ABSTRACT
The renin-angiotensin system (RAS) plays a critical role in the regulation of blood pressure. Inappropriate activation of the RAS, particularly stimulation of the ACE-Ang II-AT1 receptor axis is a key factor in hypertension and AT1R antagonists (ARBs) are first line therapies in the treatment of cardiovascular disease (CVD). Accumulating evidence suggests that the Ang II-AT1R axis may stimulate both innate and adaptive immune systems. Indeed, recent studies suggest that Ang II stimulates inflammatory events in an AT1R-independent manner by binding the MD2 accessory protein of the TLR4 complex in renal NRK-52E cells. Direct Ang II stimulation of the TLR4 complex is clinically relevant as ARBs increase circulating Ang II levels. Thus, the current study further investigated Ang II stimulation of the TLR4 pathway to release of the pro-inflammatory cytokine CCL2 under identical conditions to the TLR4 ligands LPS and palmitate in the NRK-52E cells. Although LPS (1 ng/mL) and palmitate (100 µM) stimulated CCL2 release 20-fold, Ang II (0.1-10 µM) failed to induce CCL2 release. Both the LPS and palmitate CCL2 responses were abolished by the TLR4 inhibitor Tak242 and significantly reduced by the MD2 inhibitor L48H37. Ang II (1 µM) had no additive effects on LPS (1 ng/mL) or palmitate (100 µM), and the ARB candesartan failed to attenuate CCL2 release to either agent alone. Ang II also failed to induce the release of the putative TLR4 ligand HMBG1. These studies failed to confirm that Ang II directly stimulates the MD2-TLR4 complex to induce cytokine release in NRK-52E cells.