Effects of pentoxifylline, pentifylline and gamma-interferon on proliferation, differentiation, and matrix synthesis of human renal fibroblasts.
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)-beta1 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-beta1. 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 microg/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 microg/ml) and PTF (100 microg/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-beta1 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 microg/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.
Strutz
F
F
Department of Nephrology and Rheumatology, Georg-August-University Göttingen, Göttingen, Germany.
Heeg
M
M
Kochsiek
T
T
Siemers
G
G
Zeisberg
M
M
Müller
G A
GA
D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009130:Muscle, Smooth / Q000166:cytology; D010431:Pentoxifylline / Q000494:pharmacology*; D013805:Theobromine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D007371:Interferon-gamma; C007942:pentifylline; D013805:Theobromine; D010431:Pentoxifylline