Effects of pentoxifylline, pentifylline and {gamma}-interferon on proliferation, differentiation, and matrix synthesis of human renal fibroblasts

Frank Strutz, Malte Heeg, Tobias Kochsiek, Gesa Siemers, Michael Zeisberg and Gerhard A. Müller

Department of Nephrology and Rheumatology, Georg-August-University Göttingen, Göttingen, Germany



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Kidneys that progress to end-stage renal failure are almost invariably characterized by the presence of tubulointerstitial fibrosis. Therapeutic interventions to halt the progressive deterioration of renal function are still limited. Pentoxifylline, pentifylline, and {gamma}-interferon have shown a potential benefit in the treatment of fibrotic processes in the skin and lung. Thus, the aim of the present study was the analysis of potential anti-fibrotic effects of these substances on human kidney fibroblasts in vitro.

Methods. Primary renal fibroblasts were established from human kidney biopsies and were studied in addition to two renal fibroblast cell lines. Cells were first growth arrested by withdrawal of fetal calf serum (FCS) and subsequently stimulated with 10% FCS in the presence of different concentrations of pentoxifylline (PTX), pentifylline (PTF), or {gamma}-interferon (IFN-{gamma}). Fibroblast proliferation was determined by bromodeoxyuridine incorporation and cell counts. Northern and western blot hybridizations for basic fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-ß1 were performed to analyse inhibitory effects. The effects of all three substances on matrix synthesis were evaluated by immunoblot analyses and ELISA for collagen type I and fibronectin after stimulation with TGF-ß1. Finally, differentiation into myofibroblasts was examined by double immunofluorescence staining for {alpha}-smooth-muscle actin and Hoechst dye H33258.

Results. PTX and PTF resulted in a dose- and time-dependent inhibition of proliferation in all fibroblast lines (maximum 78.9±6.2% at 500 µg/ml PTX). Conversely, IFN-{gamma} had only modest effects on fibroblast proliferation, resulting in a maximum of 36.0±6.1% inhibition at 500 U/ml. Northern blot hybridizations determined that FGF-2 mRNA levels in fibroblasts were decreased up to 73.7 and 91.5% by PTX (1000 µg/ml) and PTF (100 µg/ml), whereas IFN-{gamma} led to a reduction of 46.2% at 1000 U/ml, indicating that the inhibitory effects of all three substances may be mediated through inhibition of FGF-2 synthesis. These findings were corroborated by immunoblot analyses where again PTX and PTF had the strongest inhibitory effects. No change in TGF-ß1 mRNA levels was noted. Synthesis of cellular and secreted collagen type I was robustly inhibited by PTX and PTF, whereas IFN-{gamma} exerted the strongest inhibitory effect on fibronectin synthesis and secretion. In addition, IFN-{gamma} down-regulated the expression of {alpha}-smooth-muscle actin up to 73.3% (at 1000 U/ml) whereas PTX and PTF resulted in a down-regulation of up to 49.7±1.8 and 80.0±4.4% (at 1000 and 100 µg/ml) respectively. PTF was in all experiments about 10 times more potent than equimolar concentrations of PTX.

Conclusions. PTX and PTF exerted robust inhibitory effects on fibroblast proliferation, extracellular matrix synthesis, and myofibroblastic differentiation. Conversely, IFN-{gamma} caused strong inhibition of fibronectin synthesis and {alpha}-smooth-muscle cell actin expression but had only weak inhibitory influences on fibroblast proliferation and collagen type I synthesis. Inhibitory effects of all three substances on proliferation may be mediated through inhibition of FGF-2 synthesis.

Keywords: FGF-2; fibroblasts; {gamma}-interferon; kidney fibrosis; pentifylline; pentoxifylline



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Kidney fibrosis is a complex multicellular process characterized by the proliferation of extracellular matrix (ECM) producing cells and excessive accumulation of ECM along with a decreased rate of degradation. Though a variety of renal diseases can lead to end-stage renal disease (ESRD), almost all forms of progressive renal failure are characterized by tubulointerstitial fibrosis, tubular atrophy, and dilatation. The interstitial scarring often progresses despite resolution of the primary disease process. Thus, interstitial fibrosis seems to represent the final common pathway of disease progression irrespective of the primary cause. In fact, a number of studies have demonstrated that the extent of tubulointerstitial damage often correlates better with renal function than, for example, glomerular lesions [1,2]. Unfortunately, therapeutic interventions to restrict the progression of renal disease are still limited. Therapy in general is confined to the control of blood pressure in hypertensive patients and hyperglycaemia in diabetic subjects. Angiotensin-converting enzyme (ACE)-inhibitors added the improvement of size selectivity of the glomerular basement membrane, thus limiting proteinuria [3]. However, there is still no specific therapy to halt progressive interstitial scarring.

Renal fibrogenesis is characterized mainly by the proliferation and subsequent activation of interstitial fibroblasts to so-called myofibroblasts. Cytokines such as transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF)-2 have important roles in these processes [4]. Fibroblasts are thought to be the main effector cells in renal fibrogenesis, though other cellular elements may participate, as has been demonstrated by our group and others [5,6]. Renal interstitial fibroblasts produce increased amounts of ECM per cell when isolated from fibrotic kidneys [7]. These ECM products include fibronectin, which is often synthesized early in the process as a scaffold for other ECM components, and the collagen types I, III, and IV. Thus, potential therapeutic interventions could be directed against fibroblast proliferation and/or matrix synthesis. In addition, matrix degradation, which is often inhibited during fibrogenesis due to the upregulation of tissue inhibitor of matrix metalloproteinases (TIMP)-1 [8], could be activated. One of the substances that may effect all three processes is pentoxifylline. Pentoxifylline is a substituted methyl-xanthine that, besides its use as an enhancer of local blood flow, has shown great promise as antifibrotic therapy for skin scarring [9]. It inhibits proliferation, and synthesis of collagen, fibronectin, and glycosaminoglycans in normal, hypertrophic scar and keloid skin fibroblasts [10,11]. Similar findings were reported for fibroblasts from patients with Graves' ophthalmopathy [12] and for liver myofibroblasts [13]. Furthermore, PTX increases collagenase activity in normal skin fibroblasts [11] and reduces the differentiation rate from fat-storing cells in the liver to myofibroblasts [13]. PTX causes few side-effects and has only negligible toxic effects [14] making it a potentially useful drug for the treatment of kidney fibrosis. In fact, therapy with PTX was successful in reducing liver fibrosis in a swine model [15] and in a rat model of biliary cirrhosis [16]. Regarding the kidney, PTX inhibited collagen synthesis in mesangial cells by over 50% on the mRNA level [17] and attenuated the course of experimental mesangial proliferative glomerulonephritis [18].

Pentifylline (1-hexyl-3,7-dimethyl-xanthine, PTF) is another substituted methyl-xanthine. It is the precursor compound of PTX and has a 10-fold higher inhibitory capacity of collagen synthesis than PTX in skin fibroblasts [9]. Finally, interferons (IFN) have been described as potent inhibitors of collagen biosynthesis in dermal fibroblasts, with IFN-{gamma} being the most potent [19]. Addition of IFN-{gamma} to skin fibroblasts treated with TGF-ß abrogated the stimulatory effect of the cytokine on collagen production [20]. Application of IFN-{gamma} to rats treated with carbon tetrachloride resulted in significant reductions of ECM deposition in the liver [21]. Clinical application of IFN-{gamma} to patients with systemic sclerosis suggested significant improvements on cutaneous fibrotic abnormalities and on pulmonary fibrosis [22]. Recently, IFN-{gamma} was propagated also as a treatment for idiopathic pulmonary fibrosis [23].

The aim of the present study was to analyse and compare the effects of pentoxifylline, pentifylline, and {gamma}-interferon on proliferation, differentiation, and matrix synthesis of kidney fibroblasts in vitro.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Materials
PTX and PTF were obtained from Sigma Chemical Co., St Louis, MO. Recombinant human IFN-{gamma} derived from Escherichia coli was purchased from Boehringer Mannheim, Mannheim, Germany. The rabbit polyclonal antibody to FGF-2 from Calbiochem (La Jolla, CA) was used for immunoblots, recombinant FGF-2 from R & D Systems (Minneapolis, MN) served as control. Mouse monoclonal antibody to collagen type I from Southern Biotechnology (Birmingham, AL) was used for ELISA, mouse monoclonal antibody to collagen type I from Biotrends (Cologne, Germany) for immunoblot analyses. Polyclonal rabbit antibody to fibronectin was purchased from Sigma (St Louis, MO). The monoclonal mouse anti-human antibody against {alpha}-smooth-muscle actin (Paesel & Lorei, Wiesbaden, Germany) was used to study differentiation into myofibroblasts. For cell characterization the following additional mouse monoclonal antibodies were used: anti-cytokeratin (Dako, Carpinteria, CA), anti-vimentin (Boehringer Mannheim), anti-factor VIII (Dako), anti-human leukocyte antigen (HLA)-DR (Dako), anti-CD 44 (Pharmingen, San Diego, CA), anti-CD 54 and anti-CD 68 (Dako). Trypsin– ethylenediamine tetra-acetic acid (EDTA), Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) and fetal calf serum (FCS) were obtained from Gibco Ltd (Paisley, Scotland). Cell culture dishes were from Becton Dickinson (Franklin Lakes, NJ).

Cell culture and characterization of primary fibroblasts
Human renal fibroblast cell lines Tk 173 (obtained from a normal kidney) and Tk 188 (from a kidney with tubulointerstitial fibrosis) are transformed with the SV40-large T-antigen and have been characterized previously [24]. These cells were cultured in DMEM supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 U/ml) (all from Gibco).

Primary kidney fibroblasts were isolated from human kidney biopsy cylinders. The use of parts of the second core of the renal biopsy for cell culture was approved by the Ethics committee of the Georg-August-University. Written consent was obtained from all patients prior to kidney biopsy. For isolation of primary human kidney fibroblasts, renal biopsy cylinders were cut and immersed in DMEM medium with 20% FCS, penicillin (100 U/ml) and streptomycin (100 U/ml). This procedure promotes fibroblast specific growth. When cells had grown to confluency, cells were split 1 : 1 and were characterized by immunofluorescence for cytokeratin (Dako), vimentin (Boehringer Mannheim), {alpha}-smooth-muscle actin (Paesel & Lorei), collagen types I, III, IV (all from Southern Biotechnology), factor VIII (Dako), CD 44, CD 54, CD 68, and HLA-DR (all from Dako). Cells that were positive for vimentin, CD 44, CD 54 and the collagen types I and III, and negative for cytokeratin (cytokeratin positive cells below 5%), factor VIII and HLA-class II were considered to be fibroblasts and used for induction assays in passages 2–8.

Primary fibroblast line Tk 434 was established from a 58-year-old male (JK) with the primary diagnosis of mesangioproliferative glomerulonephritis with minimal interstitial involvement. Conversely, Tk 455 fibroblasts were cultured from the biopsy a 29-year-old female (CM) with IgA nephropathy and tubulointerstitial fibrosis involving 25% of the interstitium. After the second passage, the percentage of cytokeratin-positive cells was less than 5% in each culture. All cells were positive for vimentin, CD 44, and CD 54 by indirect immunofluorescence. The percentage of positive cells for collagen type I was 86% in Tk 434 fibroblasts and 96% in Tk 455 cells. Regarding collagen type III, the percentages were 89 and 94% respectively. The percentage of {alpha}-smooth-muscle actin-positive cells was 79.5% in Tk 434 cells and 88% in Tk 455 fibroblasts. All cells were negative for HLA-DR, factor VIII, and CD 68. Figure 1Go demonstrates the staining patterns observed by indirect immunofluorescence labelling for vimentin and collagen types I and III.



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Fig. 1. Characterization of primary renal fibroblast lines Tk 434 (A, C, and E) and Tk 455 (B, D, and F) for vimentin (A and B) and for collagen types I (C and D), III (E and F) by indirect immunofluorescence. All cells stained positive for vimentin; the percentage of collagen type I-positive cells was 86% in Tk 434 fibroblasts and 96% in Tk 455 cells; the percentage of collagen type III-positive cells was 89 and 94% respectively. Magnification: x400.

 

Proliferation assays
Proliferation studies were performed using non-radioactive bromodeoxyuridine-incorporation assays (Amersham, Arlington Heights, IL) based on the method by Gratzner [25]. Assays were performed according to the manufacturer's instructions with some modifications. 4x103 cells were plated per well in 96-well microtitre plates containing DMEM medium with supplements and incubated overnight. Cells were then made quiescent by replacement of DMEM with serum-free IMDM [26] and subsequent incubation for 24 h. After 24 h IMDM was replaced by medium containing 10% FCS to stimulate cell proliferation. Pentoxifylline was added in concentrations of 100, 500, and 1000 µg/ml (equivalent to 0.36, 1.8 and 3.6 mmol/l), pentifylline in concentrations of 10, 50, and 100 µg/ml (equivalent to 0.038, 0.19 and 0.38 mmol/l), and {gamma}-interferon in concentrations of 100, 500, and 1000 U/ml. Proliferation was measured after 24, 48, and 72 h. 10% FCS alone served as a positive control. Medium was changed to IMDM containing BrdU/FdU in a dilution of 1 : 500 4 h prior to measurements. Cells were then washed thrice in PBS and fixed in methanol containing 2% hydrogen peroxide followed by denaturation in 1 N HCl for 10 min. After three further washes in PBS containing 0.1% Tween and blocking for 30 min in PBS (0.1% Tween, 3% bovine serum albumin), 50 µl of anti-BrdU antibody was added and incubated for 45 min. Cells were again washed three times in PBS/Tween and peroxidase substrate was added (100 µl/well). Optical densities were subsequently determined photometrically at 405 nm (Dynatech MR 4000, Denkendorf, Germany). All experiments were performed in triplicate and repeated four times.

In a second set of experiments, confluent monolayers of fibroblasts were trypsinized and cells seeded at 4x103 cells/well in 96-well culture plates. After 12 h, DMEM medium was replaced by Iscove's. After further 24 h cells were stimulated with either 10% FCS alone or 10% FCS+PTX, PTF, or IFN-{gamma} and the above-mentioned concentrations for 48 h. Again, all experiments were performed in triplicate and repeated four times.

Preparation of probes and Northern blot analyses
Tk 173 fibroblasts were cultured for 24 and 48 h in the presence of 10% FCS (control) and three different concentrations of PTX, PTF, and IFN-{gamma}. Total cellular RNA was extracted from cultured cells using RNA-cleanTM (AGS, Heidelberg, Germany) according to instructions from the manufacturer. RNA concentrations were determined by absorbance at 260 nm and samples were stored at -80°C prior to use. Northern blot analyses were performed as described previously with minor modifications [27]. 40 µg of total RNA were electrophoresed on a 1.0% agarose gel containing 2.2 mol/l formaldehyde using 1xMOPS pH 7.0 as the running buffer. Photographs of ethidium-bromide-stained gels were taken under UV illumination to control for equal loading. RNA was then transferred to a nylon membrane (Hybond N, Amersham) by capillary transfer for 12 h with 20xSSC as the transfer buffer. Blots were baked at 80°C and prehybridized for 1 h at 68°C in prehybridization solution containing 0.5 mol/l Na2HPO4 buffer (pH 7.2), 0.5 M EDTA (pH 8), 25 g SDS and 1.5 g blocking reagent. FGF-2 or TGF-ß1 (both R & D systems) specific oligonucleotides were labelled with digoxigenin utilizing a kit (Boehringer Mannheim) according to the manufacturer's instructions.

Hybridizations were performed overnight at 58°C using the prehybridization solution. Washes were performed three times in a solution containing 0.5 mol/l Na2HPO4 buffer (pH 7.2), 0.5 mol/l EDTA (pH 8), and 1% SDS at 58°C. After washing, anti-DIG alkaline phosphate and CSPD substrate (both Boehringer Mannheim) were added and autoradiograms obtained. All blots were stripped and hybridized with digoxigenin labelled 18S-RNA (Ambion, Austin, TX) to control for equal loading and transfer. Quantitative analyses were performed relative to the 18S-band using a densitometer and quantitation software (Bio-Rad, Hercules, CA).

Immunoblot analyses for FGF-2
In addition, immunoblots for FGF-2 were performed as described previously [28]. Briefly, lysates from fibroblasts were obtained by lysis with a detergent based buffer after incubation with PTX (500 µg/ml), PTF (50 µg/ml), or IFN-{gamma} (500 U/ml) in 10% FCS for 0, 6, 12, 24, and 48 h. One hundred micrograms of total cellular protein were run on an 18% SDS–PAGE gel and transferred to a nitrocellulose membrane (HybondTMECLTM, Amersham) by electroblotting. 50 ng of recombinant FGF-2 served as positive control. Membranes were stained with Ponceau red to control for adequate transfer and equally loaded amounts. After blocking, the membrane was incubated with the anti-FGF-2 antibody in a concentration of 1 : 40 followed by the secondary antibody (donkey-anti-rabbit, horseradish peroxidase linked, concentration 1 : 3000, Amersham). Positive reaction products were identified by chemiluminescence (ECL, Amersham) according to the manufacturer's protocol.

In a second set of experiments, Tk 173 fibroblasts were incubated with three different concentrations of PTX (100, 500 or 1000 µg/ml), PTF (10, 50 or 100 µg/ml) and IFN-{gamma} (100, 500 or 1000 U/ml) for 48 h in the presence of 10% FCS. Immunoblots and detection of FGF-2 were performed as described.

Evaluation of matrix synthesis
The effects of PTX, PTF and IFN-{gamma} on matrix synthesis were evaluated by immunoblot analyses and ELISA of the supernatant for collagen type I and fibronectin. For immunoblots, 100 µg of total cellular protein were run on an 7.5% SDS–PAGE gel and transferred to a nitrocellulose membrane (HybondTMECLTM, Amersham) by electroblotting. Again, membranes were stained with Ponceau red to control for adequate transfer and equally loaded amounts. After blocking, the membrane was incubated with the respective antibody (anti-collagen type I in a concentration of 1 : 40, anti-fibronectin in a concentration of 1 : 20) followed by the secondary antibody (donkey-anti-rabbit, horseradish peroxidase linked, Amersham). Detection was executed as outlined above.

ELISAs of supernatants were performed as described [29] with some modifications, including the use of chemiluminescence to enhance sensitivity [30]. Eight thousand cells were plated per well and cells were again made quiescent by incubation in serum-free medium for 24 h; 1 ng/ml TGF-ß1 (R & D Systems) was then added to the wells to stimulate ECM synthesis. Next, PTX was added in concentrations of 100, 500 and 1000 µg/ml, PTF at 10, 50 and 100 µg/ml, and IFN-{gamma} at 100, 500 and 1000 U/ml, followed by incubation for 48 h. Human recombinant TGF-ß1 alone served as positive control; 50 µg/ml ascorbic acid and 50 µg/ml propionitrile (both from Sigma) were added in experiments evaluating collagen synthesis. Supernatants were transferred to a MaxisorpTMplate and incubated overnight at room temperature. Plates were subsequently dried for 2 h and blocked with 3% dried milk. Incubation with 50 µl of the primary antibody (anti-collagen type I in a concentration of 1 : 300, and anti-fibronectin at 1 : 5000) was followed by washing twice with PBS/0,1% Tween and incubation with the secondary antibody (anti-rabbit-IgG-AP and anti-goat-IgG-AP (both Boehringer Mannheim) and both at a concentration of 1 : 1000). After two additional wash steps, 100 ml CSPD-Ready-to-Use substrate (Boehringer Mannheim) was added and quantitation was performed in a luminometer (Mikrolumat CB 96P, Berthold, Bad Wildbad, Germany) using MikroWinTM software (Mikrotek, Overath, Germany). Non-specific binding was determined by incubation with the secondary antibody only. The value was normally less than 5% of the total chemiluminescence and was subtracted from each assay. All assays were performed in triplicates and repeated five times. Standardization was obtained for the collagen type I ELISA using human type I collagen (Becton Dickinson). Limit of detection was 1 ng/ml, working range 10–1000 ng/ml. Human fibronectin (Sigma) served as standard for the fibronectin ELISA. The working range of that ELISA was 0.1–10 µg/ml with a limit of detection of 10 ng/ml. All values were corrected for cell counts (103 cells) which were performed after transfer of supernatants.

Immunofluorescence staining of {alpha}-smooth-muscle actin
The effects of PTX, PTF and IFN-{gamma} on the differentiation of kidney fibroblasts into myofibroblasts were assessed by immunofluorescent staining of {alpha}-smooth-muscle actin during continuous exposure to the relevant substance for 48 h in the presence of 20% FCS. At the end of incubation, cells were washed twice with PBS and fixed with ethanol/acetic acid (50 : 50, v/v) for 20 min at 4°C. The cells were subsequently washed again and incubated for 2 h at RT with a 1 : 200 dilution of a monoclonal mouse antibody to human {alpha}-actin isoform of smooth-muscle cells. Cells were then washed again with PBS and incubated for 60 min at RT with the FITC-conjugated rabbit-anti-mouse antibody. Control cultures were incubated without application of the primary antibody. Incubation with 20% FCS alone served as positive control. Cells with the typical {alpha}-actin structure were considered to be {alpha}-actin positive whereas cells with a faint green halo were deemed negative. Additional staining with the Hoechst dye H33258 was performed to facilitate cell counting. For each group, 500 cells were determined microscopically using an Axiophot S100 microscope (Zeiss, Jena, Germany) by a blinded investigator and the percentage of {alpha}-smooth-muscle actin-positive cells was calculated. Each experiment was repeated at least three times independently. Photographs were taken by double exposure using Zeiss camera and software (MC 200 Chip).

Statistical analysis
All values are expressed as mean±SEM. ANOVA was used to determine statistical differences between treated groups and controls using Sigma-StatTM-software (Jandel Scientific, San Rafael, CA). If the analysis of variance showed a statistical difference, a post hoc Bonferroni test was performed to determine differences between treated groups and control. P values <0.05 were considered significant. All experiments were repeated 3–5 times before analysis.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Effects of PTX, PTF and IFN-{gamma} on fibroblast proliferation
All three substances resulted in a marked decrease in proliferation as determined by bromodeoxyuridine incorporation in all four studied fibroblast lines. The antiproliferative effect was more robust with the two methyl-xanthine derivatives compared to IFN-{gamma}. As expected, the effect of PTF was about 10 times as potent as the effect of PTX in equimolar concentrations. All effects were time- and dose-dependent. Figure 2Go illustrates the time-dependency in Tk 173 (A) and Tk 188 (B) fibroblasts. Medium dosages of all three substances were used for these experiments (500 µg/ml PTX, 50 µg/ml PTF and 500 U/ml IFN-{gamma}). The maximum effect could be obtained with PTX and PTF in Tk 188 fibroblasts after 72 h resulting in a decrease in proliferation of 78.9±6.2 and 76.7±6.4% respectively. Conversely, IFN-{gamma} had only moderate effects on cell proliferation, resulting in a 36.0±6.1% decrease in Tk 188 fibroblasts (Figure 2BGo). Cell counting confirmed that reduced bromodeoxyuridine incorporation was in fact paralleled by decreased cell proliferation (Figure 3Go). After 48 h of incubation with PTX at the highest dose applied, cell counts were 61.0±6.1% lower in Tk 173 (Figure 3AGo) and 45.2±6.0% lower in Tk 188 cells (Figure 3BGo). PTF had similar effects, whereas IFN-{gamma} again had only moderate antiproliferative effects, which were not strictly concentration dependent. The results on the established fibroblast lines were confirmed in the two primary lines (Tk 434 in C and Tk 455 in D). Again, PTX and PTF had similar effects on proliferation (maximum inhibition 68.1±8.7% in Tk 434 and 67.8±6.2% in Tk 455 fibroblasts with PTX at 1000 µg/ml). There was also a tendency for IFN-{gamma} to inhibit proliferation of the two primary fibroblast lines; however, mostly non-significant. Interestingly, the effects of all three substances were independent of the origin of the kidney fibroblasts. Thus, growth inhibition was similar in normal kidney and fibrotic kidney derived fibroblasts.



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Fig. 2. Effects of pentoxifylline (light shading, 500 µg/ml), pentifylline (dark shading, 50 µg/ml), and {gamma}-interferon (white, 500 U/ml) on proliferation of normal kidney derived (Tk 173, A) and fibrotic-kidney-derived human fibroblasts (Tk 188, B) as determined by bromodeoxyuridine incorporation. All experiments were performed in triplicate and results are given as means±SEM of four independent experiments relative to the positive control (10% FCS). The two methyl-xanthines exerted robust anti-mitogenic effects, whereas IFN-{gamma} displayed only modest anti-proliferative properties. *P<0.05, **P<0.01.

 


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Fig. 3. The effects of PTX and PTF but not of IFN-{gamma} on renal fibroblast proliferation are dose dependent. Three different dosages of PTX (light shading), PTF (dark shading), and IFN-{gamma} (white) were applied to Tk 173 (A), Tk 188 (B), Tk 434 (C) and Tk 455 (D) fibroblasts as indicated. Cell counts were performed and displayed in percentage relative to controls (10% FCS alone) after 48 h. *P<0.05, **P<0.01.

 

Inhibition of fibroblast proliferation by PTX and PTF may be mediated in part by decreased synthesis of FGF-2
We have recently demonstrated that FGF-2 has strong proliferative effects on renal fibroblasts and may serve as an autocrine growth factor [28]. To determine if parts of the inhibitory effects of PTX, PTF, and/or IFN-{gamma} were mediated via a reduction of FGF-2 synthesis, Northern and Western blot hybridizations were performed after incubation with 10% FCS either alone (control) or various concentrations of PTX, PTF, and IFN-{gamma}. Densitometric analysis showed a significant reduction in FGF-2 mRNA levels starting 24 h after incubation with PTX or PTF but not after incubation with IFN-{gamma} (data not shown). Figure 4Go depicts the results obtained after an incubation time of 48 h. Whereas medium concentrations of PTX and PTF resulted in a 73.7% and 91.5% reduction respectively, IFN-{gamma} led only to a 46.2% reduction in FGF-2 mRNA.



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Fig. 4. Northern blot analyses of FGF-2 expression in human kidney fibroblasts after incubation with PTX, PTF, or IFN-{gamma}. Tk 173 fibroblasts were cultured in the presence of three different concentrations of PTX, PTF, IFN-{gamma} for 48 h. Part A depicts the FGF-2 mRNA expression and the corresponding 18S-RNA levels. Part B shows the findings after densitometric analyses relative to the level of 18S-RNA.

 
The results on the mRNA levels were subsequently corroborated by immunoblot analyses. The results are depicted in Figure 5Go. Both PTX and PTF resulted in a time-dependent significant inhibition of FGF-2 protein synthesis up to 67.1±4.9% (by 500 µg/ml PTX). Conversely, IFN-{gamma} decreased intracellular FGF-2 protein only non-significantly. Moreover, the effects of PTX and PTF but not of IFN-{gamma} were dose dependent. After 48 h, FGF-2 protein was decreased by 32.4±5.3% (100 µg/ml PTX), 61.5±6.1% (500 µg/ml PTX), and 75.4±6.4% (1000 µg/ml PTX). The results for PTF were similar (data not shown) whereas again for IFN-{gamma} there was no clear dose dependency (data not shown). These results demonstrate a good correlation between a reduction in FGF-2 mRNA and protein synthesis and the anti-proliferative effects of PTX and PTF, but not IFN-{gamma}.



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Fig. 5. Immunoblot analyses for FGF-2 in Tk 173 fibroblasts after incubation with 500 µg/ml pentoxifyllin (PTX), 50 µg/ml pentifyllin (PTF) or 500 U/ml interferon-{gamma} (IFN-{gamma}) for 0, 6, 12, 24, and 48 h in the presence of 10% FCS. Part A depicts three typical blots (rFGF-2, recombinant FGF-2, which served as control). Part B summarizes the densitometric results of four different blots. Similarly to the RNA expression, FGF-2 protein levels were decreased significantly in a time-dependent manner by pentoxifylline and pentifylline but only non-significantly by IFN-{gamma}. *P<0.05, **P<0.01.

 
Since TGF-ß1 may also induce proliferation in renal fibroblasts, we also tested the influence of the three substances on the expression of TGF-ß1. No change in expression of TGF-ß1 was noted (data not shown), making it unlikely that TGF-ß1 plays a role in the mediation of the antiproliferative effects of PTX and PTF.

Effects of PTX, PTF, and IFN-{gamma} on ECM production in renal fibroblasts
To assess the effects of PTX, PTF, and IFN-{gamma} on ECM production, cellular collagen type I, and fibronectin was determined along with ELISAs of supernatants. All three substances had significant effects on ECM synthesis in renal fibroblasts. PTF had the most robust effect on intracellular collagen type I synthesis (Figure 6BGo) and IFN-{gamma} on fibronectin synthesis (Figure 6CGo). The specificity of the antibody to type I collagen was first determined by collagenase digestion and comparison with a standard (Figure 6AGo). TGF-ß1 caused an increase of intracellular type I collagen of 11.4-fold in Tk 173 and 3.1-fold in Tk 188 fibroblasts. This increase was almost completely abrogated by PTX and PTF and partly by IFN-{gamma}. The effects of TGF-ß1 on fibronectin synthesis were less pronounced (1.9-fold and 1.7-fold increase in the two fibroblast lines), and only IFN-{gamma} almost completely inhibited its effects (Figure 6CGo). The results obtained on cellular protein were paralleled by the effects on secreted proteins measured in the supernatant. Basal collagen type I and fibronectin secretions were higher in fibrotic kidney derived fibroblasts (Tk 188). TGF-ß1 caused a 8.1±1.3-fold increase in secreted type I collagen in Tk 173 and a 4.5±0.5-fold increase in Tk 188 fibroblasts (not shown). Again, TGF-ß1 induced stimulation of collagen type I secretion was inhibited by PTX and PTF in a dose dependent fashion (Figure 7AGo,BGo). Conversely, IFN-{gamma} had only modest effects in Tk 173 fibroblasts and almost no effect on Tk 188 fibroblasts. However, IFN-{gamma} did exert the strongest inhibitory effects on fibronectin synthesis (Figures 7CGo and DGo). PTX and PTF also had significant effects on fibronectin secretion that were again dose dependent. Similar to the results observed by immunoblot analyses, TGF-ß1 stimulated fibronectin synthesis by 2.0±0.3 and 1.8±0.3-fold in Tk 173 and Tk 188 kidney fibroblasts respectively.



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Fig. 6. Immunoblot analyses of collagen type I and fibronectin in Tk 173 (dark bars) and Tk 188 (grey bars) fibroblasts. Part A shows the typical three bands that were obtained after incubation with the collagen type I antibody (lane 1). Lane 2 depicts the same protein after collagenase digestion, proving that the two lower bands are collagen. Part B depicts the immunoblots in Tk 173 and Tk 188 cells in the same order as shown below graphically (control, TGF-ß1 at 1 ng/ml, PTF at 50 ng/ml, PTX at 500 ng/ml and IFN-{gamma} at 500 U/ml). Part C shows the effects on cellular fibronectin.

 


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Fig. 7. Effects of pentoxifylline (PTX), pentifylline (PTF), and interferon-{gamma} (IFN-{gamma}) on secretion of collagen type I (A and B) and fibronectin (C and D) in Tk 173 (A and C) and Tk 188 (B and D) fibroblasts. PTX and PTF have dose dependent inhibitory effects on both extracellular matrix substances whereas the effects of IFN-{gamma} are limited to fibronectin secretion. *P<0.05, **P<0.01.

 
In summary, PTX and PTF have very robust inhibitory effects on TGF-ß1 stimulated matrix synthesis whereas IFN-{gamma} inhibits fibronectin synthesis but has only marginal effects on synthesis and secretion of type I collagen.

Differentiation into myofibroblasts is inhibited by PTX, PTF, and IFN-{gamma}
Differentiation into {alpha}-smooth-muscle actin-positive, so-called myofibroblasts is thought to represent one key step during fibrogenesis. Thus, in order to determine if PTX, PTF or INF-{gamma} influenced that process in vitro, primary fibroblasts Tk 434 and Tk 455 were incubated again with three concentrations of the three substances and the number of {alpha}-smooth-muscle actin-positive cells was determined. The number of cells expressing {alpha}-smooth-muscle actin under basal conditions, i.e. culture in 20% FCS was 79.5±2.6% in Tk 434 and 88.0±1.2% in Tk 455 fibroblasts.

The number of positive cells was reduced by PTX and PTF in both primary fibroblast cell lines (Figure 8Go), though the effects were more pronounced in Tk 434 fibroblasts (49.7±1.8% by PTX and 80.0+4.4% by PTF). In Tk 455 fibroblasts, only the highest dose of each substance had a significant effect (reduction of {alpha}-smooth-muscle-positive cells by 33.9±8.1% and 32.1±3.1% by PTX and PTF respectively). Conversely, IFN-{gamma} exerted a dose-dependent effect in both fibroblast lines that resulted in a maximal reduction of 73.3±0.9% in Tk 434 cells. Figure 9Go illustrates a typical staining pattern obtained in Tk 455 fibroblasts after incubation with the three different concentrations of IFN-{gamma}.



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Fig. 8. {alpha}-Smooth-muscle actin expression in primary human kidney fibroblasts after continuous exposure to three different concentrations of pentoxifylline (PTX), pentifylline (PTF), and {gamma}-interferon (IFN-{gamma}) for 48 h. Results in Tk 434 fibroblasts are shown in A, those in Tk 455 fibroblasts in B. The cells were assessed by light microscopy after indirect immunofluorescence staining, determining the percentage of {alpha}-smooth-muscle actin-positive cells in each group. The results represent the mean values±SEM of four independent experiments. **P<0.01 and *P<0.05.

 


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Fig. 9. Indirect immunofluorescent staining for {alpha}-smooth-muscle actin in Tk 455 fibroblasts. The cells were exposed to interferon-{gamma} (IFN-{gamma}) at concentrations of 100 U/ml (B), 500 U/ml (C) or 1000 U/ml (D) for 48 h. A shows the untreated control (20% FCS). Only cells that displayed the characteristic {alpha}-smooth-muscle actin structure were considered positive. Nuclei were counterstained with Dapi to facilitate cell counting.

 
The number of {alpha}-smooth-muscle actin-positive cells was much lower in Tk 173 and Tk 188 fibroblasts, even after stimulation with 20% FCS (8.8±0.8% in Tk 173 and 12.5±1.1% in Tk 188). Though all three substances decreased the number of {alpha}-smooth-muscle actin-positive cells, the reductions were non-significant (data not shown).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Interstitial kidney fibrosis is characterized by fibroblast proliferation, phenotypic transformation in so-called ‘myofibroblasts’ and expression, synthesis, and secretion of ECM proteins. Collagen type I and fibronectin are the major components of ECM in renal fibrosis. The methyl-xanthines PTX and PTF as well as IFN-{gamma} have been reported as effective in inhibiting proliferation and collagen synthesis in dermal fibroblasts. However, fibroblasts from different organs (and even within the same organ) may display considerable heterogeneity [31]. In our study of two established transformed and two newly established primary fibroblast lines from human kidneys, we demonstrate that the two methyl-xanthines have very robust anti-mitogenic effects on fibroblast proliferation, inhibit synthesis and secretion of fibronectin and collagen type I, and prevent myofibroblastic differentiation. IFN-{gamma}, on the other hand, is even more effective than the methyl-xanthines in preventing myofibroblast formation and fibronectin synthesis. However, it has only modest effects on fibroblast proliferation and collagen type I synthesis. This observation is in contrast to a study by Varga et al. [20] on dermal fibroblasts, where IFN-{gamma} was able to reverse TGF-ß1-induced stimulation of collagen but not on fibronectin synthesis, demonstrating again that fibroblasts from different organs may react differently to the same stimuli. In addition, we did not observe any significant difference in proliferative response to any of the three tested substances in fibroblasts from a normal kidney and a fibrotic kidney, which is in contrast to the findings by Windmeier and Gressner [13], who noted that activated fibroblasts from rat livers respond to lower doses of PTX than unactivated cells.

The anti-proliferative effects of all three substances on kidney fibroblasts may be mediated via a down-regulation of FGF-2 mRNA synthesis since there was a good correlation between expression of FGF-2 mRNA and protein and proliferation. Fibroblasts have the capacity to stimulate themselves via production of cytokines in an autocrine way [8]. One of these autoregulatory factors for fibroblast growth is FGF-2 as has been recently demonstrated by our group [28]. FGF-2 is synthesized constitutively and promotes autocrine growth under basal conditions. Its expression is increased in kidneys with interstitial fibrosis by in-situ hybridization and Western blot analyses and is correlated with interstitial proliferative activity. Our study does not prove the mediatory role of FGF-2 nor can it exclude that the inhibition of other autrocrine growth factors may play a role as well in mediating the effects of PTF, PTX and IFN-{gamma}. For example, we did not evaluate the effects on PDGF synthesis, which may be mitogenic, at least for some kidney fibroblasts [32]. However, we were able to exclude that the effects were mediated via down-regulation of TGF-ß1 which is able to induce a mitogenic response in human renal fibroblasts [33]. Anti-cytokine activity of PTX has been demonstrated before. PTX inhibits production of tumour necrosis factor-alpha (TNF-{alpha}) and interleukin-1 in monocytes/macrophages and has been used for the treatment of diseases characterized by the overproduction of TNF-{alpha} such as endotoxaemia and adult respiratory distress syndrome [34].

It has been suggested that IFNs and methyl-xanthines may act through a common mechanism in inhibiting collagen synthesis, putatively via the down-regulation of transcription factor nuclear factor-1 [9]. The down-regulation of collagen production of PTX and PTF seems to be correlated with their potency as non-specific cyclic nucleotide phosphodiesterase inhibitors. Phosphodiesterase inhibitors can affect transcription via increased levels of cyclic adenosine monophosphate. However, not all studies were able to confirm that increased levels of cyclic adenosine monophosphate correlate with inhibitory effects on ECM synthesis [35] and the exact mechanisms of methyl-xanthines on ECM synthesis are currently unknown [34]. Similarly, the effects of INF-{gamma} on ECM synthesis was dependent on protein synthesis in dermal fibroblasts and may be mediated through tyrosine kinase mediated autophosphorylation but again the exact mechanisms are currently unclear [9].

Unfortunately, both classes of compounds are not unproblematic in the therapy of patients with chronic renal disease. PTX is a promising antifibrotic agent; however, the high concentrations necessary to suppress ECM synthesis and fibroblast proliferation suggest that routine oral doses of PTX would be hardly effective as antifibrotic therapy. In a recent study in patients with acute renal graft rejections for example, intake of 800 mg PTX every 8 h resulted in PTX levels of 721±726 ng/ml [36], thus, considerably lower than the level desirable according to our in vitro results. Nevertheless, the pharmacologically active metabolites of PTX such as 1-carboxypropyl-3,7-dimethyl-xanthine, reach higher levels than PTX itself and may exert similar effects [37]. Furthermore, PTX may have additional anti-inflammatory effects as was recently demonstrated by Desmoulière et al. in two animal models of liver fibrosis [38] and by Entzian et al. in experimental alveolitis [39]. Finally, its ability as a free-radical scavenger may play a role as well. Combination of PTX with {alpha}-tocopherol may provide additional antifibrotic effects and has been tried in patients with radiation-induced fibrosis [40,41].

PTF is about 10 times more potent than PTX, as was again demonstrated by our study. However, it has the disadvantage that it is not an established drug and little is known about potential side-effects. IFN-{gamma}, on the other hand, has already been used clinically as an antifibrotic agent. Whereas Hein and co-workers found a beneficial effect of IFN-{gamma} on the course of systemic sclerosis without serious side-effects [22], a study by Polisson et al. [42] demonstrated only marginal effects and frequent side-effects for most patients with systemic sclerosis. In addition, IFN-{gamma} is well characterized as a pro-inflammatory cytokine [43]. For example, stimulation of human mesangial cells with IFN-{gamma} resulted in expression of high-affinity IgG receptors, which upon activation caused increased synthesis of chemoattractants such as interleukin-8 and monocyte chemoattractant protein-1 [44]. Moreover, IFN-{gamma} may promote cell-mediated immune injury and crescent formation in experimental glomerulonephritis [45]. Thus, whereas IFN-{gamma} may exert beneficial effects on the course of fibrogenesis, it may induce de-novo glomerular inflammation, certainly an undesirable effect. Though the use of IFN-{gamma} has not been tested for the treatment of interstitial fibrosis, its application did not reduce mesangial matrix expansion in the anti-Thy 1.1 model of glomerulonephritis (despite reduction of mesangial proliferation) [46] and even promoted the development of glomerular matrix accumulation (particularly in combination with interleukin-1) in a mouse model of IgA-nephropathy [47]. Conversely, in a mouse model of liver fibrosis, Rockey and Chung [48] were able to demonstrate that continous infusion of IFN-{gamma} by osmotic pump inhibited lipocyte differentiation into myofibroblasts and reduced collagen I mRNA to 36% of controls in vivo. Similar effects were observed in pulmonary fibroblasts [49]. Thus, whereas early administration of IFN-{gamma} may be detrimental due to its pro-inflammatory properties, application during the phase of so-called post-inflammatory matrix synthesis, where inflammation plays only a minor role, may be beneficial for kidney function.

In conclusion, our studies have demonstrated that PTX, PTF, and IFN-{gamma} may exert inhibitory effects on human kidney fibroblasts in vitro. The methyl-xanthine derivatives PTX and PTF have robust effects on fibroblast proliferation, ECM synthesis, and myofibroblast formation, whereas IFN-{gamma} strongly inhibits {alpha}-smooth-muscle actin expression and fibronectin synthesis, but has only modest effects on proliferation and collagen type I synthesis. Thus, our studies indicate that particularly the two methyl-xanthines, PTX and PTF, are potentially useful reagents for treating progressive tubulointerstitial fibrosis. However, further studies, particularly animal studies, are necessary to define the status of these agents in antifibrotic therapy in the kidney.



   Acknowledgments
 
This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG) to F. Strutz (DFG Str 388/3–1). M. Zeisberg is the awardee of a graduate grant from the DFG. The authors acknowledge the excellent technical assistance of A. Renziehausen.



   Notes
 
Correspondence and offprint requests to: Frank Strutz MD, Department of Nephrology and Rheumatology, Georg-August University Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany. Back



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 Results
 Discussion
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Received for publication: 5. 7.99
Accepted in revised form: 10. 5.00