Cyclosporine A up-regulates the expression of TGF-beta1 and its receptors type I and type II in rat mesangial cells.
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-beta1 (TGF-beta1) is a potent stimulus for the synthesis of ECM proteins in MCs. We investigated whether CsA up-regulates the expression of TGF-beta1 and its receptors type I (TbetaR-I) and type II (TbetaR-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-beta1 antibodies. Time- and concentration-dependent expression of TGF-beta1, TbetaR-I and TbetaR-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-beta1 (maximum at 72 h, 500 ng/ml CsA: 2.1+/-0.5-fold, P<0.001) and TbetaR-II (maximum at 72 h, 1000 ng/ml CsA: 2.4+/-0.4-fold, P<0.005) time- and dose-dependently. TbetaR-I mRNA concentrations remained unchanged. Protein concentrations were analysed at 96 h: TGF-beta1, 220+/-32 vs 86+/-24 pg/ml, P<0.001 (500 ng/ml CsA vs control); TbetaR-I, 2.0+/-0.5-fold, P<0.005 (1000 ng/ml CsA vs control); TbetaR-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-beta1 antibodies reduced (P<0.05) CsA-induced expression of TbetaR-I (1.0+/-0.1-fold), TbetaR-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-beta1 and, via autocrine mechanisms, its receptors type I and II in rat MCs. Whereas up-regulation of PAI-1 is mediated by TGF-beta1, up-regulation of FN is-at least in part-either directly induced by CsA or mediated by factors other than TGF-beta1.
Waiser
Johannes
J
Department of Internal Medicine-Nephrology, University Hospital Charité, Campus Charité Mitte, Humboldt-University, Berlin, Germany. johannes.waiser@charite.de
Dell
Kerstin
K
Böhler
Torsten
T
Dogu
Ertan
E
Gaedeke
Jens
J
Budde
Klemens
K
Neumayer
Hans-Hellmut
HH
D030201:Activin Receptors, Type I / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016572:Cyclosporine / Q000494:pharmacology*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000235:genetics*
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D016572:Cyclosporine; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat