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 100 ng/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-beta 1) 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 alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/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-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.

Parathyroid hormone has a prosclerotic effect on vascular smooth muscle cells.

Although accelerated atherosclerosis and arteriosclerosis are common in patients with renal failure, the pathogenesis of these changes is poorly understood. Parathyroid hormone (PTH) levels are elevated in renal failure, and have been linked to uraemic vascular changes in some studies. We examined the in vitro effects of increasing doses of the 1-34 fragment of PTH on human aortic vascular smooth muscle cells (VSMCs). Factors examined were: (1) collagen production using tritiated hydroxyproline incorporation and transcription of procollagen alpha(1)(I) mRNA; (2) change in the surface area of collagen I lattices; (3) mRNA transcription of the collagen binding protein beta1 integrin; (4) proliferation using tritiated thymidine incorporation, and (5) methyl tetrazolium salt conversion to estimate live cell number after 5 days' exposure to PTH. PTH at a concentration of 200 pmol/l increased total collagen synthesis (188 +/- 25% of control, p < 0.01) as well as transcription of procollagen alpha(1)(I) mRNA (136 +/- 11% of control, p < 0.005). PTH also increased reorganisation of collagen I lattices (surface area 47 +/- 8% of well for control vs. 35.7 +/- 2.5 and 34.3 +/- 3.0% for PTH 100 and 200 pmol/l, respectively, p = 0.02) and upregulated beta1 integrin mRNA expression (160 +/- 20% of control at PTH concentration of 200 pmol/l, p < 0.05). PTH had no effect on VSMC proliferation or number at doses up to 200 pmol/l. In conclusion, PTH increases production and reorganisation of collagen by VSMCs in vitro. It is possible that more aggressive control of hyperparathyroidism in patients with renal failure may help to reduce the burden of cardiovascular disease in this patient population.

Dipyridamole inhibits in vitro renal fibroblast proliferation and collagen synthesis.

Fibroblasts are universally recognized in situations of tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. The objective of this study was to determine whether the functions of fibroblasts relevant to fibrogenesis can be modified in vitro with dipyridamole. Cells were obtained from obstructed rat renal tissue and characterized on the basis of immunohistochemical findings. Fibroblasts constituted all of the cells from passage 3. Functional parameters were measured in cells cultured with 1, 5, and 50 micromol/L dipyridamole and compared to basal parameters of cells grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum (control). Northern-blot analysis indicated that dipyridamole decreased procollagen alpha1(I) messenger ribonucleic acid expression (P <.05, 50 micromol/L vs control), results that were reflected in a reduction in total collagen secretion as measured on the basis of hydroxyproline incorporation (P <.001, 50 micromol/L vs control). Mitogenesis, as measured on the basis of incorporation of tritiated thymidine, was decreased in a dose-dependent fashion by dipyridamole. Likewise, 50 micromol/L dipyridamole reduced cell-population growth to 16.8% +/- 2.1% of basal growth over 3 days (P <.001 vs control). Effects of dipyridamole on population growth were prevented by coincubation with a protein kinase G inhibitor peptide (P <.001 vs 50 micromol/L dipyridamole; P = not significant vs control). No such effect was observed with inhibitors for protein kinase A (H-89) and protein kinase C (bisindolylmaleimide I). Consistent with a protein kinase G-dependent mechanism, immunofluorescence staining indicated that dipyridamole increased basal expression of the inducible form of nitric oxide synthase. In conclusion, the results of this study demonstrate that at clinically relevant concentrations, dipyridamole inhibits profibrotic activities of renal fibroblasts. Effects on mitogenesis are mediated through a cyclic guanosine monophosphate-protein kinase G effector pathway.

Lovastatin downregulates renal myofibroblast function in vitro.

Interstitial fibrosis is recognised as the best histological predictor of progressive renal disease. Myofibroblasts contribute to this process through several functions including hyperproliferation, collagen and collagenase synthesis and reorganisation of extracellular matrix. Recent limited in vitro studies suggest that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors may reduce renal injury not only through their lipid-lowering effects but also by antagonising myofibroblast function. This study therefore examined the effects of lovastatin on the above interstitial myofibroblast behaviours in vitro. Primary cultures of rat renal cortical myofibroblasts were grown by explantation and characterised by immunohistochemistry. Dose response effects of lovastatin (0, 15, 30 microM) in DMEM and 10% FCS were examined on myofibroblast kinetics, total collagen synthesis, collagen I lattice contraction and actin filament rearrangement. Lovastatin decreased myofibroblast proliferation and growth. Likewise, collagen I lattice contraction and actin filament rearrangement were partially inhibited when lovastatin was added at 30 microM. In addition, lovastatin decreased both collagen and collagenase synthesis. Our results suggest that myofibroblast function may be downregulated by lovastatin in vitro. Although a decrease in myofibroblast activity may offer potential benefit in the prevention of progressive scarring, further studies will be necessary to determine the relative importance of these functions.