1258 often to an extent far greater than their effect on blood pressure [9–11]. IgA nephropathy (IgAN) is now recognized to be the most common form of glomerulonephritis worldwide. It has been reported that more than 30% of cases progress to end-stage renal failure within 20 years [12, 13]. In pediatric IgAN, glomerular lesion is characterized by an increased number of mesangial cells (MC) and accumulation of mesangial extracellular matrix (ECM), and this pathological change is thought to be an early and active lesion for IgAN [14]. It is reported that treatment with ACEi and/or ARB provides antiproteinuric and renoprotective effects in normotensive pediatric patients with IgAN, suggesting that intrarenal action of ang II may play a critical role in the progression of pediatric IgAN [15, 16]. Recently, a real-time polymerase chain reaction (RT-PCR) study using isolated glomeruli with IgAN revealed an increased expression of RAS components such as renin, angiotensinogen (AGT), and ACE, possibly followed by the production of ang II, an effector molecule of RAS, suggesting that the glomerular RAS activation might occur under this disease setting [17]. In this study, AGT messenger ribonucleic acid (mRNA) levels were positively correlated with the mRNA expres- sion of renin, ACE, and a fibrogenic cytokine transforming growth factor-β (TGF-β). AGT is the sole substrate for the RAS pathway, and it has been reported that AGT is present within the kidney and ang II is produced locally, indepen- dent of the circulating RAS [18]. Interestingly, the increased expression of intrarenal AGT is directly involved in the disease progression in several rat models of renal injury [19–22]. However, little information is available about protein expression in an RAS cascade from AGT to ang II and the association between glomerular RAS activation and pathological changes in childhood IgAN. Therefore, in this study, the glomerular protein expres- sion of RAS components such as AGT, ACE, ang II, and AT1R, as well as their association with the index of glomerular injury were examined to clarify the role of the RAS cascade, especially that of AGT in glomerular lesions of pediatric IgAN. Subsequently, we investigated the amplification mechanism of glomerular AGT expression leading to an increase of glomerular RAS activity using cultured human glomerular endothelial cells (GEC). Materials and methods Kidney tissue Renal tissues used for this study were obtained from 23 children with IgAN and eight with minor glomerular abnormalities (MGA) by biopsy. Diagnosis of renal diseases was made based on studies of tissue by light, electron, Pediatr Nephrol (2008) 23:1257–1267 and immunofluorescence microscopy according to the classification of the World Health Organization (WHO) [23]. This classification defined diffuse mesangial prolifer- ation as >80% of glomeruli showing moderate or severe mesangial cell proliferation, i.e. more than three cells per peripheral mesangial area, and focal mesangial proliferation as up to 80% of glomeruli showing the same changes described above. IgAN was diagnosed as a mesangial proliferative glomerulonephritis with predominant IgA de- posit in the mesangium. Patients diagnosed with IgAN showed diffuse mesangial proliferation (15) or focal mesangial proliferation (8). Patients diagnosed with MGA showed normal glomerular morphology and negative immu- nofluorescence but had mild proteinuria or microscopic hematuria. All subjects with other disorders, including Henoch-Schönlein purpura, systemic lupus erythematosus, and chronic liver disease, were excluded. The control tissue (5) consisted of normal cortex taken from kidneys resected for localized neoplasms. Blood pressure, serum creatinine, and creatinine clearance were normal in all participants at the time of biopsy. Patients had not been previously treated with either ACEi, ARB, glucocorticosteroids, or immunosuppres- sive agents. The study protocol was approved by the ethics committee of the University of Tokushima Graduate School, and written informed consent was obtained from all patients and/or their parents in all cases. The clinical laboratory parameters, including age, term from onset of urinary abnormalities to renal biopsy, blood pressure, proteinuria, hematuria index, serum creatinine, and 24-h creatinine clearance were determined at the time of biopsy. Based on examination of urine samples, the severity of hematuria was classified as follows: 0, 0–5 erythrocytes/high-power field (HPF); 1+, 5–30 erythrocytes/HPF; 2+, >30 erythrocytes/ HPF; 3+, macroscopic hematuria, as previously described [24]. Clinical data are presented in Table 1. Histological scoring of glomerular cell proliferation, glomerulosclerosis, and tubulointerstitial changes For light-microscopic examination, the biopsied tissues were fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections (3 μm) were stained with periodic acid- Schiff reagent. All glomeruli in each section (usually 10–26) were coded and read by a blinded observer. The number of cells in at least four equatorially cut glomeruli in each section was counted, and the average number of cells was used as an indicator of glomerular cell proliferation, as previously described [25]. The glomerulosclerosis (GS) score was deter- mined using a previously described method with a modifi- cation [26]. The percentage of each glomerulus occupied by mesangial ECM was estimated and assigned a code as follows: 0, absent; 1+, 1–5%; 2+, 5–25%; 3+, 25–50%; 4+, >50%; and the mean value per specimen was calculated [27].