1264 Fig. 7 Effects of angiotensin II (ang II) on the expression of angiotensinogen (AGT) messenger ribonucleic acid (mRNA) by cultured human glomerular endothelial cells (GEC). Human GEC were incubated with 10−7 M ang II for the indicated time, extracted, and analyzed for AGT mRNA levels by real-time polymerase chain reaction (RT-PCR) (a). Values are means ± standard deviation (SD). *P<0.05 and †P<0.01 vs time 0. Human GEC were incubated for 3 h with vehicle control or with indicated doses of ang II, extracted, and analyzed for AGT mRNA levels by RT-PCR (b). The graph represents the mean ± SD of at least three independent experiments. *P<0.05 and †P<0.01 vs control at 12 and 24 h after stimulation (Fig. 8a). Consistent with the results of RT-PCR analysis, a dose-dependent increase of human GEC-AGT protein to ang II stimulation was observed between 10−11 M and 10−7 M (Fig. 8b). Discussion Enhanced intrarenal RAS has been implicated in the development of hypertension and the progression of chronic renal diseases, including IgAN [1, 2, 17]. Ang II, the major effector molecule of the RAS cascade, is a key mediator of glomerular hypertension as well as glomerular sclerosis through the induction of TGF-β [5, 6]. However, glomerular expression of RAS components and their association with local RAS activity and injury has not been extensively studied in human subjects, especially pediatric patients with IgAN. In this study, we demonstrated for the first time that both glomerular expression of AGT protein, a sole substrate for RAS cascade, and ang II were strikingly increased with GEC and MC in nephritic glomeruli of pediatric IgAN. As Pediatr Nephrol (2008) 23:1257–1267 it is already known that ang II is locally produced within the glomerulus and RAS is present in glomerular cell types such as GEC, MC, and podocyte [19, 20, 34], the increased expression of ang II seen in pediatric IgAN most likely reflects enhanced glomerular RAS activation under this disease setting. We also demonstrated that there was a positive correlation between levels of glomerular AGT, ang II, and ang II- inducible factors (TGF-β and α-SM actin) and glomerular injury (cell proliferation and ECM accumulation), suggesting that the activated glomerular RAS cascade contributes to the development and progression of glomerular injury in pediatric IgAN. There are several studies investigating glomerular gene expression of RAS components in IgAN [17, 24, 35, 36]. Most studies have shown an increased expression of renin, AGT, and ACE genes and decreased expression of AT1R gene in nephritic glomeruli of IgAN. Furthermore, it has been shown that a negative feedback loop exists between ang II and its receptor, AT1R, in glomerular cells [36]. Therefore, if there was a proportional increase in ang II as a result of increased upstream components of RAS, a proportional Fig. 8 Effects of angiotensin II (ang II) on the expression of angiotensinogen (AGT) protein by cultured human glomerular endothelial cells (GEC). Human GEC were incubated with 10−7 M ang II for the indicated time, extracted, and then subjected to Western blot analysis for AGT and β-actin (a). Western blot analysis demonstrated two specific immunoreactive bands between 50 and 60 kD. Data for AGT protein levels were normalized by β-actin protein levels. The graph represents the mean ± standard deviation (SD) of at least three independent experiments. †P<0.01 vs time 0. Human GEC were incubated for 12 h with vehicle control or with indicated doses of ang II, extracted, and analyzed, as described in a (b). Data are means ± SD of at least three independent experiments. *P<0.05 vs control