Thrombin Is a Pro-Fibrotic Factor for Rat Renal Fibroblasts in vitro

Abstract

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 α-smooth muscle actin; αSMA), expression of procollagen α1(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 α1(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 αSMA. 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. Conclusion: 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.

Actions of thrombin in the interstitium

“…Thrombin binds tightly to fibroblasts [63] and activates fibroblasts via cleavage of PAR 1 receptors [64]. Hewitson et al [65] demonstrated that thrombin functions as an in vitro agonist for renal fibroblasts. These investigators observed an increase in fibroblast proliferation in culture, an increase in procollagen a1(I) expression and increased fibroblast contraction of collagen gels in the presence of thrombin.…”

Gene expression profiling in the remnant kidney model of wild type and kinin B1 and B2 receptor knockout mice

“…47 The effect of the B 2 R gene knockout in 20-week remnant kidneys seems to be more profound, as B 2 R-KO 20-week remnant kidneys display strong overexpression for a number of mediators of inflammation, immune, and acute phase response (Fga, Fgb, Fgg, Hp, Hpxn, IgJ588, Orm1, Orm2, SerpinA3N, Cnlfsf2b). Additionally, some genes with potential importance in renal fibrosis, including haptoglobin (Hp), 48 serum amyloid A2 (Saa2), 49 Fga, Fgb and Fgg, 50 Edn1, 51 and Ren1 52 were overexpressed in remnant kidneys of SNXB2R-KO mice. Overall this suggests that the B 2 R is an endogenous protector in this chronic renal disease model.…”

Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis

“…This dissociation of the role of thrombin activity from hemostasis has important and immediate clinical implications for individuals with IPF, given recent findings that warfarin treatment is not beneficial in IPF (32), and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients. In addition, these findings may have widespread relevance for pathological fibrosis, as the thrombin/PAR1/α v β 6 /TGF-β axis has also been implicated in the development of fibrosis in other organ systems, including the liver and kidneys (33)(34)(35)(36)(37)(38).…”