Cyclosporine A up-regulates the expression of TGF-ß1 and its receptors type I and type II in rat mesangial cells

Johannes Waiser, Kerstin Dell, Torsten Böhler, Ertan Dogu, Jens Gaedeke, Klemens Budde and Hans-Hellmut Neumayer

Department of Internal Medicine–Nephrology, University Hospital Charité, Campus Charité Mitte, Humboldt-University, Berlin, Germany



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Chronic cyclosporine A (CsA) nephropathy is a well described side effect of CsA treatment. CsA has been shown to induce the synthesis of extracellular matrix (ECM) proteins in mesangial cells (MCs) in vitro, and glomerulosclerosis in vivo. Transforming growth factor-ß1 (TGF-ß1) is a potent stimulus for the synthesis of ECM proteins in MCs. We investigated whether CsA up-regulates the expression of TGF-ß1 and its receptors type I (TßR-I) and type II (TßR-II) in cultured rat MCs, and whether this effect translates into enhanced matrix protein accumulation.

Methods. Resting MCs were incubated in the presence or absence of CsA and anti-TGF-ß1 antibodies. Time- and concentration-dependent expression of TGF-ß1, TßR-I and TßR-II were measured at both the mRNA (competitive reverse transcription PCR) and protein level (enzyme-linked immunosorbent assay (ELISA) and western blotting). Fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) synthesis were measured by ELISA.

Results. Compared with untreated controls, CsA stimulated mRNA production of TGF-ß1 (maximum at 72 h, 500 ng/ml CsA: 2.1±0.5-fold, P<0.001) and TßR-II (maximum at 72 h, 1000 ng/ml CsA: 2.4±0.4-fold, P<0.005) time- and dose-dependently. TßR-I mRNA concentrations remained unchanged. Protein concentrations were analysed at 96 h: TGF-ß1, 220±32 vs 86±24 pg/ml, P<0.001 (500 ng/ml CsA vs control); TßR-I, 2.0±0.5-fold, P<0.005 (1000 ng/ml CsA vs control); TßR-II, 2.5±0.7-fold, P<0.05 (1000 ng/ml CsA vs control). CsA (500 ng/ml) also enhanced the production of FN (1.6-fold, P<0.05) and PAI-1 (2.0-fold, P<0.05). Co-incubation with neutralizing anti-TGF-ß1 antibodies reduced (P<0.05) CsA-induced expression of TßR-I (1.0±0.1-fold), TßR-II (1.3±0.1-fold) and PAI-1 (1.3-fold), but not FN production (1.6-fold).

Conclusions. Pharmacologically relevant concentrations of CsA time- and dose-dependently up-regulate the expression of TGF-ß1 and, via autocrine mechanisms, its receptors type I and II in rat MCs. Whereas up-regulation of PAI-1 is mediated by TGF-ß1, up-regulation of FN is—at least in part—either directly induced by CsA or mediated by factors other than TGF-ß1.

Keywords: cyclosporine A; glomerulosclerosis; mesangial cells; TGF-ß



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cyclosporine A (CsA) is one of the most important factors in assuring the success of renal transplantation [1]. However, treatment with CsA is associated with chronic CsA nephrotoxicity [2]. Perico et al. demonstrated that CsA induces glomerulosclerosis after renal transplantation in rats [3]. Functionally, each oral dose of CsA is followed by a transient and dose-related reduction in renal plasma flow and glomerular filtration rate that results from acute reversible renal hypoperfusion due to afferent renal arteriolar vasoconstriction. Structurally, CsA causes glomerulosclerosis by up-regulation of extracellular matrix (ECM) protein expression in various cell types, including mesangial cells (MCs) [4]. Pathogenetically, the development of CsA-induced glomerulosclerosis seems to be a result of both a state of persistent tissue ischaemia and a direct stimulatory effect on the synthesis of ECM proteins.

Existing evidence supports an important role for the multifunctional cytokine transforming growth factor-ß (TGF-ß) in the pathogenesis of chronic CsA nephropathy [5]. TGF-ß is a potent stimulus for the synthesis of ECM proteins in various cell types [6], including MCs [7]. Numerous cell types including MCs produce TGF-ß and have receptors for it. TGF-ß is released as an inactive precursor, in a complex bound with a latency-associated peptide. Its activity is controlled at different levels. Plasmin, thrombospondin and reactive oxygen species (ROS) have been shown to release active TGF-ß from its complex. In addition, active TGF-ß can be inhibited by local factors such as decorin. In humans, three isoforms of TGF-ß have been described. TGF-ß1 is the one most implicated in tissue fibrosis [6]. Three classes of TGF-ß receptors are known, but only two of these are involved in signalling pathways [8]. TßR-III is a membrane protein that modulates the binding of TGFß-1 to the signalling receptors. TßR-II binds TGF-ß1 and then forms a hetero-oligomeric complex with TßR-I. Activated TßR-II transphosphorylates the glycine- and serine-rich domain of the TßR-I kinase, thereby activating TßR-I. TßR-I then associates with and phosphorylates a class of transcription factors known as Smad proteins.

In vitro, CsA has been shown to stimulate the synthesis of TGF-ß1 in T-lymphocytes [9], in proximal tubular cells and in tubulo-interstitial fibroblasts [10]. In rats, CsA enhances the expression of TGF-ß1, particularly in the tubulo-interstitial and vascular compartments [11]. In mice, CsA augments intrarenal expression of TGF-ß1 [12]. Extending these results, Khanna et al. showed in vivo that TGF-ß1 mimics CsA-induced nephrotoxicity, while treatment with anti-TGF-ß1 antibodies abrogates CsA nephrotoxicity [13]. The potential role of TGF-ß in chronic CsA nephrotoxicity has been illustrated further by clinical studies. Pankewycz et al. observed a higher level of TGF-ß protein expression in renal allograft biopsy specimens from patients with CsA nephrotoxicity as compared with patients with acute cellular rejection or acute tubular necrosis [14]. In renal biopsy tissue from patients with non-renal transplant CsA nephropathy, Langham et al. recently showed an increased expression of TGF-ß1-inducible ßig-H3 protein in distal convoluted tubular epithelium and parietal glomerular epithelium [15]. These findings underline the fact that TGF-ß1 is a key fibrogenic cytokine in the development of CsA nephropathy. Yamamoto et al. found a correlation between the expression of all three TGF-ß isoforms and pathological accumulation of ECM proteins in glomerular diseases characterized by ECM accumulation [16]. Hence, CsA seems to stimulate the formation of ECM proteins through the induction of TGF-ß protein expression, thus promoting the progression of renal fibrosis. Like many other cell types, MCs are able to produce TGF-ß [17]. To date, it has not been investigated whether pharmacologically relevant doses of CsA modulate the expression of TGF-ß1 and its main receptors in MCs. Because this pathway may play an important role in the pathogenesis of CsA-induced glomerulosclerosis, we addressed this question.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Cell culture
Primary cultures of rat MCs were obtained from outgrowths of collagenase-treated isolated rat renal glomeruli as described previously [18]. Briefly, kidneys from male Sprague–Dawley rats (Tierzucht Schönwalde, Berlin, Germany) weighing 250 g were excised. Renal cortices were minced and glomeruli were isolated by serial sieving. After incubation with bacterial collagenase (type IV, 750 µg/ml; Sigma chemical Co., St Louis, MO, USA) for 30 min at 37°C, washed glomerular remnants were resuspended in complete Dulbecco's Modified Eagle's Medium (DMEM; Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated foetal calf serum (FCS; Biochrom), 100 U/ml penicillin (Sigma), 100 µg/ml streptomycin (Sigma), 2 mM L-glutamine (Biochrom) and 0.2 IU/ml insulin. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. These cells were considered MCs using standard morphological and immunohistochemical criteria [18]. Subcultures revealed stable morphological and functional features.

Test conditions
For the experiments, MCs (1.5x105/ml) between the 4th and 12th passage were used. First, MCs were starved for 24 h in serum- and insulin-free complete DMEM to make them quiescent. Afterwards, MCs were cultured in serum- and insulin-free DMEM in the presence of CsA or vehicle. CsA was dissolved in EtOH (vehicle) at a final EtOH concentration of 0.1% (v/v). The following CsA concentrations were used: 100, 500 and 1000 ng/ml. Incubation times (TGF-ß1, TßR-I and TßR-II) were 8, 24, 48 and 72 h for mRNA measurements, and 96 h for protein measurements, respectively. TßR-I protein concentrations were additionally measured after 24, 48 and 72 h incubation.

Primers, antigens, antibodies and blocking peptides
Reverse transcription–polymerase chain reaction (RT–PCR) primers were synthesized by TIB MOLBIOL (Berlin, Germany). As confirmed by a DDBJ/EMBL/Genbank search for homology to other genes (BLAST at NCBI, Bethesda, MD, USA), all primers proved to be specific. The primer sequences are shown in Table 1Go.


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Table 1.  Primer sequences for the construction of internal cDNA control reference standards and for competitive PCR

 
For western blotting, all antibodies and blocking peptides were purchased from Santa Cruz Biotechnology (Heidelberg, Germany): polyclonal rabbit anti-TßR-I (1 µg/ml), polyclonal rabbit anti-TßR-II (1 µg/ml) and horseradish-peroxidase-conjugated goat anti-rabbit (0.4 µg/ml). The specificity of primary antibodies was confirmed by using non-specific rabbit IgG (1 µg/ml) as primary antibody. In additional control experiments, primary antibodies were pre-saturated with a 10-fold concentration of the appropriate synthetic blocking peptide (TßR-I and TßR-II) to verify specificity.

Fibronectin (FN) enzyme-linked immunosorbent assay (ELISA) was performed using the following. Antigen: purified rat FN (200 µg/ml; Sigma); competition antibody: rabbit anti-FN, IgG (1 µg/ml; Dako Diagnostica GmbH, Hamburg, Germany); detection antibody: peroxidase-conjugated goat anti-rabbit, IgG (0.4 µg/ml; Dianova, Hamburg, Germany); substrate: O-phenylenediamine-dihydrochloride (OPD) tablet sets (Sigma FastTM; Sigma), used according to the manufacturer's instructions.

Plasminogen activator inhibitor type-1 (PAI-1) ELISA was performed using the following. Antigen: recombinant rat PAI-1 (10 µg/ml; American Diagnostica, Greenwich, CT, USA); competition antibody: rabbit anti-PAI-1, IgG (1.6 µg/ml; American Diagnostica); detection antibody: peroxidase-conjugated goat anti-rabbit, IgG (0.4 µg/ml; Dianova); substrate: OPD tablet sets (Sigma FastTM; Sigma), used according to the manufacturer's instructions.

For blocking of TGF-ß1 activity, a neutralizing mouse monoclonal antibody against TGF-ß1 was used (IgG1, 10 µg/ml; R&D Systems Inc., Wiesbaden, Germany). A non-specific mouse monoclonal antibody (IgG1, 10 µg/ml; R&D Systems Inc.) served as isotype control.

RNA extraction and reverse transcription
Total RNA from cultured MCs was extracted with Trizol® according to the manufacturer's protocol (Life Technologies, Grand Island, NY, USA). RNA was reverse transcribed into single-stranded wild-type cDNA (ss-wt-cDNA) according to the following protocol. The standard reaction mixture (20 µl) contained 1 µg RNA template, 2.5 µM random hexamer primers, 2 µl PCR buffer II (10x: 100 mM Tris–HCl, pH 8.3, 500 mM KCl), 5 mM MgCl2, 1 mM dNTP mixture, 2.5 U/µl MuLV reverse transcriptase and 1 U/µl RNase inhibitor in DEPC water. Reagents were preincubated for 10 min at 25°C. Reverse transcription was performed for 45 min at 42°C and then stopped by heating to 95°C for 5 min. All reagents for reverse transcription were purchased from Perkin Elmer (Branchburg, NJ, USA).

Generation of internal cDNA competitive reference standards (CRS)
After reverse transcription, ss-wt-cDNA was subjected to PCR with modified primer pairs (Table 1Go). The downstream primers were complementary to the mRNA strands. Their 3' ends were linked to an additional sequence, complementary to a region 50–70 base pairs (bp) upstream. This resulted in a 50–70 bp deletion in the final PCR product. The upstream primers were identical in sequence to the mRNA strands except that their 5' end was coupled with a T7 promotor sequence. The PCR reagent mixture (20 µl) contained 2 µl ss-wt-cDNA, 1 µM of each primer, 2 µl PCR buffer II (10x), 2–2.5 mM MgCl2 according to the optimized PCR profile, 0.5 mM dNTP and 1.25 U Ampli-Taq-Gold® polymerase (Perkin Elmer) in distilled water. After an initial 10 min denaturation step at 95°C, PCR with 35 cycles was carried out. Each cycle consisted of a 30 s denaturation step at 94°C and a 45 s annealing step (TGF-ß1 at 71°C, TßR-I at 62°C, TßR-II at 65°C and GAPDH at 70°C), followed by an extension at 72°C for 1 min. After the last cycle the extension step was prolonged to 7 min. Afterwards, the PCR products were separated on a 2% agarose gel and stained with ethidium bromide. In order to ensure identical PCR conditions for the template and the CRS, a ss-cDNA CRS was generated. Therefore, ds-cDNA bands were excised from the gel, eluted using the Qiagen Gel Extraction Kit® (Qiagen, Hilden, Germany) and transcribed into cRNA with the Riboprobe T7 system® (Promega, Madison, WI, USA). The amount of extracted cRNA was determined by UV spectrophotometry at 260 nm and 0.5 µg cRNA were reverse transcribed into the internal ss-cDNA CRS. Finally, the concentration of ss-cDNA was determined.

Competitive PCR
Constant amounts of ss-wt-cDNA (2 µl) together with serial dilutions (1:5) of ss-cDNA CRS (TGF-ß1, 0.25 µg; TßR-I and -II, 1 µg; GAPDH, 2 µg) were co-amplified in 20 µl of PCR mixture utilizing a single set of primers (Table 1Go). The PCR mixture contained 1 µM of each primer, 2 µl PCR buffer II (10x), 2–2.5 mM MgCl2 according to the optimized PCR-profile, 0.5 mM dNTP and 1.25 U Ampli-Taq-Gold® polymerase (Perkin Elmer) in distilled water. After starting at 95°C for 10 min, amplification was performed with the following temperature profile: 30 s denaturation at 94°C, 30 s annealing at 60°C (TGF-ß1, TßR-II and GAPDH) or 54°C (TßR-I), followed by 1 min of extension at 72°C. After the last cycle an additional extension step at 72°C for 7 min was carried out.

PCR products were then directed to agarose gel electrophoresis (2%), visualized by ethidium bromide staining and photographed with a digital camera (Kodak DC 40, Rochester, NY, USA). Two different bands were distinguishable due to the smaller size of the cDNA CRS as compared with target cDNA. Band intensities were densitometrically analyzed using the 1D Image Analysis Software® (Kodak Digital ScienceTM). Results are expressed as relative amounts of target mRNA (x-fold) from CsA-treated MCs as compared with control MCs, incubated in the absence of CsA [19]. The values of target mRNA were normalized to the expression of GAPDH mRNA.

ELISA
The amount of protein in culture supernatants was measured using specific ELISA systems. Supernatants of MCs were harvested, centrifuged at 10 000 g for 7 min to remove any cell debris and stored at -80°C. Total TGF-ß1 production was measured after acid activation using a commercially available kit (Duo-Set®; R&D Systems Inc.) according to the manufacturer's instructions. FN and PAI-1 synthesis were measured with modified inhibitory ELISA assays according to published standard protocols [20]. Optical density was determined at 450 nm.

SDS–PAGE and western blotting of TßR-I and TßR-II
MCs were washed twice with PBS and solubilized in lysis buffer (50 mM Tris–HCl, pH 8.0, containing 150 mM NaCl, 10 µg/ml phenylmethylsulfonylfluoride (PMSF), 10 µg/ml aprotinin and 1% Triton X). After incubation for 1 h on ice, samples were centrifuged at 10 000 g for 10 min at 4°C and the supernatants were collected. Total protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA) and then adjusted to 30 µg/sample. Samples were mixed (5:1) with 6x sample buffer (7 ml 4x Tris–HCl (0.5 M Tris base, pH 6.8), 1 g sodium dodecyl sulfate (SDS), 3.8 g glycerol (30%), 0.5 M dithiothreitol (DTT) and 1.2 mg bromophenol blue), boiled for 5 min at 95°C, separated on a 12% SDS polyacrylamide Laemmli mini-gel and then transferred to a polyvinyl membrane (Serva/Boehringer, Ingelheim, Heidelberg, Germany) in a mini-electrotransfer unit (Hoefer, Freiburg, Germany) at 100 V, 400 mA, for 1.5 h in 192 mM glycine, 25 mM Tris–HCl, pH 8.3 and 20% methanol. Blots were blocked overnight at 4°C in 5% non-fat dry milk in 1x Tris-buffered saline (TBS) plus 0.1% Tween 20 (TBS-T). The membranes were then probed with polyclonal rabbit anti-TßR-I (overnight) or with polyclonal rabbit anti-TßR-II (1.5 h). After washing four times (10 min) with TBS-T, the blots were incubated for 1 h at room temperature with horseradish-peroxidase-conjugated goat anti-rabbit secondary antibodies. After washing (three times with TBS-T, once with TBS), the immunoreactive bands were visualized with the enhanced chemiluminescence (ECL) system according to the manufacturer's recommendations (Amersham, Little Chalfont, UK). Finally, immunoblots were exposed to a Hybond-ECL film (Amersham). Quantitative calculation of western blots was carried out by densitometric analysis of the band intensities using the 1D Image Analysis Software® (Kodak Digital ScienceTM). Negative controls were obtained by pre-saturating the primary antibodies with a 10-fold concentration of the appropriate synthetic blocking peptide (TßR-I and TßR-II) and by using normal rabbit IgG as non-specific primary antibody. Results are expressed as relative amounts of protein compared with control templates from MCs incubated in the absence of CsA.

In order to separate membrane-bound from cytosolic proteins, MCs were homogenized in 1 ml of homogenization buffer (100 mmol/l KHCO3 pH 7.4, 250 mmol/l sucrose, 1 mmol/l EDTA, 10 µg/ml PMSF and 10 µg/ml aprotinin). After treatment with a Dounce homogenizator (Merck, Darmstadt, Germany), the homogenates were centrifuged at 8000 g (10 min, 4°C). The supernatant was then centrifuged at 100 000 g (1 h, 4°C). The 100 000 g supernatant was referred to as the cytosolic fraction. The pellet, representing the membrane protein fraction, was resuspended in 50 µl of homogenization buffer. Total protein concentration of both fractions was measured by the BCA method. Finally, both fractions were subjected to western blotting as described above.

Flow cytometry
Cell viability was determined by flow cytometry following staining with propidium iodide (PI; PharMingen, Hamburg, Germany). Briefly, after 96 h incubation, MCs were harvested by trypsin–EDTA digestion. After washing with PBS (4°C), 106 MCs were incubated in the presence of PI (50 µg/ml, 106 cells/10 µl, 15 min on ice). Sample fluorescence of 5x103 MCs was analyzed by flow cytometry (FACS-Calibur; Becton Dickinson GmbH, Heidelberg, Germany) within 1 h.

Statistical analysis
Results are expressed as means±SD of at least three separate experiments. Statistical differences between groups were assessed by ANOVA (analysis of variance), followed by a Bonferroni (post hoc) test. A P value <0.05 was considered statistically significant.



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Effect of CsA on the synthesis of TGF-ß1
The effect of CsA on the synthesis of TGF-ß1 mRNA in cultured rat MCs was assessed by competitive RT–PCR using an internal cDNA CRS. Time- and dose-dependent mRNA synthesis was investigated. TGF-ß1 mRNA synthesis started to increase after 48 h incubation (48 vs 24 h at 500 ng/ml CsA: 1.6±0.3- vs 1.1±0.1-fold, P<0.01) (Figure 1AGo–CGo). The maximum TGF-ß1 mRNA concentration was observed at 72 h with 500 ng/ml CsA (2.1±0.5-fold, P<0.001 compared with control). Higher CsA concentrations did not increase TGF-ß1 mRNA synthesis further. In contrast, the amount of TGF-ß1 mRNA tended to decrease when MCs were incubated in the presence of 1000 ng/ml CsA (72 h) (1000 ng/ml vs 500 ng/ml CsA: 1.7±0.3- vs 2.1±0.5-fold, P=not significant (NS)).



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Fig. 1.  Effect of CsA on TGF-ß1 expression in rat MCs. *P<0.01, **P<0.001 vs control (without CsA). (A) Time dependency of TGF-ß1 mRNA expression (relative amount compared with untreated MCs) at 500 ng/ml CsA. (B) Dose dependency of TGF-ß1 mRNA expression (relative amount compared with untreated MCs) after 72 h incubation. (C) Example of two RT-PCR blots for TGF-ß1: upper blot without CsA, lower blot in the presence of CsA (500 ng/ml). The lower row of each blot shows 1:5 dilutions of ss-cDNA competitive reference standard products. The upper row of each blot shows wild-type mRNA products. (D) Dose dependency of total TGF-ß1 protein expression after 96 h incubation. CRS, competitive reference standard; M, marker (100 bp DNA ladder).

 
To determine whether CsA affects the production of TGF-ß1 protein in MCs, total TGF-ß1 concentrations in MC supernatants were measured 24 h after the maximum mRNA concentration was reached, i.e. after 96 h incubation, by ELISA (Figure 1DGo). In agreement with the mRNA expression pattern, the maximum TGF-ß1 protein concentration was observed at 500 ng/ml CsA (500 ng/ml CsA vs control: 220.0±31.9 vs 85.6±23.6 pg/ml, P<0.001). As before, higher CsA concentrations did not result in higher TGF-ß1 protein concentrations (1000 vs 500 ng/ml CsA: 197.2±33.9 vs 220.0±31.9 pg/ml, P=NS). Co-incubation (96 h) of MCs with CsA and neutralizing antibodies against TGF-ß1 reduced measurable TGF-ß1 concentrations to baseline levels (500 ng/ml CsA: 95.2±24.4 pg/ml, P=NS vs control; 1000 ng/ml CsA: 97.1±6.0 pg/ml, P=NS vs control). Co-incubation with CsA and isotype control antibodies did not affect CsA-induced TGF-ß1 expression (data not shown).

Effect of CsA on the expression of TGFß receptor type I
The effect of CsA on the expression of TßR-I mRNA was investigated as described above. Compared with untreated MCs, no significant increase in mRNA synthesis was detectable at any timepoint or at any CsA concentration (Figure 2AGo and BGo). After 72 h incubation at 1000 ng/ml CsA, the observed TßR-I mRNA concentration did not differ from controls (0.9±0.3-fold, P=NS).



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Fig. 2.  Effect of CsA on TßR-I expression in rat MCs. *P<0.05, **P<0.005 vs control (without CsA). (A) Time dependency of TßR-I mRNA expression (relative amount compared with untreated MCs) at 1000 ng/ml CsA. (B) Dose dependency of TßR-I mRNA expression (relative amount compared with untreated MCs) after 72 h incubation. (C) Dose dependency of TßR-I protein expression after 96 h incubation (relative amount compared with untreated MCs). (D) Western blot for TßR-I.

 
TßR-I protein expression was determined after 24, 48, 72 and 96 h incubation by western blotting. After 96 h, TßR-I protein expression turned out to be dose-dependently elevated, compared with untreated controls (Figure 2CGo and DGo). Maximum protein expression was observed at 1000 ng/ml CsA (2.0±0.5-fold, P<0.005 vs control). An increased TßR-I protein expression was also observed at 500 ng/ml CsA (1.6±0.4-fold, P<0.05 vs control). At any earlier timepoint, incubation with CsA did not cause significant changes in TßR-I expression (data not shown). Incubation of MCs (96 h) in the presence of CsA (1000 ng/ml) together with antibodies directed towards TGF-ß1 reduced the expression of TßR-I down to baseline levels (1.0±0.1-fold, P=NS vs control).

Separation of cytosolic from membrane-bound proteins (resting MCs) revealed that a much higher proportion of TßR-I protein was present in the cytosolic fraction (94%) as compared with the membrane fraction (6%). Incubation of MCs with CsA (1000 ng/ml, 96 h) increased both cytosolic (1.6±0.2-fold; P<0.05 vs control) and membrane-bound TßR-I expression (4.2±0.4-fold; P<0.01 vs control).

Effect of CsA on the synthesis of TGF-ß receptor type II
Synthesis of TßR-II mRNA and protein was investigated as described for TßR-I. Maximum TßR-II mRNA expression (2.4±0.4-fold, P<0.005 vs control) was observed at 1000 ng/ml CsA after 72 h incubation (Figure 3AGo and BGo). At that timepoint, TßR-II mRNA expression was also increased in the presence of 500 ng/ml CsA (1.7±0.5-fold, P<0.05 vs control). At any earlier timepoint, no significant changes in TßR-II mRNA concentration were detectable (48 h, 1000 ng/ml CsA: 1.2±0.3-fold, P=NS vs control).



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Fig. 3.  Effect of CsA on TßR-II expression in rat MCs. *P<0.05, **P<0.005 vs control (without CsA). (A) Time dependency of TßR-II mRNA expression (relative amount compared with untreated MCs) at 1000 ng/ml CsA. (B) Dose dependency of TßR-II mRNA expression (relative amount compared with untreated MCs) after 72 h incubation. (C) Dose dependency of TßR-II protein expression after 96 h incubation. (D) Western blot for TßR-II (relative amount compared with untreated MCs).

 
TßR-II protein synthesis was measured 24 h after the maximum mRNA concentration was observed, i.e. after 96 h incubation. Compared with untreated MCs, TßR-II protein concentration was significantly elevated in the presence of 1000 ng/ml CsA (2.5±0.7-fold, P<0.05) and 500 ng/ml CsA (1.6±0.3-fold, P<0.05) (Figure 3CGo and DGo). Incubation (96 h) of MCs in the presence of CsA (1000 ng/ml) together with neutralizing antibodies against TGF-ß1 reduced the expression of TßR-II (1.3±0.1-fold, P<0.05 vs control).

Following separation of cytosolic from membrane-bound proteins (resting MCs), we found TßR-II expression only in the membrane fraction. No TßR-II protein was found in the cytosolic fraction. After incubation with CsA (1000 ng/ml, 96 h) an increased expression of membrane-bound TßR-II was observed (1.7±0.2-fold, P<0.01 vs control). As before, no TßR-II protein was found in the cytosolic fraction.

Effect of CsA on the synthesis of fibronectin and PAI-1
The effect of CsA (500 and 1000 ng/ml) on the synthesis of FN and PAI-1 was measured after 96 h incubation with modified inhibitory ELISA assays. Compared with untreated controls, CsA (500 and 1000 ng/ml) induced a 1.6-fold increase in FN synthesis (Figure 4AGo) and a 2.0-fold increase in PAI-1 synthesis (Figure 4BGo). Co-incubation together with neutralizing anti-TGF-ß1 antibodies significantly reduced CsA-induced PAI-1 production (Figure 4BGo), but not FN production (Figure 4AGo).



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Fig. 4.  Rat MCs were incubated (96 h) with or without CsA (500 or 1000 ng/ml) in the presence or absence of neutralizing anti-TGF-ß1 antibodies (10 µg/ml). *P<0.05 vs control (without CsA), #P<0.05 vs MCs incubated with CsA alone (same concentration). (A) Fibronectin synthesis. (B) PAI-1 synthesis. mAb, monoclonal antibody.

 

Effect of CsA on cell viability
Necrotic cells take up PI. PI staining of MCs followed by flow cytometric analysis revealed that serum deprivation (0% FCS) slightly increased the percentage of necrotic cells (3.5%, P<0.05 vs 10% FCS) (Figure 5Go). Incubation of resting MCs (0% FCS) in the presence of CsA (500 or 1000 ng/ml) did not cause additional changes in the percentage of necrotic cells (increase <1%, P=NS vs resting MCs incubated without CsA) (Figure 5Go).



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Fig. 5.  Effect of serum deprivation (0% FCS) and CsA (500 or 1000 ng/ml) on cell viability. After 96 h incubation, the percentage of necrotic MCs was determined by staining with propidium iodide followed by flow cytometry. (A) 10% FCS, 23.3%; (B) 0% FCS, 27.0%; (C) 0% FCS+500 ng/ml CsA, 28.2%; (D) 0% FCS+1000 ng/ml CsA, 27.6%. M1, fraction of positively stained necrotic MCs; M2, fraction of negatively stained viable MCs.

 



   Discussion
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 Materials and methods
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 Discussion
 References
 
The introduction of CsA about 20 years ago has substantially improved 1-year graft survival rates after organ transplantation [1]. However, graft survival beyond 1 year post-transplantation has not improved in a similar fashion [21]. Chronic CsA nephrotoxicity has turned out to be one of the major drawbacks of CsA treatment, limiting the clinical utility of this immunosuppressive compound [2]. Pathologically, chronic CsA nephrotoxicity is characterized by afferent arteriolopathy, tubular atrophy, striped tubulointerstitial fibrosis and glomerulosclerosis. The lesions of chronic CsA nephrotoxicity are not necessarily dose-related [2]. Current evidence indicates that increased expression of soluble mediators, such as angiotensin II, endothelin-1 and TGF-ß, is involved in the pathogenesis of chronic CsA nephrotoxicity [22]. CsA was shown to stimulate ECM protein production in MCs in vitro [4] and to cause glomerulosclerosis in vivo [3]. Furthermore, CsA induces the synthesis of TGF-ß in various cell types [911]. MCs are able to produce TGF-ß [17] and TGF-ß is a potent stimulus for ECM protein synthesis in MCs [7]. These observations prompted us to investigate whether pharmacologically relevant doses of CsA modulate the expression of TGF-ß1 and its main receptors in rat MCs in vitro.

We found that CsA time- and dose-dependently induced the expression of TGF-ß1 and its receptors type I and type II in cultured rat MCs. At the protein level, an increased expression of TGF-ß1 and its receptors was found at CsA concentrations comparable to the range of CsA blood concentrations in allograft recipients. Apart from TßR-I, all protein concentrations were determined 24 h after the maximum mRNA concentration was reached. Thus, the rationale for choosing 96 h incubation time for protein measurements was based on the fact that maximum mRNA concentrations of TGF-ß1 and TßR-II were observed after 72 h incubation. Concerning TGF ß-1 and TßR-II, the parallel course of mRNA and protein expression indicates that CsA acts at the transcriptional level. In contrast, the fact that CsA stimulated the expression of TßR-I protein without affecting mRNA levels suggests a post-transcriptional regulatory mechanism. In order to rule out that these effects were influenced by changes in cell viability, MCs were subjected to PI staining followed by flow cytometric analysis. We found that CsA treatment did not affect the viability of resting MCs.

Kleeff et al. recently demonstrated that TGF-ß1 up-regulates the expression of TßR-I and -II in COLO-357 cells [23]. We co-incubated MCs with CsA and neutralizing TGF-ß1 antibodies to find out whether such an autocrine mechanism also exists in CsA-treated MCs. Our results make clear that comparable to COLO-357 cells, TGF-ß1 mediates the expression of TßR-I and TßR-II in CsA-treated MCs.

Separation of membrane-bound and cytosolic proteins from untreated, resting MCs revealed that TßR-I was mainly present in the cytosolic fraction, whereas TßR-II was detectable in the membrane fraction only. With respect to TßR-II, incubation with CsA increased the expression of membrane-bound TßR-II without inducing any cytosolic TßR-II expression. Concerning TßR-I, CsA treatment caused a greater increase in membrane-bound TßR-I expression as compared with cytosolic TßR-I expression, thus increasing the proportion of membrane-bound TßR-I from 6 to 15%.

In order to evaluate whether up-regulation of the TGF-ß system translates into an increased ECM accumulation, we assessed the synthesis of FN and PAI-1. Compared with untreated MCs, the expression of both FN and PAI-1 was significantly increased in CsA-treated MCs. Whereas the effect of CsA on PAI-1 synthesis was reversible by co-incubation with neutralizing anti-TGF-ß1 antibodies, FN expression remained unchanged. These results indicate that: (i) activation of the TGF-ß system in MCs plays a role in CsA-induced glomerulosclerosis; (ii) other profibrotic mediators also seem to be involved in the pathogenesis of this process.

The clinical relevance of the effects described here becomes clear by comparison with similar experiments, in which the effect of CsA on the production of TGF-ß1 was investigated in other cell types. Ahuja et al. [9] reported that TGF-ß1 mRNA levels were 2- to 4-fold higher in activated human T-lymphocytes pre-treated with CsA (10–1000 ng/ml) compared with controls. In the same system, TGF-ß1 protein secretion was increased by 40–80% in the presence of CsA. Wolf et al. [10] showed that CsA dose-dependently stimulated the expression of TGF-ß1 in proximal tubular cells at both the mRNA and the protein level (1000 ng/ml: 4- and 2-fold, respectively). In comparison, we found a 2.5-fold increase in the expression of TGF-ß1 protein (500 ng/ml CsA). Thus, the amount of TGF-ß1 produced by MCs after stimulation with CsA was within the same range as that shown previously for T lymphocytes and proximal tubular cells. Additionally, we were able to show that CsA up-regulates protein expression of TßR-I and TßR-II (2- and 2.5-fold, respectively, at 1000 ng/ml CsA). The fact that both the effector molecule and its receptors are up-regulated at the protein level may result in an additive effect through autocrine mechanisms [17]. Taken together, these observations indicate that CsA-induced up-regulation of TGF-ß1 and its receptors in various cell types at least in part mediate CsA-induced glomerulosclerosis. This conclusion is supported by the results of Khanna et al., who showed in vivo that histopathological changes caused by CsA treatment can be prevented by anti-TGF-ß1 antibodies [13]. Campistol [22] pointed out that chronic CsA nephrotoxicity is caused by a combination of acute haemodynamic changes (functional nephrotoxicity) and direct ‘toxic’ effects. Consequently, functional and structural CsA nephrotoxicity should not be regarded as distinct entities but as interrelated processes that influence each other. In addition to this, TGF-ß also plays an important role in the pathogenesis of chronic allograft nephropathy [24]. Therefore, CsA-induced overexpression of TGF-ß also seems to be a pathogenetic link between chronic CsA nephropathy and chronic allograft nephropathy [25].

Di Paolo et al. [26] investigated the influence of CsA on the expression of TGF-ß1 in human MCs in culture. They found an increased expression of TGF-ß1 mRNA (2- to 3-fold) and protein (3-fold) in MCs treated with 2.5 µg/ml CsA. Unfortunately, they did not investigate the effect of lower, pharmacologically more relevant CsA concentrations. Recently, Fernoni et al. [27] investigated the effect of CsA on murine MCs isolated from glomerulosclerosis-prone mice (ROP) and from glomerulosclerosis-resistant mice (C57). In MCs from both strains, CsA treatment (1 µg/ml) did not affect TGF-ß1 mRNA expression and protein levels. Hutchinson et al. showed that genotypic variations in the TGF-ß1 gene, which result in different levels of TGF-ß1 production, are associated with the development of graft fibrosis after lung transplantation, cardiac transplant vasculopathy following cardiac transplantation, chronic rejection after liver transplantation and chronic allograft nephropathy after kidney transplantation [28]. Taken together, these findings demonstrate that species-specific differences, as well as inter-individual differences in humans, exist in levels of TGF-ß1 production.

Tacrolimus was shown to cause similar pathologic changes compared with CsA [29]. The nephrotoxic side effects of both CsA and tacrolimus appear to result from calcineurin inhibition. In rats treated with tacrolimus, increased expression of TGF-ß1 and matrix proteins was found [30]. In addition, tacrolimus stimulates TGF-ß1 expression in mammalian lymphoid as well as non-lymphoid cells [31]. Whether or not tacrolimus turns out to have qualitatively and quantitatively similar effects in MCs compared with those described here remains to be investigated.

In conclusion, it may be beneficial to reduce the expression of TGF-ß1 in patients with chronic CsA nephrotoxicity or chronic allograft nephropathy [32,33]. However, TGF-ß1 also has important immunosuppressive properties. Because low TGF-ß1 concentrations may increase alloreactivity and thus promote acute or chronic allograft rejection, it remains unclear whether down-regulation of TGF-ß1 and its receptors in renal allograft recipients is generally desirable.



   Acknowledgments
 
This work was supported by a grant from the Else Kröner-Fresenius Foundation. We are indebted to Pia Hambach and Dr Harm Peters for their help with fibronectin and PAI-1 ELISA assays.



   Notes
 
Correspondence and offprint requests to: Dr Johannes Waiser, Medizinische Klinik mit Schwerpunkt Nephrologie, Universitätsklinikum Charité, Campus Charité Mitte, Schumannstrasse 20/21, 10117 Berlin, Germany. Email: johannes.waiser{at}charite.de Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 8.01
Accepted in revised form: 13. 3.02