Relaxin down regulates renal fibroblast function and promotes matrix remodelling in vitro

BACKGROUND - Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS - Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS - H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS - The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression