Pediatr Nephrol (2008) 23:1257–1267 Expression of RAS components, TGF-β, and α-SM actin in patients with MGA or IgAN The glomerular localization of RAS components was studied by immunohistochemistry in renal biopsies from patients with either IgAN or MGA. Immunostaining of AGT was detected mainly in proximal tubules and weakly in GEC lining capillary walls in MGA specimens, as in normal tissues (Fig. 2a). This renal localization of AGT was consistent with rat renal localization of AGT, as previously described [33]. Of note, the staining level of AGT was enhanced in a typical endocapillary pattern, including changes of GEC and MC in IgAN patients (Fig. 2b). No positive immunostaining of AGT was seen when a chicken antibody against AGT, absorbed by preincubation with excess human purified AGT, was used as the primary antibody or when the primary antibody was omitted (Fig. 2c). The double immunostaining in IgAN showed 1261 that AGT was primarily detected on GEC, to a lesser extent on MC, and only faintly on podocytes, supporting the glomerular localization of AGT in an immunoperoxidase technique (Fig. 2d–l). ACE and ang II expression were examined by immuno- fluorescence, as specific staining was not detected by the immunoperoxidase technique. Faint immunofluorescence of ACE and ang II was observed in glomeruli from patients with MGA, as in normal tissues (Fig. 3a,c). In IgAN patients, staining for ACE tended to increase in an endocapillary pattern, but that of ang II was enhanced in GEC and MC of nephritic glomeruli (Fig. 3b,d). A similar staining level of AT1R was found in glomeruli, particularly MC, in specimens from patients with MGA or IgAN, and in control kidney tissues (Fig. 3e,f). A semiquantitative analysis of glomerular staining for AGT, ACE, ang II, and AT1R revealed that the level of glomerular AGT (mean score 1.89) and ang II (mean score Fig. 2 Glomerular expression of angiotensinogen (AGT) and its localization in the biopsies. Kidney sections were immunostained with anti-AGT antibody in patients with minor glomerular abnormalities (MGA) (a) or immunoglobulin A nephropathy (IgAN) (b) (original magnification × 200). Positive immunostaining of AGT could not be detected when a chicken antibody against AGT, absorbed by preincubation with excess human purified AGT, was used as the primary antibody (c). To identify glomerular localization of AGT, double immunofluorescence staining of AGT (green; d, g, j) with either glomerular endothe- lial cells (GEC) marker CD31 (red; e), activated mesangial cells (MC) (red; h), or podocyte marker (red; k) in IgAN was performed. Merged images show that AGT clearly colocalized on GEC (f), weakly on activated MC (i), and only faintly on podocyte (l) in IgAN (×400)