Thrombin is a pro-fibrotic factor for rat renal fibroblasts in vitro.
BACKGROUND
Generation of thrombin occurs in response to parenchymal injury. Thrombin not only converts plasma fibrinogen into an insoluble fibrin clot, but also potentially augments inflammation through receptor-mediated activity. This study examines whether thrombin may potentially exacerbate fibrosis by upregulating the function of interstitial fibroblasts in vitro.


METHODS
Fibroblasts were isolated by explant outgrowth culture of rat kidneys. Subcultured cells were grown in DMEM+10% FCS supplemented with 0.1-0.5 U/ml thrombin. Functional parameters examined included kinetics (thymidine incorporation and change in cell number), differentiation (Western blotting for alpha-smooth muscle actin; alphaSMA), expression of procollagen alpha1(I) (Northern blotting) and contraction of collagen I lattices. RT-PCR was used to characterise expression of protease-activated receptors (PAR) previously implicated in thrombin's cellular effects.


RESULTS
Cell population growth was increased 66 +/- 41 and 47 +/- 41% by 0.1 and 0.5 U/ml thrombin respectively (both p < 0.05 vs. basal). Likewise, 0.5 U/ml thrombin increased corrected procollagen alpha1(I) expression 2.4-fold (p < 0.05 vs. basal) and exacerbated the ability of fibroblasts to contract collagen matrix (p < 0.05 vs. basal). These effects were not associated with any change in expression of the myofibroblast marker alphaSMA. Effects on cell number were inhibited by treatment with (D)-Phe-Pro-Arg-chloromethylketone HCl (PPACK) suggesting that functional effects were mediated by serine protease activity. PAR-1 was the only fully functional known thrombin receptor expressed by these cells.


CONCLUSIONS
Thrombin is a potential unrecognised fibroblast agonist in renal disease. Further studies of thrombin and its receptors may yield valuable insights into the pathogenesis of interstitial fibrosis.
Hewitson
T D
TD
Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Australia. Tim.Hewitson@mh.org.au
Martic
M
M
Kelynack
K J
KJ
Pagel
C N
CN
Mackie
E J
EJ
Becker
G J
GJ
D000199:Actins / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D005355:Fibrosis; D005782:Gels; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044463:Receptor, PAR-1 / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D015415:Biomarkers; D005782:Gels; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D015842:Serine Proteinase Inhibitors; D003094:Collagen; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone