NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs

Abstract

We previously showed that luminal flow stimulates thick ascending limb (TAL) superoxide (O(2)(-)) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O(2)(-) is NADPH oxidase (Nox). However, the specific Nox isoform involved is unknown. Of the three isoforms expressed in the kidney-Nox1, Nox2, and Nox4-we hypothesized that Nox4 is responsible for flow-induced O(2)(-) production in TALs. Measurable flow-induced O(2)(-) production at physiological flow rates of 0, 5, 10, and 20 nl/min was 5 ± 1, 9 ± 2, 36 ± 6, and 66 ± 8 AU/s, respectively. RT-PCR detected mRNA for all three Nox isoforms in the TAL. The order of RNA abundance was Nox2 > Nox4 >>> Nox1. Since all three isoforms are expressed in TALs and pharmacological inhibitors are not selective, we used rats transduced with siRNA and knockout mice. Nox4 siRNA knocked down Nox4 mRNA expression by 63 ± 7% but did not reduce Nox1 or Nox2 mRNA. Flow-induced O(2)(-) was 18 ± 9 AU/s in TALs transduced with Nox4 siRNA compared with 77 ± 9 AU/s in tubules transduced with scrambled siRNA. Flow-induced O(2)(-) was 81 ± 5 AU/s in Nox2 knockout mice compared with 83 ± 13 AU/s in wild-type mice. In TALs transduced with Nox1 siRNA, flow-induced O(2)(-) was 82 ± 7 AU/s. We conclude that Nox4 mediates flow-induced O(2)(-) production in TALs.

Fig. 1.

Superoxide (O₂⁻) production by the thick ascending limb in response to various luminal flow rates. P < 0.0003 for 10 nl/min and P < 0.0001 for 20 nl/min (compared with 0 nl/min); n = 4 to 6.

Fig. 2.

RT-PCR analysis of NADPH oxidase (Nox) isoforms expressed in the thick ascending limb. MW = 100-bp DNA molecular weight ladder.

Fig. 3.

Adenovirus-delivered Nox4 siRNA specificity in the thick ascending limb.

Fig. 4.

Flow-induced O₂⁻ in Nox4 siRNA-transduced thick ascending limbs. Neg con siRNA, transduction with nonsilencing AdscrsiRNA. Nox4 siRNA, transduction with AdNox4siRNA.

Fig. 5.

Flow-stimulated O₂⁻ production in wild-type and Nox2 knockout (−/−) mice.

Fig. 6.

Flow-induced O₂⁻ in Nox1 siRNA-transduced thick ascending limbs. Con, nontransduced. Nox1 siRNA, transduction with AdNox1siRNA.