may be essential for antigen presentation [12]. De novo expression of HLA-DR and ICAM-1 by tubular epithelial cells during allograft rejection is well known [9,37,38]. Furthermore, the expression of these molecules by IFN-stimulated tubular cells in culture has been described [9,12].
Since ICAM-1 and HLA-DR are co-expressed in transplant rejection and other inflammatory conditions in the human kidney, we studied whether MPA affects the stimulated expression of these antigens. In vitro, MPA has been shown to prevent the binding of activated human lymphocytes to activated endothelial cells, demonstrating its ability to inhibit recruitment and subsequent actions at the potential sites of rejection [7,40]. MPA has been described to block the glycosylation of glycoproteins involved in the adhesion of leukocytes to their target cells [5,28]. In contrast, MPA induced ICAM-1 expression on human endothelial cells, may be due to a marked GTP depletion and UTP increase, since both are responsible for the synthesis of glycoproteins [40]. MPA enhances TNF-induced VCAM-1 and E-selectin expression on endothelial cells by selectively increasing the mRNA-stability of these cell adhesion molecules [41]. In kidney-transplanted rats MPA reduced the number of cells positive for HLA-DR molecules [19]. Thomson et al. found no effect on the expression of cell activation molecules like HLA-DR on stimulated lymphocytes in vitro by MPA [42]. No significant effect of MPA on the cytokine-induced antigen expression of HLA-DR and ICAM-1 could be proven in our study. On the other hand, a significant inhibition of HLA-DR expression, but not ICAM-1 by dexamethasone was detectable. Other in vitro studies are in good agreement with these results, and showed that
IFN-induced HLA-DR expression can be inhibited by dexamethasone, whereas ICAM-1 expression was not significantly reduced [12,13].
These data demonstrate that apart from its immunosuppressive and antiproliferative effects on lymphocytes, MPA differentially affects the renal tubular epithelium by the inhibition of cell proliferation and the upregulation of cytokine-induced chemokine production. The clinical relevance of the synergistic upregulation of tubular RANTES synthesis seems to be minimized because MPA is used in combination with other immunosuppressive agents like steroids and is not as a single therapy. Furthermore, we showed that the elevated expression of HLA and ICAM-1 is not downregulated by MPA. In conclusion, the immunosuppressive actions of MPA appear to be independent from the effect on the tubular epithelium.
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Acknowledgments |
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References |
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