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 (O2−) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O2−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 O2−production in TALs. Measurable flow-induced O2−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 O2−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 O2−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 O2−was 82 ± 7 AU/s. We conclude that Nox4 mediates flow-induced O2−production in TALs.