NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

Fu Y, Zhang R, Lu D, Liu H, Chandrashekar K, Juncos LA, Liu R. NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa. Am J Physiol Regul Integr Comp Physiol 298: R707-R712, 2010. First published January 6, 2010; doi:10.1152/ajpregu.00762.2009.-Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O-2(-)) and angiotensin II (ANG II). We have reported that NaCl-induced O-2(-) in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O-2(-) in MD is unknown. Thus we determined the pathways by which ANG II increased O-2(-) in the MD by measuring O-2(-) in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O-2(-) levels to increase by more than twofold (P < 0.01). This increase was blocked by losartan (AT(1) receptor blocker) but not PD-123319 (AT(2) receptor antagonist). Apocynin (a NOX inhibitor) decreased O-2(-) by 86% (P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O-2(-) was due to the NOX2 isoform; a short interfering (si) RNA against NOX2 blunted ANG II-induced increases in O-2(-), whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O-2(-) production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O-2(-) production in the MD primarily via AT(1)-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.