Th Thy1.1 antibody at day 0 (a) and day 8 (b, c, and d). Axl and -smooth muscle actin are distributed within the same site from the glomerulus (yellow in d) in an expanded mesangial pattern. Some outer web sites in the glomerular capillary wall (arrows) and a few Bowman’s capsular epithelial cells are only positive for Axl. Original magnification, 200.1428 Yanagita et al AJP April 2001, Vol. 158, No.Figure two. Inhibitory effects of warfarin on Thy1 GN. Effects of warfarin remedy on glomerular cell proliferation (A) and glomerular expression of OX-7 (B). Representative glomeruli of day 0 (a), day 8 of Thy1 GN (b), and day 8 of Thy1 GN with warfarin treatment (0.five mg/ml) (c) are shown. A: PAS staining. B: Immunofluorescent staining for OX-7. Original magnification, 200. C: PCNA expression in glomeruli of Thy1 rats. PCNA-positive cell numbers per glomerular cross-section are counted as described in Components and Procedures. Closed squares, nonABL2 Proteins site treated Thy1 rats; closed circles, Thy1 rats treated with 0.25 mg/L of warfarin; open circles, Thy1 rats treated with 0.five mg/L of warfarin. , P 0.001 versus nontreated Thy1 rats. D: Expression of extracellular matrix protein in glomeruli of Thy1 rats at day eight. Collagen sort I (a), sort III (b), variety IV (c), fibronectin (d), and laminin B2 (e) staining scores per glomerular cross-section are counted as described in Materials and Strategies. Open bar, manage rats (day 0); closed bar, nontreated Thy1 rats; hatched bar, Thy1 rats treated with 0.25 mg/L of warfarin; dotted bar, Thy1 rats treated with 0.five mg/L of warfarin in D and E. , P 0.001 versus nontreated Thy1 rats. E: Urinary albumin excretion standardized by urinary creatinine of Thy1 rats at day eight. , P 0.001 versus nontreated Thy1 rats.Low-Dose Warfarin Inhibits Glomerular Cell Proliferation in VivoBecause expression of Gas6 and Axl was induced considerably in parallel with illness severity of Thy1 GN, the Gas6/Axl pathway seems to play an essential role in the improvement of glomerulonephritis. Thus, we examined whether inhibiting this pathway may possibly be valuable in treating this experimental glomerulonephritis. We administered warfarin in drinking water at various concentrations (0, 0.25, or 0.5 mg/ml). Serum concentrations of warfarin in these rats had been 0.28 0.05 mol/L (0.25 mg/L) and 1.23 0.four mol/L (0.five mg/L) (Table 1), which had been inside the serum concentrations that inhibit mesangial cell proliferation in vitro. Substantial prolongation of prothrombin times, anemia (Table 1), or bleeding tenTable 1.dency was not observed in rats through the whole Toll-like Receptor Proteins web period of warfarin remedy. Mesangial cell proliferation and mesangial matrix expansion on day eight in Thy1 GN was substantially reduced by warfarin therapy (Figure 2A). Expression of OX-7 was also lowered in glomeruli of Thy1 GN treated with warfarin (Figure 2B, c). To examine the impact of warfarin on glomerular cell proliferation, the number of PCNApositive cells were counted. The number of PCNA-positive cells inside the glomeruli of rats treated with warfarin was considerably reduced in a dose-dependent manner at every point studied (Figure 2C). To examine the participation of infiltrating macrophages inside the variety of PCNA-positive cells per glomerulus, double immunostaining of PCNA and CD68 was performed. The number of PCNA/CD68-positive cells was 0.03 0.18 at day 0,Serum Concentrations of Warfarin, Prothrombin Time, and Hematocrit of Thy1 Rats Treated with Warfarin 0 0 12.63 48.4 0.51 1.0 0.25 0.28 13.33 49.