Zheleznova Nadezhda N, Yang Chun, Cowley Allen W
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin.
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Am J Physiol Renal Physiol. 2016 Aug 1;311(2):F450-8. doi: 10.1152/ajprenal.00187.2016. Epub 2016 Jun 8.
Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress, and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 in the generation of H2O2 and O2 (·-) in the renal medullary thick ascending limb of Henle (mTAL) of SS rats in response to increasing luminal flow (from 5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the mTAL of SS rats. Responses of SS rats were compared with those of SS rats with knockout of Nox4 (SS(Nox4-/-)) or functional mutation of p67phox (SS(p67phox-/-)). Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined using the fluorescent dye peroxyfluor 6-AM (PF6-AM). The rate of mitochondrial H2O2 production [as determined by mitochondria peroxy yellow 1 (mitoPY1)] was also significantly reduced in SS(Nox4-/-) compared with SS rats, but not in SS(p67phox-/-) rats. In contrast, intracellular superoxide (O2 (·-)) production (the ratio of ethidium to dihydroethidium) in the mTAL of SS(Nox4-/-) rats was nearly identical to that of SS rats in response to luminal flow, indicating that Nox4 made no measurable contribution. mTAL O2 (·-) production was reduced in SS(p67phox-/-) compared with SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases in intracellular and mitochondrial H2O2, which are dependent on the presence of Nox4, and that p67phox/Nox2 accounts solely for increases in O2 (·-) production.
Nox4和Nox2是肾脏中含量最丰富的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶，在盐敏感型（SS）高血压大鼠中，它们会导致高血压、肾脏氧化应激和损伤。本研究聚焦于Nox4和p67phox/Nox2在SS大鼠髓袢升支粗段（mTAL）中，对管腔流量增加（从5 nl/min至20 nl/min）时过氧化氢（H2O2）和超氧阴离子（O2(·-)）生成的作用。研究发现，Nox4和p67phox/Nox2基因在SS大鼠的mTAL中表达。将SS大鼠的反应与Nox4基因敲除的SS大鼠（SS(Nox4-/-)）或p67phox功能突变的SS大鼠（SS(p67phox-/-)）进行比较。使用荧光染料过氧氟6-乙酰甲酯（PF6-AM）测定发现，Nox4是管腔流量增加时细胞内H2O2生成增加的主要来源。与SS大鼠相比，SS(Nox4-/-)大鼠线粒体H2O2生成速率[通过线粒体过氧黄1（mitoPY1）测定]也显著降低，但SS(p67phox-/-)大鼠则不然。相反，SS(Nox4-/-)大鼠mTAL中细胞内超氧化物（O2(·-)）生成（溴化乙锭与二氢溴化乙锭的比率）在管腔流量增加时与SS大鼠几乎相同，这表明Nox4对此无显著作用。在5 nl/min的低管腔流量下，与SS大鼠相比，SS(p67phox-/-)大鼠mTAL中O2(·-)生成减少，当灌注流量变为20 nl/min时逐渐增加。我们得出结论，mTAL管腔流量增加会导致细胞内和线粒体内H2O2增加，这依赖于Nox4的存在，而p67phox/Nox2仅导致O2(·-)生成增加。
