Abstract 565: Angiotensin Ii Type 1 Receptor Activation is Required for Angiotensin Ii and Interleukin 6-induced Augmentation of Angiotensinogen Expression in Mouse Renal Proximal Tubular Cells

Abstract

In angiotensin II (AngII) dependent hypertension, an inappropriate elevation of intrarenal Ang II leads to the progression of hypertension and kidney injury. Intrarenal angiotensinogen (AGT), produced mainly by renal proximal tubular cells (PTC), is a crucial factor in regulation of intratubular Ang II. Increases in intrarenal AGT are associated with increases in intrarenal immune cells and interleukin 6 (IL-6) levels which contribute to Ang II-dependent AGT augmentation. These results suggest that Ang II and IL-6 synergize to increase AGT levels during progression of Ang II-dependent hypertension. However, the interactions among Ang II, Ang II type 1 receptors (AT1R) and IL-6 on AGT expression and the mechanisms have not been delineated. In this study, mouse PTC (WT-PTC) and AT1aR knockout strains (AT1aRKO-PTC) were used to test the existence of this synergism between Ang II and IL-6. Basal AGT mRNA expression levels in PTC were determined and compared with that in mouse mesangial cells which is another established AGT-expressing kidney cells. AGT mRNA expression levels were measured by real-time RT-PCR and normalized based on GAPDH levels. WT-PTC had 21.1-fold (± 2.0) higher basal AGT mRNA levels than mesangial cells, indicating markedly high AGT production by the PTC. Further, WT-PTC and AT1aRKO-PTC were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Stimulation with either Ang II or IL-6 alone did not significantly alter AGT mRNA expression in both WT-PTC and AT1RaKO-PTC. Co-stimulation with Ang II and IL-6 increased AGT mRNA levels (1.59 ± 0.04, ratio to control) in WT-, but not AT1aRKO-PTC. To test for involvement of the NF-κB signaling pathway in the synergism between Ang II and IL-6, activation of NF-κB as an AT1R signaling pathway was evaluated by western blot analysis. Furthermore, WT-PCT were treated with an NF-κB inhibitor during the co-stimulation. In WT-PTC, IκB levels were decreased by the co-stimulation (0.61 ± 0.04, ratio to control) indicating NF-κB activation. Ten μM Parthenolide, an NF-κB inhibitor, attenuated the AGT mRNA increase induced by the co-stimulation. These results suggest that simultaneous AT1R activation acts synergistically with IL-6 to increase AGT expression in mouse PTC and that the signaling pathway involves NF-κB.