Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice.

NAD(P)H oxidase (Nox)2 and Nox4 are the isoforms of Nox expressed in the macula densa (MD). MD-derived superoxide (O₂⁻), primarily generated by Nox2, is enhanced by acute ANG II stimulation. However, the effects of chronic elevations in ANG II during ANG II-induced hypertension on MD-derived O₂⁻ are unknown. We infused a slow pressor dose of ANG II (600 ng·min⁻¹·kg⁻¹) for 2 wk in C57BL/6 mice and found that mean arterial pressure was elevated by 22.3 ± 3.4 mmHg (P < 0.01). We measured O₂⁻ generation in isolated and perfused MDs and found that O₂⁻ generation by the MD was increased from 9.4 ± 0.9 U/min in control mice to 34.7 ± 1.8 U/min in ANG II-induced hypertensive mice (P < 0.01). We stimulated MMDD1 cells, a MD-like cell line, with ANG II and found that O₂⁻ generation increased from 921 ± 91 to 3,687 ± 183 U·min⁻¹·10⁵ cells⁻¹, which was inhibited with apocynin, oxypurinol, or NS-398 by 46%, 14%, and 12%, respectively. We isolated MD cells using laser capture microdissection and measured mRNA levels of Nox. Nox2 and Nox4 levels increased by 3.7 ± 0.17- and 2.6 ± 0.15-fold in ANG II-infused mice compared with control mice. In MMDD1 cells treated with Nox2 or Nox4 small interfering (si)RNAs, ANG II-stimulated O₂⁻ generation was blunted by 50% and 41%, respectively. In cells treated with p22(phox) siRNA, ANG II-stimulated O₂⁻ generation was completely blocked. In conclusion, we found that a subpressor dose of ANG II enhances O₂⁻ generation in the MD and that the sources of this O₂⁻ are primarily Nox2 and Nox4.