Pediatr Nephrol (2008) 23:1257–1267 Table 1 Clinical data of patients MGA minor glomerular abnormalities, IgAN immuno- globulin A nephropathy Data are means ± standard deviation * P<0.01 vs MGA Number Age (years) Term from onset of urinary abnormalities to renal biopsy (years) Systolic/diastolic blood pressure (mmHg) Proteinuria (g/day) Hematuria index Serum creatine (mg/dl) 24-h creatine clearance (ml/min per 1.73 m2) To evaluate the level of tubulointerstitial change, three to five fields of the cortical interstitium in each section were examined, as previously described [28]. In each field, tubulointerstitial change, like tubular atrophy and interstitial broadening, were graded as follows: 0, representing involve- ment of <5%; 1+, involvement between 5 and 20%; 2+, involvement between 20 and 40%; 3+, involvement of >40%. Antibodies Chicken IgY-rich fraction against highly purified full-length human plasma AGT (anti-AGT antibody) was used for immunohistochemistry [29]. Mouse monoclonal anti-ACE antibody, rabbit polyclonal anti-ang II serum, and rabbit polyclonal anti-AT1R antibody were purchased from Chemicon International (Temecula, CA, USA), IgG corpo- ration (Nashville, TN, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Mouse monoclonal anti-α-smooth-muscle (α-SM) actin antibody (1A4) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was used to detect activated human MC. Rabbit polyclonal anti-TGF-β antibody was a kind gift from Dr. Kawachi (Niigata University, Niigata, Japan). Immunohistochemistry The glomerular expression of AGT or AT1R was detected by an immunoperoxidase technique, as previously described [27]. Briefly, the sections (3 μm) were incubated with the primary antibodies overnight at 4°C. After rinsing, the sections were incubated with either biotinylated anti-chicken IgG (for AGT) (Vector Labs, Burlingame, CA, USA) or biotinylated anti-rabbit IgG (for AT1R) (Vector Labs). After rinsing, the sections were incubated with avidin-biotin- peroxidase complex (ABC Elite; Vector Labs), followed by 3,3′-diaminobenzidine (Dojindo, Kumamoto, Japan). Each section was counterstained with Mayer’s hematoxylin (Wako, Tokyo, Japan), dehydrated, and coverslipped. In double-staining experiments, 3-μm frozen sections stained with chicken anti-AGT antibody, and fluorescein- MGA 8 13.1±1.9 1.5±1.2 112.1±9.5/62.6±7.5 0.07±0.04 0.93±0.37 0.54±0.14 100.5±23.5 1259 IgAN 23 13.0±3.1 0.9±0.7 111.0±10.4/61.3±7.9 0.37±0.26* 1.89±0.70* 0.54±0.15 103.8±29.9 isothiocyanate (FITC)-labeled donkey anti-chicken IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were further incubated with either mouse anti-CD31 antibody (DAKO, Glostrup, Denmark), mouse monoclonal anti-α-SM actin antibody (1A4), or mouse mono- clonal synaptopodin antibody (G1D4; Progen Biotechnik, Heidelberg, Germany), followed by an appropriate tetra- methylrhodamine-isothiocyanate (TRITC)-coupled donkey anti-mouse antibody (Jackson Immuno-Research Laborato- ries). Indirect immunofluorescent staining was performed using the following primary antibodies: anti-ACE antibody, anti-ang II antiserum, anti-α-SM actin antibody, and anti- TGF-β antibody. FITC-labeled donkey anti-rabbit IgG anti- body, donkey anti-mouse IgG, and TRITC-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) were used as the secondary antibodies. Frozen sections were incubated with primary antibodies for 1 h, and then secondary antibodies, appropriately. To evaluate the level of glomerular staining with each antibody, we performed semiquantitative analysis. The immunostaining of AGT was graded as follows: 0, very weak or absent capillary or mesangial staining; 1+, weak capillary staining with 1–25% focally increased capil- lary staining; 2+, 25–50% of glomerular tuft demonstrating strong capillary and mesangial staining; 3+, 50–75% of glomerular tuft demonstrating strong capillary and mesangial staining; 4+, 75% or more of the glomerular tuft demonstrated strong capillary and mesangial staining, as previously described [25]. The immunostaining of ACE, ang II, AT1R, TGF-β, and α-SM actin was graded as follows: 0, no staining; 1+, weak staining; 2+, mild staining; 3+, moderate staining; 4+, marked staining. At least four equatorially sectioned glomeruli in each section were evaluated blindly by two separate investigators. The individual immuno- staining score was determined by calculating the average value per single glomerulus [27]. Cell culture Cultured human GEC derived from normal human kidneys were purchased from Cell System (Kirkland, WA, USA).