Liu Ruisheng, Garvin Jeffrey L, Ren YiLin, Pagano Patrick J, Carretero Oscar A
Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA.
Am J Physiol Renal Physiol. 2007 Jun;292(6):F1867-72. doi: 10.1152/ajprenal.00515.2006. Epub 2007 Mar 6.
Superoxide (O(2)(-)) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O(2)(-) production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O(2)(-) production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O(2)(-) production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O(2)(-) production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O(2)(-), we used tempol (10(-4) M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O(2)(-), we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O(2)(-) production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O(2)(-) production was blocked. To test whether depolarization of the macula densa induces O(2)(-) production, we artificially induced depolarization by adding valinomycin (10(-6) M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O(2)(-) production. We conclude that increasing luminal NaCl induces O(2)(-) production during tubuloglomerular feedback. O(2)(-) generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.
超氧阴离子（O₂⁻）通过清除致密斑处的一氧化氮来增强球管反馈。然而，致密斑处O₂⁻产生的信号通路尚不清楚。我们推测，引发球管反馈的肾小管氯化钠浓度升高会通过NAD(P)H氧化酶诱导致密斑产生O₂⁻，而NAD(P)H氧化酶会因致密斑去极化而被激活。我们分离并对兔Henle袢升支粗段及附着的致密斑进行了微灌流。使用荧光染料二氢乙锭来检测致密斑处O₂⁻的产生。当管腔氯化钠浓度从10 mM切换到80 mM时，即引发最大球管反馈反应的情况，O₂⁻的产生显著增加。为确保氧化乙锭/二氢乙锭比率的变化是由于O₂⁻的变化引起的，我们使用了tempol（10⁻⁴ M），一种稳定的可透过细胞膜的超氧化物歧化酶模拟物。在存在tempol的情况下，当我们将氯化钠浓度从10 mM切换到80 mM时，氧化乙锭/二氢乙锭比率的增加被阻断。为确定O₂⁻的来源，我们使用了NAD(P)H氧化酶抑制剂阿朴吗啡。当在阿朴吗啡存在下将管腔氯化钠浓度从10 mM切换到80 mM时，O₂⁻的产生被抑制了80%。为观察增加管腔氯化钠的作用是否涉及钠 - 钾 - 2氯共转运体，我们用呋塞米抑制它们。当在呋塞米存在下将管腔氯化钠浓度从10 mM切换到80 mM时，O₂⁻的产生被阻断。为测试致密斑去极化是否诱导O₂⁻的产生，我们通过向管腔灌流液中添加缬氨霉素（10⁻⁶ M）和25 mM氯化钾来人工诱导去极化。单独的去极化显著增加了O₂⁻的产生。我们得出结论，在球管反馈过程中增加管腔氯化钠会诱导O₂⁻的产生。致密斑产生的O₂⁻主要来源于NAD(P)H氧化酶，并由去极化诱导产生。
