1260 Human GEC were cultured on culture flask coated with type 1 collagen, as described previously [30]. Human GEC were used at the sixth to eighth passage in our experiments. At about 70–80% confluency, GEC growth was arrested by incubation for 48 h in serum-free medium and then treated with ang II at increasing concentrations (0, 10−11, 10−9, 10−7 M) for 3–12 h to detect mRNA or protein, respec- tively. In addition, a time-course study was undertaken to assess changes in AGT mRNA expression at 0, 1, 3, 6, 12, and 24 h and protein expression at 0, 6, 12, and 24 h with 10−7 M ang II-treated GEC. Cells were harvested for RNA extraction and total cell extract preparation. Western blot Human GEC cultures were dissolved in radioimmunopre- cipitation assay (RIPA) buffer. Protein samples (25 μg) were separated by 12.5% sodium dodecyl sulfate poly- acrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). The membranes were probed with anti-AGT antibody (1:1,000) and then incubated with a horseradish peroxidase-conjugated rabbit anti-chicken/ turkey IgG (H+L) (1:2,000; Zymed Laboratories, San Francisco, CA, USA). Equivalent protein loading for each lane was confirmed by stripping and reblotting each membrane for β-actin (1:10,000; Sigma-Aldrich). Immu- noreactive proteins were detected with an enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL, USA). The antibodies were tested on human purified AGT (Calbiochem, Merck, Darmstadt, Germany) as control, where two bands between 50 and 60 kD were detected [31]. Bands were quantitated by ImageJ 1.33u (National Institute of Health), and the fold expression was indicated as the relative protein level. RT-PCR Total RNA (500 ng) extracted from human cell cultures was reverse transcripted using the SYBR ExScript RT-PCR Kit (TaKaRa, Kyoto, Japan). The complementary DNAs (cDNAs) were amplified by RT-PCR with primers for human AGT (forward 5′-TCTCCCCGGACCATCCA-3′ and reverse 5′-TGCTCAATTTTTGCAGGTTCAG-3′) and glyceraldehyde-3 phosphate dehydrogenase (G3PDH) (forward 5′-GAAGGTGAAGGTCGGAGT-3′ and reverse 5′-TGGCAACAATATCCACTTTACCA-3′) as an internal control, using iCycler Thermal Cycler (BioRad, Hercules, CA, USA) [17, 32]. Melting curve analysis and PAGE confirmed the specificity of the amplification products. Semiquantification data were analyzed with iCycler analysis software and expressed as the ratio between starting the quantity mean of the target and the G3PDH gene. Pediatr Nephrol (2008) 23:1257–1267 Statistical analysis Results are presented as means ± standard deviation (SD). StatView (SAS institute) was used for statistical analyses. Between-group differences were assessed by the unpaired t test. The correlation of two variations was evaluated using Pearson’s or Spearman’s correlation coefficients. Western blot and RT-PCR analyses were performed at least three times, and representative results are shown in the figures. The mRNA expression or protein synthesis of AGT in cultured cells was analyzed by t test. Values of P< 0.05 were considered statistically significant. Results Clinical data and histologic changes in patients Clinical data in patients with MGA or IgAN are listed in Table 1. There were no significant differences between MGA and IgAN patients for age, follow-up term from onset of urinary abnormalities, blood pressure, serum creatinine, and creatinine clearance at the time of biopsy. Mean urine protein excretion was significantly higher in IgAN patients than that in MGA patients (P < 0.01). The extent of glomerular cellularity, ECM accumulation, and tubulointer- stitial changes were semiquantified in both patient groups and are shown in Fig. 1. The glomerular cell number and GS score were significantly higher in IgAN patients compared with those in MGA patients (MGA vs IgAN; mean cell number: 66.1 vs 77.9, P<0.05; mean GS score: 0.73 vs 1.58, P<0.01). Tubular atrophy and interstitial broadening were faintly present in IgAN patients but not in MGA patients. However, there was no significant difference in tubulointerstitial score change between the two groups (mean score: 0.00 in MGA vs 0.26 in IgAN, P=0.188). Fig. 1 The scorings of glomerular cell number (a), glomerulosclerosis (b), and tubulointerstitial changes (c) in the biopsies. Glomerular cell count and glomerulosclerotic indexes except tubulointerstitial changes significantly increased in immunoglobulin A nephropathy (IgAN) (■) compared with minor glomerular abnormalities (MGA) (□). *P<0.05 vs MGA; †P<0.01 vs MGA