Tumor necrosis factor-α suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells

Satou R, Miyata K, Katsurada A, Navar LG, Kobori H. Tumor necrosis factor-alpha suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells. Am J Physiol Cell Physiol 299: C750-C759, 2010. First published June 30, 2010; doi:10.1152/ajpcell.00078.2010.-Angiotensinogen (AGT) expression in renal proximal tubular cells (RPTCs) and intrarenal tumor necrosis factor-alpha (TNF-alpha) levels are increased in hypertension and renal diseases However, the contribution of TNF-alpha to AGT expression in RPTCs has not been established. Therefore, the objective of the present study was to determine influence of TNF-alpha on AGT expression in RPTCs. Human kidney-2 (HK-2) cells, immortalized human RPTCs, were treated with several concentrations of TNF-alpha up to 24 h. AGT mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. Activation of nuclear factor-kappa B (NF-kappa B) by TNF-alpha was evaluated by Western blot analysis, immunocytochemistry, and electrophoretic mobility shift assay (EMSA). TNF-alpha suppressed AGT mRNA expression in a dose-and time-dependent manner. Maximum AGT mRNA reduction was caused by 40 ng/ml of TNF-alpha (0.52 +/- 0.09, ratio to control, at 24 h) and at 24 h (0.66 +/- 0.05, ratio to control, by 10 ng/ml TNF-alpha). TNF-alpha reduced AGT protein accumulation in the medium between 8 and 24 h (0.62 +/- 0.13 by 40 ng/ml TNF-alpha, ratio to control). TNF-alpha activated and induced translocalization of p50 and p65, which are NF-kappa B subunits. Elevated formation of p50/p65 and p50/p50 dimers by TNF-alpha were observed by EMSA and supershift assay. Gene silencing of p50, but not p65, attenuated the effect of TNF-alpha on reduction of AGT expression in RPTCs. These results indicate that TNF-alpha suppresses AGT expression through p50/p50 homodimer formation in human RPTCs, suggesting a possible counteracting mechanism that limits excessive intrarenal AGT production.