Effects of probucol on phenotypic changes in diabetic nephropathy with or without Smad3 deletion. (a) Time course of changes in urinary albumin excretion (as the ratio of albumin to creatinine) in the three groups treated with probucol - normal control (CTL) mice (▴), diabetic mice (db/db mice) (●), and Smad3-knockout diabetic mice (Smad3+/−; db/db mice) (▪) (n = 10 for normal control mice; n = 10 for db/db mice; n = 6 for Smad3+/−; db/db mice; NS, not significant, *p < 0.05 versus db/db mice, t test). (b) Time course of changes in body weight in the above three groups (NS, not significant, *p < 0.05 versus db/db mice, t test). (c) Biochemical data in the above three groups (results are expressed as the mean ± S.E., *p < 0.05 versus db/db mice). (d) Representative photomicrographs of PAS and PAM staining in the above three groups. (e) Mesangial sclerotic fraction in the above three groups was determined as percentage of mesangial matrix area per total glomerular surface area. All glomeruli were analyzed for each sample (*p < 0.01 versus probucol-treated normal control mice, **p < 0.01 versus probucol-treated db/db mice, t test). (f) Representative photomicrographs of immunohistochemical staining of ECM proteins (Col4, Col1 and Col3) in the above three groups. (g) The expression level of Col4, Col1, and Col3 in the glomeruli in the above three groups. They were analyzed by qPCR and normalized to the expression of Rn18s. The values are expressed as the mean ± S.E. (NS, not significant, *p < 0.05 versus probucol-treated normal control mice, **p < 0.05 versus probucol-treated db/db mice, t test). C, D, and S3D stand for normal control mice, db/db mice, and Smad3+/−; db/db mice, respectively.