Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4

Abstract

Angiotensin II (ANG II) stimulates production of superoxide (O(2)(-)) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O(2)(-) production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O(2)(-) production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O(2)(-) production in wild-type and NOX2 knockout mice (KO). ANG II increased O(2)(-) production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O(2)(-) production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O(2)(-) production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O(2)(-) production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O(2)(-) production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O(2)(-) production by TALs.

Fig. 1.

Effect of 1 nM ANG II on net O₂⁻ production in isolated rat thick ascending limbs. RLU, relative light units. Tubules were exposed to ANG II for 5 min (*P < 0.03; n = 6).

Fig. 2.

A: effect of 100 mM apocynin on net ANG II-induced O₂⁻ production in isolated thick ascending limbs. Tubules were exposed to apocynin for 10 min and ANG II for 5 min before acquisition of data. (*P < 0.02; n = 6). B: effect of 100 mM apocynin on basal O₂⁻ production in isolated thick ascending limbs. Tubules were exposed to apocynin for 10 min and ANG II for 5 min before acquisition of data (NS; n = 6).

Fig. 3.

Protein expression of each NADPH oxidase isoform in 50 μg of total protein from isolated thick ascending limbs. Each Western blot was probed with isoform-specific antibodies. NOX, NADPH oxidase.

Fig. 4.

Effect of 1 nM ANG II on net O₂⁻ production in isolated thick ascending limbs from wild-type or NOX2 knockout mice. Tubules were exposed to ANG II for 5 min before acquisition of data. (n = 9).

Fig. 5.

Effect of adenovirus delivery of NOX4 short hairpin (sh)RNA on ANG II-induced O₂⁻ production in isolated thick ascending limbs. Tubules were isolated 6 or 7 days after transduction with NOX4 shRNA and treated with 1 nM ANG II for 5 min before acquisition of data (*P < 0.005; n = 7).

Fig. 6.

Effect of adenovirus delivery of NOX4 shRNA on NOX4 mRNA measured 6 or 7 days following transduction. (*P < 0.0001; n = 7).

Fig. 7.

Western blot showing the effect of 1 nM ANG II for 5 min at 37° on NOX4 protein expression using an isoform-specific antibody.

Fig. 8.

Effect of 100 mM apocynin on net ANG II-induced O₂⁻ production in isolated thick ascending limbs transduced with NOX4 shRNA. Tubules were exposed to apocynin for 10 min and ANG II for 5 min before acquisition of data (n = 8).