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. 2010 Mar;298(3):R707-12.
doi: 10.1152/ajpregu.00762.2009. Epub 2010 Jan 6.

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

Yiling Fu  1 , Rui Zhang, Deyin Lu, Haifeng Liu, Kiran Chandrashekar, Luis A Juncos, Ruisheng Liu
Affiliations

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

Yiling Fu et al. Am J Physiol Regul Integr Comp Physiol. 2010 Mar.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
Effect of AT1 (losartan) and AT2 receptor blockade (PD-123319) on ANG II-induced O2− production in MMDD1 cells. O2− production increased significantly in MMDD1 cells stimulated with ANG II. Losartan blocked ANG II-induced O2−, whereas PD-123319 did not, indicating that ANG II was acting via its AT1 receptor. Values are means ± SE (n = 7). *P < 0.05 vs. control. #P < 0.05 vs. ANG II.
Fig. 2.
Fig. 2.
Contributions of NADPH oxidase (NOX), xanthine oxidase, and cyclooxygenase-2 (COX-2) to ANG II-induced O2− production in MMDD1 cells. Blocking NOX with apocynin blocked ANG II-induced increases in O2−, whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a COX-2 inhibitor) had no significant effect. Values are means ± SE (n = 15). *P < 0.05 vs. control. #P < 0.05 vs. ANG II.
Fig. 3.
Fig. 3.
Efficacy and specificity of the NOX2 and NOX4 isoform short interfering (si)RNAs in knocking down NOX2 and NOX4 mRNA. A: NOX2 mRNA levels in macula densa cells treated with scrambled, anti-NOX2, or anti-NOX4 siRNA. B: NOX4 mRNA levels in macula densa cells treated with scrambled, anti-NOX2, or anti-NOX4 siRNA. Results are expressed as a percentage of the scrambled siRNA control levels. Values are means ± SE (n = 4). *P < 0.05 vs. scrambled RNA.
Fig. 4.
Fig. 4.
Effect of the NOX2 and NOX4 siRNAs on ANG II-induced O2− production in MMDD1 cells. ANG II-induced O2− in NOX2/siRNA-treated MMDD1 cells was significantly less than in NOX4/siRNA-treated cells. Values are means ± SE (n = 5 each). *P < 0.05, ANG II vs. its control for each siRNA group. #P < 0.05, NOX2/siRNA + ANG II vs. NOX4/siRNA + ANG II.
Fig. 5.
Fig. 5.
Effect of the Rac1 inhibition on ANG II-induced O2− production in NOX2/siRNA- and NOX4/siRNA-treated MMDD1 cells. Inhibiting Rac1 did not further decrease ANG II induced O2− production in NOX2/siRNA-treated MMDD1 cells but significantly blunted it in NOX4/siRNA-treated cells. Values are means ± SE (n = 5). *P < 0.05 vs. NOX4/siRNA + ANG II.
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