Hypertension and Vascular Research Div., Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA.
Am J Physiol Renal Physiol. 2012 Oct 15;303(8):F1151-6. doi: 10.1152/ajprenal.00181.2012. Epub 2012 Aug 15.
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.
我们之前已经表明，管腔液流通过拉伸上皮细胞和增加 NaCl 转运来刺激升支粗段（TAL）超氧化物（O(2)(-)）的产生，并报道了诱导 O(2)(-)的主要来源是 NADPH 氧化酶（Nox）。然而，涉及的具体 Nox 同工型尚不清楚。在肾脏中表达的三种同工型-Nox1、Nox2 和 Nox4-中，我们假设 Nox4 负责 TAL 中诱导的 O(2)(-)的产生。在 0、5、10 和 20 nl/min 的生理流速下，可测量的诱导 O(2)(-)的产生分别为 5 ± 1、9 ± 2、36 ± 6 和 66 ± 8 AU/s。RT-PCR 在 TAL 中检测到所有三种 Nox 同工型的 mRNA。RNA 丰度的顺序为 Nox2 > Nox4 >>> Nox1。由于所有三种同工型都在 TAL 中表达，并且药理学抑制剂不具有选择性，因此我们使用转导了 siRNA 的大鼠和敲除小鼠进行研究。Nox4 siRNA 将 Nox4 mRNA 的表达敲低了 63 ± 7%，但没有降低 Nox1 或 Nox2 mRNA。转导了 Nox4 siRNA 的 TAL 中诱导的 O(2)(-)为 18 ± 9 AU/s，而转导了乱序 siRNA 的小管中为 77 ± 9 AU/s。Nox2 敲除小鼠的诱导的 O(2)(-)为 81 ± 5 AU/s，而野生型小鼠为 83 ± 13 AU/s。在转导了 Nox1 siRNA 的 TAL 中，诱导的 O(2)(-)为 82 ± 7 AU/s。我们得出结论，Nox4 介导了 TAL 中诱导的 O(2)(-)的产生。
