摘要
第一部分：醛固酮诱导致密斑细胞产生超氧阴离子
目的：
近年来,氧化应激在高血压等心血管疾病发生发展过程中的作用引起人们广泛关注。有研究报道心肌梗死和心衰病人体内醛固酮水平明显增高,会导致心脏炎症发生,该过程伴有心肌细胞氧化应激损伤和环氧合酶-2(Cyclooxygenase 2, COX-2)等大量炎性介质的释放。在脑和肠系膜动脉中,醛固酮能够通过释放一氧化氮(Nitric oxide, NO)介导血管舒张,同时还能够激活NAD(P)H氧化酶(Nicotinamide adenine dinucleotide phosphate-oxidase)使细胞内超氧阴离子(Superoxide, O2-)生成增多。在对体外培养的动脉上皮细胞系研究中也发现醛固酮通过刺激NAD(P)H (Nicotinamide adenine dinucleotide phosphate-oxidase)氧化酶增加02-的生成。另外,有人提出在临床治疗中及时纠正氧化／抗氧化失衡,可显著降低慢性肾衰病人并发心血管疾病的危险性。
管球反馈(Tubuloglomerular glomerular feedback, TGF)是调节肾血流动力学、NaCl排泄和血压的重要机制。当流经肾致密斑血流量增加时,通过TGF反应可引起肾小球入球小动脉收缩,从而使肾小球率过滤降低。致密斑(Macula densa, MD)细胞释放一氧化氮可以调节TGF功能,而同样由MD合成的O2-则可使NO失活。我们在近期研究中发现NAD(P)H氧化酶NOX-2和NOX-4亚型在大鼠MD和MMDD1(Macula dense like cell line)细胞中都有表达。虽然醛固酮能诱导心脏和血管发生氧化或炎症过程,但其在MD细胞中的作用机制还尚不清楚。
盐皮质激素受体(Mineralocorticoid receptor,MR)可被醛固酮激活,该受体在机体中分布广泛,其中包括肾远曲小管和集合管等,但在MD细胞中是否有表达却尚无报道。
我们推测醛固酮可能通过激活MD细胞上MR而增加02-合成,此过程是由COX-2增加和NOX-2、NOX-4激活介导的。该研究有助于进一步了解醛固酮引起的氧化应激在高血压和高血压相关靶器官(心脏和肾脏等)损伤中的作用,为临床治疗提供新的干预靶点。
材料方法
实验细胞系
本实验中所采用的MMDD1细胞是具有致密斑细胞特性的肾小管上皮细胞系。
超氧阴离子(02-)检测
我们使用lucigenin增强化学发光法检测MMDD1细胞中02-的量。操作步骤主要为,磷酸盐缓冲液(Phosphate buffer solution, PBS)冲洗细胞两次,1 mL胰酶消化1 min,离心,去上清后,加入12 mL Krebs/Hepes缓冲液制备细胞悬浊液。然后分别进行以下五种处理：无处理组；醛固酮(10-8mol/L)组；醛固酮+COX-2阻断剂(NS398 10-6 mol/L)组；醛固酮+NAD(P)H阻断剂(apocynin 10-5 mol/L)组；醛固酮+COX-2+NAD(P)H阻断剂(醛固酮10-8 mol/L, NS398 10-6 mol/L和apocynin 10-5 mol/L)组。同时每组加入lucigenin (5×10-6 mol/L)37℃水浴30min后上机检测。PCR
使用RNeasy Mini kit提取MMDD1细胞和小鼠心肌细胞tRNA。逆转录合成cDNA后,按以下步骤扩增COX-2、NOX-2、NOX-4和MR目的片段。1)1μL cDNA产物,1μL特异性引物,2μL 10×PCR缓冲液,1μL dNTP 2.5 mmol/L, 0.2μL Super Taq酶(5 U/μL),NOX-2和NOX-4的PCR体系中加入1.2μL 50%甘油,最后加入去RNAase水使体系总体积达到20μL。2) PCR条件为：94℃3min,循环条件为94℃20 sec,59.4℃(COX-2),56.6℃(NOX-2),52.9℃(NOX-4) 30 sec, 72℃30 sec并循环40次。最后延长72℃8 min。3) MR受体的PCR体系为50μL其中包括1μL MMDD1 cDNA或是1μL小鼠心肌cDNA、MR引物和Taq多聚酶。PCR条件为,95℃2 min,循环条件为95℃40 sec,退火温度为58℃40sec,72℃55 sec并循环40次。最后延伸72℃8 min。电泳分离产物,观察拍照。β-actin作为内参。Real-time PCR
醛固酮处理MMDD1细胞30 min后,使用RNeasy Micro kit (Qiagen)提取细胞tRNA,然后逆转录成cDNA。最后使用C1000TM Thermal Cycler real-time PCR仪参照说明书进行实验。检测COX-2、NOX-2和NOX-4 mRNA。β-actin作为内参。siRNA转染
使用COX-2 siRNA (Ambion,美国)转染MMDD1细胞,以沉默COX-2基因。转染使采用siPORTTM Amine Transfection Agent (Ambion,美国)作为转染辅助物。Scrambled siRNA(Invitrogen,美国)作为阴性对照。实验流程为：在转染之前24 h,准备MMDD1细胞,将细胞分到6孔细胞培养盘中(2×105细胞／孔),并且使用无抗生素的培养液培养。待细胞数为80%左右时,进行转染实验。转染时,将COX-2 siRNA 10 nmol/L和Amine Transfection Agent加入到培养细胞中共孵育18 h。然后,PBS冲洗细胞一次后更换正常细胞培养液,转染24 h后检测siRNA转染效率。Western blot
RIPA缓冲液(加入蛋白酶抑制剂混合物)离心提取蛋白之后,使用Nanodrop分光光度计检测蛋白浓度,光波长度设定为280 nM。蛋白样本经电泳和转膜后,1%脱脂奶粉TTBS室温封闭一小时。根据研究目的,分别使用以下一抗,包括：1)MR受体一抗混合rMR 1-18 clone 1D5 (1:50)和MRN 365 clone2D6(1：100)。2)兔来源抗COX-2抗体(1：500)；兔来源抗NOX-2抗体(1：1000)；3)兔来源抗NOX-4抗体(HRP)(1：1000)；4)小鼠来源抗GAPDH抗体(1：5000),在4℃孵育过夜。然后TTBS多次冲洗,室温下辣根过氧化物酶(Horseradish peroxidase, HRP)标记的二抗(1：10000)孵育1 h (NOX-4除外),ECL显影。Western blot条带采用VersaDoc image analysis system (Bio-Rad)进行分析。GAPDH作为内参蛋白。
统计分析
我们采用了单因素方差分析(ANOVA)和post-hoc Fisher LSD检验对数据进行统计分析,所有数据均采用均数±标准误表示,以P<0.05为显著性差异界值。
结果1. MMDD1细胞上存在MR受体。本实验从mRNA水平和蛋白水平证实MMDD1细胞上有盐皮质激素受体(MR)表达。
2.醛固酮通过激活MMDD1细胞上MR受体产生超氧阴离子O2-。醛固酮(10-8mol/L)处理细胞30 min后,O2-生成量从1260.9±50.9 RLU·s-1·105 cells-1增加到2463.5±145.2 RLU·s-1·105 cells-1。而细胞经MR受体拮抗剂eplerenone预孵育后再加入醛固酮,结果显示eplerenone完全阻断醛固酮诱发MMDD1细胞增多超氧阴离子02-的效果,02-生成量为1264.4±50.0 RLU·s-1·105 cells-1。
3. RT-PCR结果显示MMDD1细胞上有COX-2、NOX-2和NOX-4 mRNA表达。
4.醛固酮增加MMDD1细胞中COX-2、NOX-2和NOX-4蛋白表达。平行比较不同浓度醛固酮(10-9 mol/L和10-8 mol/L)对MMDD1细胞中COX-2、NOX-2和NOX-4蛋白表达量影响,发现10-8mol/L浓度的醛固酮,蛋白上调作用最为显著。醛固酮(10-8mol/L)处理细胞30 min后,MMDD1细胞中COX-2、NOX-2和NOX-4蛋白表达明显增多,其增加量分别为对照组的1.78±0.05倍、2.31±0.07倍和2.33±0.14倍。
5.醛固酮通过COX-2、NOX-2和NOX-4影响MMDD1细胞超氧阴离子O2-生成。我们在观察不同阻断剂对醛固酮引起MMDD1细胞02-合成增多的影响中发现,NS-398 (10-6 mol/L) (COX-2阻断剂),apocynin (10-5 mol/L) (NAD(P)H氧化酶阻断剂)或是同时加入NS-398 (10-6 mol/L)和apocynin (10-5 mol/L)预处理细胞都能够完全阻断醛固酮刺激MMDD1细胞内02-生成增多的作用。
6.siRNA对MMDD1细胞中COX-2表达的影响。COX-2 siRNA显著降低MMDD1细胞中COX-2 mRNA表达。Scrambled siRNA对COX-2 mRNA则无明显作用。COX-2 siRNA对NOX-2 mRNA和NOX-4 mRNA表达无影响。以上结果表明该COX-2 siRNA对MMDD1细胞中COX-2沉默作用具有特异性和高效性。
7.COX-2 siRNA对醛固酮诱导MMDD1细胞超氧阴离子O2-合成增多的影响。使用醛固酮(10-8 mol/L)刺激MMDD1细胞30 min,细胞中COX-2、NOX-2和NOX-4 mRNA表达明显增多。而使用COX-2 siRNA选择性沉默COX-2后,再用醛固酮刺激MMDD1细胞,结果显示醛固酮增加COX-2、NOX-2和NOX-4 mRNA水平的作用被阻断。
为进一步验证COX-2在醛固酮诱导细胞产生超氧阴离子O2-过程中的作用,我们使用COX-2 siRNA预处理MMDD1细胞后,再检测其中超氧阴离子O2-水平。结果显示,醛固酮刺激MMDD1细胞内超氧阴离子O2-释放增多,而COX-2 siRNA预处理细胞则逆转醛固酮该效果。
结论
在MMDD1细胞中,醛固酮作用于MR受体,刺激细胞内COX-2表达增多,激活NAD(P)H氧化酶亚基NOX-2和NOX-4,从而引起细胞合成超氧阴离子O2-增加。
第二部分：PKCa在醛固酮诱导致密斑细胞产生超氧阴离子中的作用
目的
醛固酮能激活结肠和肾小管(远端小管、收集小管和集合管等)上皮细胞上盐皮质激素受体(Mineralocorticoid receptors, MR),引起电解液流量增加；它还能诱导心脏炎症和心肌氧化应激的发生。在论文第一部分我们主要阐述了醛固酮通过激活COX-2和NAD(P)H氧化酶导致MMDD1细胞生成O2-产物增多。但醛固酮该促氧化作用由何种细胞信号通路传导尚不清楚。
蛋白激酶C (Protein kinase C, PKC)是介导细胞对类固醇类激素发生快速反应的一种信号转导蛋白。有研究报道醛固酮能增加肾皮质集合管细胞中的钠离子转运,该作用是经由PKCa信号通路介导。因此,我们推测PKCa可能参与了醛固酮通过刺激NOX-2和NOX-4导致MD细胞中的O2-产物增加的促氧化作用,并在本次研究中对该假设进行验证。
材料方法
同第一部分。
结果
1. NAD(P)H氧化酶抑制剂阻断醛固酮刺激MMDD1细胞产生O2-的作用。醛固酮(10-8 mol/L)刺激MMDD1细胞30 min后,O2-生成量明显增多,从1293±106 RLU·s·-1·105 cells-1升高到2349±222 RLU·s-1·105 cells-1。同时加入醛固酮和NAD(P)H氧化酶抑制剂apocynin处理MMDD1细胞30 min,醛固酮刺激MMDD1细胞合成O2-增多的效果被阻断。
2.PKC抑制剂减弱醛固酮对MMDD1细胞促氧化作用。醛固酮(10-8mol/L)处理MMDD1细胞30 min后,O2-生成量从1184±54 RLU·s-1·105 cells-1增加到1982±138 RLU·s-1·105 cells-1。同时加入醛固酮和PKC抑制剂CC (Chelerythrine chloride)刺激MMDD1细胞30 min,醛固酮诱导MMDD1细胞O2-合成增多的效果明显减弱,表明PKC抑制剂阻断醛固酮促氧化作用。
3. PKCα特异性抑制剂减弱醛固酮促氧化作用。为验证PKC亚型是否参与醛固酮促氧化作用,我们在实验中使用PKCα特异性抑制剂Go6976 (Go).醛固酮(10-8 mol/L)明显增加MMDD1细胞合成O2-的作用。而同时加入醛固酮和Go6976 (Go)处理细胞30 min后,醛固酮诱导MMDD1细胞O2-合成增多的效果被阻断,表明PKCa参与醛固酮该促氧化作用。
4. PKCαsiRNA阻断醛固酮对MMDD1细胞促氧化作用。为进一步证实PKCα在醛固酮促氧化途径中的作用。我们在实验中使用PKCαsiRNA,经验证PKCαsiRNA能有效沉默PKCαmRNA。醛固酮(10-8 mol/L)刺激后明显增加MMDD1细胞合成O2-量,而使用PKCαsiRNA预处理细胞18h则逆转醛固酮该诱导作用。
5. PKCαsiRNA阻断醛固酮诱导的MMDD1细胞中NOX-2和NOX-4表达上调的作用。醛固酮(10-8 mol/L)刺激30 min后使MMDD1细胞内NOX-2和NOX-4蛋白表达明显上调,而使用PKCαsiRNA预处理则阻断醛固酮该诱导作用。该结果表明醛固酮刺激MMDD1细胞内NOX-2和NOX-4蛋白表达增加是通过PKCα通路介导。
结论
醛固酮诱导MMDD1细胞O2-产物生成增多的作用是由PKCα- NAD(P)H氧化酶途径介导的。MD细胞中O2-和NO浓度平衡在维持正常肾脏TGF中起到重要作用。
Part I Aldosterone stimulates superoxide production in macula densa cells
OBJECTIVE
In recent years, the role of excessive oxidative stress in the development of cardiovascular disease including hypertension has been highlighted. In patients with myocardial infarction or heart failure, aldosterone has been shown to promote cardiac inflammation. This is accompanied by increases in myocardial oxidative stress and release of inflammatory markers that include cyclooxygenase-2 (COX-2). Aldosterone also causes nitric oxide (NO)-mediated vasodilation and superoxide (O2-) release due to activation of NAD(P)H oxidase in cerebral and mesenteric arterioles. In cultured aortic endothelial cells, aldosterone has also been shown to induce O2-generation by activating NAD(P)H oxidase. Today there is no doubt that the correction of the oxidant/antioxidant imbalance in patients with chronic renal failure is an important approach for the reduction of the risk of those patients to develop cardiovascular disorders.
Tubuloglomerular glomerular feedback (TGF) is a critical mechanism for regulation of renal hemodynamics, NaCl excretion and blood pressure. Increasing tubular flow to the macula densa (MD) initiates a TGF signal causing constriction of the afferent arteriole thus decreasing GFR. NO released from the MD modulates the TGF response, and O2- release from the MD will inactivate NO. Recently, we found that NOX-2 and NOX-4 isoforms of NAD(P)H oxidase are expressed in the rat MD and MMDD1 cells. Although the role of aldosterone as a prooxidant and a proinflammatory agent has been established in the heart and in blood vessels, the prooxidant role of aldosterone in the MD is poorly understood. In addition, aldosterone activates mineralocorticoid receptors (MR), which have been found throughout the body but of particular interest to this paper, in the distal tubules, connecting tubules, and collecting ducts of the kidney. The presence of MR in the MD cells has not been reported. Our hypothesis is that aldosterone increases O2-production from MD cells acting through MR, and this is accompanied by increases in COX-2 production and NAD(P)H oxidase via NOX-2 and NOX-4 production. These studies will greatly enhance our understanding of the role of aldosterone in regulating TGF and renal hemodynamics.
MATERIALS AND METHODS
MMDD1 Cells
We used MMDD1 cells, a renal epithelial cell line with properties of macula densa cells. These cells were derived from SV40 transgenic mice and have been shown to express well-known macula densa markers, eg, COX-2, nNOS, ROMK (Renal Outer Medullary Potassium channel), and NKCC2(Na+-K+-2Cl-co-transporter).
Measurement of O2- with Lucigenin
We measured O2- production in the MMDD1 cells using a lucigenin-enhanced chemiluminescence assay. Briefly, MMDD1 cells (lOcm-dish) were washed by PBS 2 times, trypsinized from the dish and kept in 12 ml Krebs/Hepes buffer. The Krebs/Hepes buffer was evenly divided into following five groups with different antagonists:1). non-treated; 2). aldosterone 10-8 mol/L; 3). aldosterone 10-8 mol/L and NS398 10-6 mol/L; 4).aldosterone 10-8 mol/L and apocynin 10-5 mol/L; 5). aldosterone 10-8 mol/L, NS398 10-6 mol/L and apocynin 10-5 mol/L. Then lucigenin (5×10-6 mol/L) was added to each of the samples which were incubated for 30 min at 37℃with oxygen bubbling. From each group, a 0.5 ml sample was transferred into a 1.6-mL polypropylene 8×50 mm tubes (Evergreen Scientific), and then using a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany), O2- was measured following the manufacturer's instructions. Luminescence was measured for 10 sec with a delay of 5 sec.
RT-PCR for MMDD1 Cells
Total RNA from the MMDD1 cells and mouse heart cells (as a positive control for the MR receptor) was extracted with an RNeasy Mini kit following the manufacturer's instructions. Oneμg of total RNA was reverse transcribed for 1 hour at 42℃using 1μL random primer 3μg/μL and the Ambion RETROscript kit following the manufacturer's instructions. In the protocol of RT-PCR for COX-2, NOX-2 and NOX-4, measured by specific subunit for each NOX isoform, the resultant RT product was then amplified by PCR by adding 1μL of the RT reaction,1μmol/L of the gene-specific primers,2μL 10×complete PCR buffer,1μL dNTP mix 2.5mmol/L each,0.2μL Super Taq 5U/μL,(1.2μL glycerol 50% only for NOX-2 and NOX-4), and adding nuclear-free water to achieve a volume of 20μL. The mixed samples were heated to 94℃for 3 min and cycled at 94℃for 20 sec,59.4℃(COX-2),56.6℃(NOX-2),52.9℃(NOX-4) for 30 sec, and 72℃for 30 sec for 40 cycles. Final extension was for 8 min at 72℃. In the protocol for RT-PCR for the MR receptor, the mixed samples using the described primers and using Titanium Taq polymerase were heated to 95℃for 2 min and cycled at 95℃for 40 sec, annealing temperature 58℃for 40 sec, and 72℃for 55 sec for 40 cycles. Final extension was for 8 min at 72℃. The amplified products of RT-PCR were run on 1.4%(0.5% for MR receptor) agarose gels containing ethidium bromide 10 mg/mL and visualized under UV light.β-actin, as a housekeeping gene, was set up as an internal loading control.
Real-time PCR
Real-time PCR was used to quantify mRNA level of the COX-2, NOX-2 and NOX-4 responses to the aldosterone. Total RNA was isolated using the RNeasy Micro kit, and complementary DNA synthesis was carried out as described in the method for RT-PCR. Real-time PCR was performed in a C1000TM Thermal Cycler real-time PCR machine. The (3-actin was used as a housekeeping gene.
Preparations for siRNA
COX-2 siRNA was used a pre-designed product from Ambion. siRNA transfection was performed using a siPORTTM Amine Transfection Agent according to the manufacturer's instructions. Scrambled siRNA (Invitrogen) were synthesized and used as negative controls. At 24 hours before transfection, MMDD1 cells were transferred onto 6-well plates (2×105 cells per well) with antibiotic-free medium. The cells were transfected with 10 nmol/L COX-2 siRNA duplex using Amine Transfection Agent for 18 hours in medium devoid of antibiotics. This procedure does not affect cell viability, measured with calcein as we described previously. The MMDD1 cells were washed once with PBS and grown in complete medium. Gene silencing was monitored by measuring RNA after incubation for 24 hours. We added aldosterone (10-8 mol/L) into each cell culture well and incubated at 37℃for 30 min in a cell incubator before harvesting the MMDD1 cells.
Western blot for MMDD1 cells
MMDD1 cells proteins were extracted with RIPA buffer plus a protease inhibitor cocktail. Protein concentration was measured using a Nanodrop instrument. Cells were homogenized in RIPA buffer, centrifuged and protein measured using a Nanodrop spectrophotometer measuring absorption at 280 nm. Proteins extracted from MMDD1 cells in the amount of 100,50 and 20μg were separated by 8% polyacrylamide gel electrophoresis and transferred to PVDF membranes, respectively. After the transfer, membranes were blocked at room temperature for 1h with 1% fat-free dry milk in TTBS. The membranes were blocked and probed with the following primary antibodies, respectively:1) MR--mixture of rMR 1-18 clone 1D5 (1:50 dilution) and MRN 365 clone 2D6 (1:100 dilution, overnight incubation); 2) COX-2 antibody (1:500 dilution); NOX-2 antibody (1:1000 dilution); 3) NOX-4 antibody (HRP)(1:1000 dilution);4) GAPDH (1:5000)(4℃, overnight incubation), then followed by the secondary antibody (1:10,000 dilution) (1 h incubation, room temperature). The bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and captured with a VersaDoc image analysis system (Bio-Rad).
Statistics
Data were analyzed as repeated measures over time or compared to a common control. We tested only the effects of interest, using analysis of variance (ANOVA) for repeated measures and a post-hoc Fisher LSD test. The changes were considered to be significant if P< 0.05. Data are presented as mean±SEM.
RESULTS
1. Mineralocorticoid receptors exist on the MMDD1 cells
RT-PCR results for the MR on MMDD1 cells. We used mouse myocardial cells as a positive control. Western blot results of MR protein compared to the positive control. The results for the first time demonstrate the existence of mRNA and protein for MR in MMDD1 cells.
2. Aldosterone acts through the MR to produce superoxide
We measured O2- levels in aldosterone-treated MMDD1 cells with and without the MR receptor inhibitor (eplerenone 10-5 mol/L) to confirm that the effect of aldosterone is mediated by MR. Eplerenone completely prevented aldosterone-induced increases in O2- in the MMDD1 cells. In the presence of MR antagonist eplerenone (10-5 mol/L), aldosterone-induced O2- production was blocked. Eplerenone itself had no significant effect on O2- levels in MMDD1 cells.
3. COX-2, NOX-2 and NOX-4 are expressed in MMDD1 cells
Because the MMDD1 cell line may have some subtle differences with macula densa cells isolated from the in vivo, we made sure the MMDD1 cells expressed COX-2, NOX-2 and NOX-4. The representative blot depicted in COX-2, NOX-2 and NOX-4 are expressed in the MMDD1 cells, and this result also verifies the efficacy of all of our primers to detect COX-2, NOX-2 and NOX-4.
4. Aldosterone enhances COX-2, NOX-2 and NOX-4 mRNA and protein expressions
We used different aldosterone concentrations (1 nmol/L and 10 nmol/L,) to stimulate the MMDD1 cells for 30 min. The result shows that exposing MMDD1 cells to aldosterone at different concentration enhances COX-2, NOX-2 and NOX-4. Aldosterone at 10-8 mol/L had the greater effect. Therefore, we used this concentration of aldosterone in the following experiment.
We measured protein levels of COX-2, NOX-2 and NOX-4 with specific antibodies in MMDD1 cells stimulated with aldosterone (10-8 mol/L) for 30 min. The result in shows that aldosterone significantly increased protein levels of COX-2, NOX-2 and NOX-4.
5. COX-2, NOX-2 and NOX-4 are a major source of aldosterone-induced O2-
We next identified the major sources of aldosterone-induced O2 in MMDD1 cells. For this, we measured O2- levels in aldosterone-treated cells (10-8 mol/L) in the presence of the following inhibitors:NS-398 (10-6 mol/L) (inhibits COX-2), apocynin (10-5 mol/L) (inhibits NOX) or in the presence of both NS-398 (10-6 mol/L) and apocynin (10-5 mol/L). Apocynin, NS398 or both NS398 and apocynin completely prevented aldosterone-induced increases in O2- in the MMDD1 cells. In the presence of either NS-398 or apocynin, aldosterone-induced increases in O2- were blocked. These data suggested that COX-2, NOX-2 and NOX-4 were the primary sources of O2- produced by the MMDD1 cells during aldosterone stimulation.
6. A siRNA knocked down COX-2 in the MMDD1 cells
To study the function of COX-2 in aldosterone-induced O2- generation, we used a siRNA to silence COX-2. COX-2 siRNA significantly reduced its target COX-2 mRNA. Scrambled siRNA had no significant effect on COX-2 mRNA. Therefore, the results show that the COX-2 siRNA effectively knocked down the COX-2 mRNA. Next, we determined if this siRNA had any effect on NOX-2 or NOX-4 mRNA levels. COX-2 siRNA did not affect the NOX-2 mRNA expression or the NOX-4 mRNA expression. The scrambled siRNA had no effect on the NOX-2 mRNA expression or the NOX-4 mRNA expression. These data demonstrate the efficiency and specificity of COX-2 siRNA.
7. COX-2 siRNA blocked the effect of aldosterone on COX-2, NOX-2 and NOX-4 mRNAs and O2- generation.
To study the role and their interactions between COX-2 and NOX-2 or NOX-4 in aldosterone-induced increases, we knocked down COX-2 and measured NOX-2 and NOX-4 levels. Aldosterone (10"8 mol/L) stimulated COX-2, NOX-2 and NOX-4 mRNA expression. In the MMDD1 cells treated with COX-2 siRNA, aldosterone-induced increases in NOX-2 and NOX-4 mRNAs were blocked, COX-2 mRNA was reduced.
To study the role of COX-2 in aldosterone-induced O2- generation, we knocked down COX-2 and measured O2-. Aldosterone (10-8 mol/L) enhances O2-.In the MMDD1 cells treated with COX-2 siRNA, aldosterone-induced increases in O2-generation were blocked.
CONCLUSIONS
These data indicate a novel signaling pathway for aldosterone-induced O2-generation in the MD cells. Aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O2-.
Part II The prooxidant effect of aldosterone in macula densa cells is mediated by PKCa
OBJECTIVE
Aldosterone activates mineralocorticoid receptors (MR) in the colon and renal epithelial cells, especially the principal cells and intercalated cells of the late distal tubule, collecting tubules and collecting ducts, and an increase in electrolyte flux occurs. However, aldosterone also causes cardiac inflammation and increases in myocardial oxidative stress. We have recently studied the mechanisms by which aldosterone increases O2- production in MMDD1 cells, a renal epithelial cell line with properties of macula densa cells. This prooxidant effect occurred because of increases in COX-2 and NAD(P)H oxidase. However, the signaling method by which aldosterone effects changes in these prooxidants is unknown.
PKC is a signal transduction protein that mediates rapid responses to steroid hormones. A recent study has found that aldosterone triggers both early and late increases in sodium transport in renal cortical collecting duct cells, and the PKCa signaling pathway mediated the effects of aldosterone on the collecting duct cells. However, whether the PKCa signaling pathway plays an important role in the aldosterone prooxidant effect on renal MD cells is not known. We hypothesize that aldosterone increases O2- production in MD cells by activating PKCa which stimulates NAD(P)H oxidase via NOX-2 and NOX-4 production. Our results indicate that MD PKCa and NAD(P)H oxidase are important mediators of the aldosterone prooxidant effect on MD cells.
METHODS
The method followed that of part I.
RESULTS
1. NAD(P)H oxidase inhibition decreased aldosterone-stimulated O2- production.
To confirm that aldosterone stimulates O2- production in MMDD1 cells, we exposed MMDD1 cells to 10 nM aldosterone for 30 min and measured O2- production. Aldosterone increased O2- production from a control value of 1293±106 RLU·s-1·105 cells-1 to 2349±222 RLU·s-1·105 cells-1.
To test if the NAD(P)H oxidase system is involved in mediating the increase in O2- production in MMDD1 cells during exposure to aldosterone, we added the NAD(P)H oxidase inhibitor apocynin. Aldosterone response in the presence of apocynin was markedly reduced.
2. General inhibition of PKC attenuated the aldosterone prooxidant effect
To determine if PKC is involved in aldosterone-induced O2- production, we exposed the cells to 10-7 M chelerythrine chloride (CC), a non-selective PKC inhibitor in the media. Aldosterone increased O2- production from a control value of 1184±54 RLU·s-1·105 cells-1 to 1982±138 RLU·s-1·105 cells-1. Addition of CC markedly blunted the aldosterone-induced O2- production. This suggests that one or more of the isoforms of PKC is a mediator of the prooxidant effect of aldosterone.
3. Specific inhibition of PKCa attenuated the aldosterone prooxidant effect
To determine if PKCa is involved in aldosterone-induced O2- production, we exposed the cells to PKCa specific inhibitor,10-7 M Go6976 (Go). Aldosterone increased O2- production in MMDD1 cells. Addition of the specific PKCa inhibitor, Go, nearly completely inhibited any effect of aldosterone on O2- production. This suggests an important role of PKCa in the aldosterone prooxidant response.
4. PKCa siRNA inhibited the aldosterone prooxidant effect
PKCa siRNA was used to test the hypothesis that PKCa is a mediator of aldosterone-stimulated O2- production. PKCa siRNA markedly decreased PKCa mRNA of MMDD1 cells as determined with real-time PCR. This indicates that our PKCa siRNA was a suitable inhibitor of PKCa expression.
To provide further evidence that PKCa is involved in aldosterone-induced O2-production, we exposed the cells to PKCa siRNA. Aldosterone caused a large increase in MMDD1 O2- production. Addition of a scrambled siRNA did not inhibit the aldosterone-induced O2- production. However, addition of a specific siRNA for PKCa markedly inhibited O2- production in aldosterone-treated cells.
5. PKCa siRNA inhibited the increase in NOX-2 and NOX-4 protein
To determine if PKCa enhanced NOX-2 and NOX-4 protein expression, we exposed MMDD1 cells to aldosterone with and without PKCa siRNA. Aldosterone increased NOX-2 and NOX-4 protein. Addition of PKCa siRNA completely inhibited the aldosterone-induced increase in NOX-2 and NOX-4 protein. These data suggest that the aldosterone-induced increases in NOX-2 and NOX-4 protein were mediated by increases in PKCa.
CONCLUSIONS
Aldosterone-induced increases in MD O2- production are mediated by PKCa, PKCa stimulates MD NOX-2 and NOX-4 which generate O2-. The resulting balance between O2- and NO in the MD is important in regulating TGF.
引文
1. Akasaki T, Ohya Y, Kuroda J, Eto K, Abe I, Sumimoto H and Iida M. Increased expression of gp91 phox homologues of NAD(P)H oxidase in the aortic media during chronic hypertension:involvement of the renin-angiotensin system. Hypertens Res 29:813-820,2006.
2. Al-Dujaili EA and Edwards CR. Development and application of a direct radioimmunoassay for plasma aldosterone [proceedings]. J Endocrinol 77:13P-14P,1978.
3. Aldieri E, Riganti C, Polimeni M, Gazzano E, Lussiana C, Campia I and Ghigo D. Classical inhibitors of NOX NAD(P)H oxidases are not specific. Curr Drug Metab 9:686-696,2008.
4. Alvarez Y, Perez-Giron JV, Hernanz R, Briones AM, Garcia-Redondo A, Beltran A, Alonso MJ and Salaices M. Losartan reduces the increased participation of cyclooxygenase-2-derived products in vascular responses of hypertensive rats. J Pharmacol Exp Ther 321:381-388,2007.
5. Alzamora R, Brown LR and Harvey BJ. Direct binding and activation of protein kinase C isoforms by aldosterone and 17beta-estradiol. Mol Endocrinol 21:2637-2650,2007.
6. Ando K and Fujita T. Role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the development of hypertensive organ damage. Clin Exp Nephrol 8:178-182, 2004.
7. Araujo M and Welch WJ. Cyclooxygenase 2 inhibition suppresses tubuloglomerular feedback:roles of thromboxane receptors and nitric oxide. Am J Physiol Renal Physiol 296: F790-F794,2009.
8. Arima S, Kohagura K, Xu HL, Sugawara A, Abe T, Satoh F, Takeuchi K and Ito S. Nongenomic vascular action of aldosterone in the glomerular microcirculation. J Am Soc Nephrol 14:2255-2263,2003.
9. Bartz RR and Piantadosi CA. Clinical review:oxygen as a signaling molecule. Crit Care 14:234,2010.
10. Berl T, Katz FH, Henrich WL, de TA and Schrier RW. Role of aldosterone in the control of sodium excretion in patients with advanced chronic renal failure. Kidney Int 14:228-235, 1978.
11. Beswick RA, Dorrance AM, Leite R and Webb RC. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension 38: 1107-1111,2001.
12. Blanco-Rivero J, Cachofeiro V, Lahera V, Aras-Lopez R, Marquez-Rodas I, Salaices M, Xavier FE, Ferrer M and Balfagon G. Participation of prostacyclin in endothelial dysfunction induced by aldosterone in normotensive and hypertensive rats. Hypertension 46: 107-112,2005.
13. Blantz RC and Pelayo JC. A functional role for the tubuloglomerular feedback mechanism. Kidney Int 25:739-746,1984.
14. Callera GE, Tostes RC, Yogi A, Montezano AC and Touyz RM. Endothelin-1-induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms. Clin Sci (Lond) 110:243-253,2006.
15. Chandramohan G, Bai Y, Norris K, Rodriguez-Iturbe B and Vaziri ND. Effects of dietary salt on intrarenal angiotensin system, NAD(P)H oxidase, COX-2, MCP-1 and PAI-1 expressions and NF-kappaB activity in salt-sensitive and -resistant rat kidneys. Am J Nephrol 28:158-167,2008.
16. Chen X, Touyz RM, Park JB and Schiffrin EL. Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke-prone SHR. Hypertension 38:606-611,2001.
17. Cheng HF and Harris RC. Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways. J Biol Chem 277:45638-45643,2002.
18. Christ M, Meyer C, Sippel K and Wehling M. Rapid aldosterone signaling in vascular smooth muscle cells:involvement of phospholipase C, diacylglycerol and protein kinase C alpha. Biochem Biophys Res Commun 213:123-129,1995.
19. Cifuentes ME and Pagano PJ. Targeting reactive oxygen species in hypertension. Curr Opin Nephrol Hypertens 15:179-186,2006.
20. Cottone S, Mule G, Guarneri M, Palermo A, Lorito MC, Riccobene R, Arsena R, Vaccaro F, Vadala A, Nardi E, Cusimano P and Cerasola G. Endothelin-1 and F2-isoprostane relate to and predict renal dysfunction in hypertensive patients. Nephrol Dial Transplant 24:497-503,2009.
21. Darko D, Dornhorst A, Kelly FJ, Ritter JM and Chowienczyk PJ. Lack of effect of oral vitamin C on blood pressure, oxidative stress and endothelial function in Type Ⅱ diabetes. Clin Sci (Lond) 103:339-344,2002.
22. DeMarco VG, Habibi J, Whaley-Connell AT, Schneider RI, Heller RL, Bosanquet JP, Hayden MR, Delcour K, Cooper SA, Andresen BT, Sowers JR and Dellsperger KC. Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat. Am J Physiol Heart Circ Physiol 294:H2659-H2668,2008.
23. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA and Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279:L429-L438,2000.
24. Deng A, Wead LM and Blantz RC. Temporal adaptation of tubuloglomerular feedback: effects of COX-2. Kidney Int 66:2348-2353,2004.
25. Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS and Lele RD. Free radicals and antioxidants in human health:current status and future prospects. J Assoc Physicians India 52:794-804,2004.
26. Diaz Encarnacion MM, Warner GM, Gray CE, Cheng J, Keryakos HK, Nath KA and Grande JP. Signaling pathways modulated by fish oil in salt-sensitive hypertension. Am J Physiol Renal Physiol 294:F1323-F1335,2008.
27. Diep QN, Amiri F, Touyz RM, Cohn JS, Endemann D, Neves MF and Schiffrin EL. PPARalpha activator effects on Ang Ⅱ-induced vascular oxidative stress and inflammation. Hypertension 40:866-871,2002.
28. Duffy SJ, Gokce N, Holbrook M, Hunter LM, Biegelsen ES, Huang A, Keaney JF, Jr. and Vita JA. Effect of ascorbic acid treatment on conduit vessel endothelial dysfunction in patients with hypertension. Am J Physiol Heart Circ Physiol 280:H528-H534,2001.
29. El JA, Valente AJ, Lechleiter JD, Gamez MJ, Pearson DW, Nauseef WM and Clark RA. Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med 44:868-881,2008.
30. EI-Benna J, Dang PM, Gougerot-Pocidalo MA, Marie JC and Braut-Boucher F. p47phox, the phagocyte NADPH oxidase/NOX2 organizer:structure, phosphorylation and implication in diseases. Exp Mol Med 41:217-225,2009.
31. Ergul A, Johansen JS, Stromhaug C, Harris AK, Hutchinson J, Tawfik A, Rahimi A, Rhim E, Wells B, Caldwell RW and Anstadt MP. Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes:role of endothelin-1. J Pharmacol Exp Ther 313:70-77,2005.
32. Faraci FM and Didion SP. Vascular protection:superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol 24:1367-1373,2004.
33. Feuerstein GZ and Ruffolo RR, Jr. Carvedilol, a novel vasodilating beta-blocker with the potential for cardiovascular organ protection. Eur Heart J 17 Suppl B:24-29,1996.
34. Forstermann U. Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal. Biol Chem 387:1521-1533,2006.
35. Franco MC, Kawamoto EM, Gorjao R, Rastelli VM, Curi R, Scavone C, Sawaya AL, Fortes ZB and Sesso R. Biomarkers of oxidative stress and antioxidant status in children born small for gestational age:evidence of lipid peroxidation. Pediatr Res 62:204-208, 2007.
36. Fridovich I. Superoxide anion radical (02-.), superoxide dismutases, and related matters. J Biol Chem 272:18515-18517,1997.
37. Fu Y, Katsuya T, Matsuo A, Yamamoto K, Akasaka H, Takami Y, Iwashima Y, Sugimoto K, Ishikawa K, Ohishi M, Rakugi H and Ogihara T. Relationship of bradykinin B2 receptor gene polymorphism with essential hypertension and left ventricular hypertrophy. Hypertens Res 27:933-938,2004.
38. Fu Y, Zhang R, Lu D, Liu H, Chandrashekar K, Juncos LA and Liu R. NOX2 is the primary source of angiotensin Ⅱ-induced superoxide in the macula densa. Am J Physiol Regul Integr Comp Physiol 298:R707-R712,2010.
39. Gao WD, Liu Y and Marban E. Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium. Circulation 94:2597-2604,1996.
40. Gekle M, Golenhofen N, Oberleithner H and Silbernagl S. Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+and stimulation of a plasma membrane proton conductance. Proc Natl Acad Sci USA 93:10500-10504,1996.
41. Gomez-Sanchez CE, de Rodriguez AF, Romero DG, Estess J, Warden MP, Gomez-Sanchez MT and Gomez-Sanchez EP. Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 147:1343-1348,2006.
42. Good DW. Nongenomic actions of aldosterone on the renal tubule. Hypertension 49:728-739,2007.
43. Greene EL, Kren S and Hostetter TH. Role of aldosterone in the remnant kidney model in the rat. JClin Invest 98:1063-1068,1996.
44. Griendling KK, Sorescu D and Ushio-Fukai M. NAD(P)H oxidase:role in cardiovascular biology and disease. Cire Res 86:494-501,2000.
45. Guichard C, Pedruzzi E, Fay M, Ben MS, Coant N, Daniel F and Ogier-Denis E. [The Nox/Duox family of ROS-generating NADPH oxidases]. Med Sci (Paris) 22:953-959, 2006.
46. Gupte SA, Kaminski PM, George S, Kouznestova L, Olson SC, Mathew R, Hintze TH and Wolin MS. Peroxide generation by p47phox-Src activation of Nox2 has a key role in protein kinase C-induced arterial smooth muscle contraction. Am J Physiol Heart Circ Physiol 296:H1048-H1057,2009.
47. Harvey BJ and Higgins M. Nongenomic effects of aldosterone on Ca2+in M-1 cortical collecting duct cells. Kidney Int 57:1395-1403,2000.
48. Hatzitolios A, Iliadis F, Katsiki N and Baltatzi M. Is the anti-hypertensive effect of dietary supplements via aldehydes reduction evidence based? A systematic review. Clin Exp Hypertens 30:628-639,2008.
49. He H, Podymow T, Zimpelmann J and Burns KD. NO inhibits Na+-K+-2C1-cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1). Am J Physiol Renal Physiol 284:F1235-F1244,2003.
50. Heitzer T, Wenzel U, Hink U, Krollner D, Skatchkov M, Stahl RA, MacHarzina R, Brasen JH, Meinertz T and Munzel T. Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension:evidence for an involvement of protein kinase C. Kidney Int 55:252-260,1999.
51. Heitzmann D, Derand R, Jungbauer S, Bandulik S, Sterner C, Schweda F, El WA, Lalli E, Guy N, Mengual R, Reichold M, Tegtmeier Ⅰ, Bendahhou S, Gomez-Sanchez CE, Aller MI, Wisden W, Weber A, Lesage F, Warth R and Barhanin J. Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis. EMBO J 27:179-187,2008.
52. Hermann M, Shaw S, Kiss E, Camici G, Buhler N, Chenevard R, Luscher TF, Grone HJ and Ruschitzka F. Selective COX-2 inhibitors and renal injury in salt-sensitive hypertension. Hypertension 45:193-197,2005.
53. Heumuller S, Wind S, Barbosa-Sicard E, Schmidt HH, Busse R, Schroder K and Brandes RP. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 51:211-217,2008.
54. Hirono Y, Yoshimoto T, Suzuki N, Sugiyama T, Sakurada M, Takai S, Kobayashi N, Shichiri M and Hirata Y. Angiotensin Ⅱ receptor type 1-mediated vascular oxidative stress and proinflammatory gene expression in aldosterone-induced hypertension:the possible role of local renin-angiotensin system. Endocrinology 148:1688-1696,2007.
55. Hirono Y, Yoshimoto T, Suzuki N, Sugiyama T, Sakurada M, Takai S, Kobayashi N, Shichiri M and Hirata Y. Angiotensin Ⅱ receptor type 1-mediated vascular oxidative stress and proinflammatory gene expression in aldosterone-induced hypertension:the possible role of local renin-angiotensin system. Endocrinology 148:1688-1696,2007.
56. Hollenberg NK, Stevanovic R, Agarwal A, Lansang MC, Price DA, Laffel LM, Williams GH and Fisher ND. Plasma aldosterone concentration in the patient with diabetes mellitus. Kidney Int 65:1435-1439,2004.
57. Hong HJ, Hsiao G, Cheng TH and Yen MH. Supplemention with tetrahydrobiopterin suppresses the development of hypertension in spontaneously hypertensive rats. Hypertension 38:1044-1048,2001.
58. Hong NJ, Silva GB and Garvin JL. PKC-alpha mediates flow-stimulated superoxide production in thick ascending limbs. Am J Physiol Renal Physiol 298:F885-F891,2010.
59. Houston MC. Nutraceuticals, vitamins, antioxidants, and minerals in the prevention and treatment of hypertension. Prog Cardiovasc Dis 47:396-449,2005.
60. Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, Sekiguchi N, Kobayashi K, Sumimoto H, Utsumi H and Nawata H. Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes:role of vascular NAD(P)H oxidase. J Am Soc Nephrol 14:S227-S232,2003.
61. Iwashima F, Yoshimoto T, Minami I, Sakurada M, Hirono Y and Hirata Y. Aldosterone induces superoxide generation via Racl activation in endothelial cells. Endocrinology 149: 1009-1014,2008.
62. Johnson F and Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med 26:340-352,2005.
63. Jung O, Schreiber JG, Geiger H, Pedrazzini T, Busse R and Brandes RP. gp91phox-containing NADPH oxidase mediates endothelial dysfunction in renovascular hypertension. Circulation 109:1795-1801,2004.
64. Kagota S, Tada Y, Kubota Y, Nejime N, Yamaguchi Y, Nakamura K, Kunitomo M and Shinozuka K. Peroxynitrite is Involved in the dysfunction of vasorelaxation in SHR/NDmcr-cp rats, spontaneously hypertensive obese rats. J Cardiovasc Pharmacol 50: 677-685,2007.
65. Kawahara T and Lambeth JD. Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes. BMC Evol Biol 7:178,2007.
66. Kimura S, Mullins JJ, Bunnemann B, Metzger R, Hilgenfeldt U, Zimmermann F, Jacob H, Fuxe K, Ganten D and Kaling M. High blood pressure in transgenic mice carrying the rat angiotensinogen gene. EMBO J 11:821-827,1992.
67. Kinugawa S, Tsutsui H, Hayashidani S, Ide T, Suematsu N, Satoh S, Utsumi H and Takeshita A. Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice:role of oxidative stress. Circ Res 87:392-398,2000.
68. Kourie JI. Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol 275:C1-24,1998.
69. Lahera V, Cachofeiro V, Balfagon G and Rodicio JL. Aldosterone and its blockade:a cardiovascular and renal perspective. ScientificWorldJournal 6:413-424,2006.
70. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM and Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 40:511-515,2002.
71. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE and Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111:1201-1209,2003.
72. Lassegue B and Griendling KK. Reactive oxygen species in hypertension; An update. Am JHypertens 17:852-860,2004.
73. Lavi S, Yang EH, Prasad A, Mathew V, Barsness GW, Rihal CS, Lerman LO and Lerman A. The interaction between coronary endothelial dysfunction, local oxidative stress, and endogenous nitric oxide in humans. Hypertension 51:127-133,2008.
74. Le MC, Ouvrard-Pascaud A, Capurro C, Cluzeaud F, Fay M, Jaisser F, Farman N and Blot-Chabaud M. Early nongenomic events in aldosterone action in renal collecting duct cells:PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response. J Am Soc Nephrol 15:1145-1160,2004.
75. Li H, Witte K, August M, Brausch I, Godtel-Armbrust U, Habermeier A, Closs El, Oelze M, Munzel T and Forstermann U. Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats. J Am Coll Cardiol 47:2536-2544,2006.
76. Li Q, Zhang Y, Marden JJ, Banfi B and Engelhardt JF. Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury. Biochem J 411:531-541, 2008.
77. Liu R, Carretero OA, Ren Y and Garvin JL. Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback. Kidney Int 67:1837-1843,2005.
78. Liu R, Carretero OA, Ren Y, Wang H and Garvin JL. Intracellular pH regulates superoxide production by the macula densa. Am J Physiol Renal Physiol 295:F851-F856, 2008.
79. Liu R, Garvin JL, Ren Y, Pagano PJ and Carretero OA. Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase. Am J Physiol Renal Physiol 292:F1867-F1872,2007.
80. Liu R and Persson AE. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCI concentration. J Physiol 563:895-901,2005.
81. Liu R, Pittner J and Persson AE. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration. J Am Soc Nephrol 13: 2688-2696,2002.
82. Liu R, Ren Y, Garvin JL and Carretero OA. Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole. Kidney Int 66:268-274,2004.
83. Lob HE, Marvar PJ, Guzik TJ, Sharma S, McCann LA, Weyand C, Gordon FJ and Harrison DG. Induction of hypertension and peripheral inflammation by reduction of extracellular superoxide dismutase in the central nervous system. Hypertension 55:277-83, 6p,2010.
84. Lombes M, Farman N, Oblin ME, Baulieu EE, Bonvalet JP, Erlanger BF and Gasc JM. Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldosterone. Proc Natl Acad Sci U S A 87: 1086-1088,1990.
85. Losel R, Schultz A and Wehling M. A quick glance at rapid aldosterone action. Mol Cell Endocrinol 217:137-141,2004.
86. Lu D, Fu Y, Lopez-Ruiz A, Zhang R, Juncos R, Liu H, Manning RD, Jr., Juncos LA and Liu R. Salt-sensitive splice variant of nNOS expressed in the macula densa cells. Am J Physiol Renal Physiol 298:F1465-F1471,2010.
87. Ma A, Qi S and Chen H. Antioxidant therapy for prevention of inflammation, ischemic reperfusion injuries and allograft rejection. Cardiovasc Hematol Agents Med Chew 6:20-43, 2008.
88. Manning RD, Jr., Tian N and Meng S. Oxidative stress and antioxidant treatment in hypertension and the associated renal damage. Am J Nephrol 25:311-317,2005.
89. Matsushita K, Morrell CN, Mason RJ, Yamakuchi M, Khanday FA, Irani K and Lowenstein CJ. Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor. J Cell Biol 170:73-79,2005.
90. Meyer JW and Schmitt ME. A central role for the endothelial NADPH oxidase in atherosclerosis. FEBS Lett 472:1-4,2000.
91. Mihailidou AS, Bundgaard H, Mardini M, Hansen PS, Kjeldsen K and Rasmussen HH. Hyperaldosteronemia in rabbits inhibits the cardiac sarcolemmal Na(+)-K(+) pump. Circ Res 86:37-42,2000.
92. Mistry HD, Wilson V, Ramsay MM, Symonds ME and Broughton PF. Reduced selenium concentrations and glutathione peroxidase activity in preeclamptic pregnancies. Hypertension 52:881-888,2008.
93. Morawietz H, Weber M, Rueckschloss U, Lauer N, Hacker A and Kojda G. Upregulation of vascular NAD(P)H oxidase subunit gp91phox and impairment of the nitric oxide signal transduction pathway in hypertension. Biochem Biophys Res Commun 285: 1130-1135,2001.
94. Nistala R, Wei Y, Sowers JR and Whaley-Connell A. Renin-angiotensin-aldosterone system-mediated redox effects in chronic kidney disease. Transl Res 153:102-113,2009.
95. Nistala R, Whaley-Connell A and Sowers JR. Redox control of renal function and hypertension. Antioxid Redox Signal 10:2047-2089,2008.
96. Ohara N, Kasama K, Naito Y, Nagata T, Saito Y, Kuwagata M and Okuyama H. Different effects of 26-week dietary intake of rapeseed oil and soybean oil on plasma lipid levels, glucose-6-phosphate dehydrogenase activity and cyclooxygenase-2 expression in spontaneously hypertensive rats. Food Chem Toxicol 46:2573-2579,2008.
97. Okoshi MP, Yan X, Okoshi K, Nakayama M, Schuldt AJ, O'Connell TD, Simpson PC and Lorell BH. Aldosterone directly stimulates cardiac myocyte hypertrophy. J Card Fail 10:511-518,2004.
98. Paliege A, Pasumarthy A, Mizel D, Yang T, Schnermann J and Bachmann S. Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 290:R694-R700, 2006.
99. Paliege A, Pasumarthy A, Mizel D, Yang T, Schnermann J and Bachmann S. Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 290:R694-R700, 2006.
100. Paravicini TM and Touyz RM. NADPH oxidases, reactive oxygen species, and hypertension:clinical implications and therapeutic possibilities. Diabetes Care 31 Suppl 2: S170-S180,2008.
101. Pechanova O and Simko F. The role of nitric oxide in the maintenance of vasoactive balance. Physiol Res 56 Suppl 2:S7-S16,2007.
102. Portaluppi F, Boari B and Manfredini R. Oxidative stress in essential hypertension. Curr Pharm Des 10:1695-1698,2004.
103. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Criendling KK and Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97:1916-1923,1996.
104. Rebsamen MC, Perrier E, Gerber-Wicht C, Benitah JP and Lang U. Direct and indirect effects of aldosterone on cyclooxygenase-2 and interleukin-6 expression in rat cardiac cells in culture and after myocardial infarction. Endocrinology 145:3135-3142,2004.
105. Rivera J, Sobey CG, Walduck AK and Drummond GR. Nox isoforms in vascular pathophysiology:insights from transgenic and knockout mouse models. Redox Rep 15:50-63,2010.
106. Rudolph AE, Rocha R and McMahon EG. Aldosterone target organ protection by eplerenone. Mol Cell Endocrinol 217:229-238,2004.
107. Ruffolo RR, Jr. and Feuerstein GZ. Pharmacology of carvedilol:rationale for use in hypertension, coronary artery disease, and congestive heart failure. Cardiovasc Drugs Ther 11 Suppl 1:247-256,1997.
108. Sachse A and Wolf G. Angiotensin Ⅱ-induced reactive oxygen species and the kidney. J Am Soc Nephrol 18:2439-2446,2007.
109. Saito Y, Nakamura T and Kurabayashi M. [Oxidative stress and mild hypertension]. Nippon Rinsho 66:1525-1529,2008.
110. Satoh M, Fujimoto S, Haruna Y, Arakawa S, Horike H, Komai N, Sasaki T, Tsujioka K, Makino H and Kashihara N. NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy. Am J Physiol Renal Physiol 288:F1144-F1152,2005.
111. Schiffrin EL. Effects of aldosterone on the vasculature. Hypertension 41:312-318,2006.
112. Schnermann J and Levine DZ. Paracrine factors in tubuloglomerular feedback:adenosine, ATP, and nitric oxide. Annu Rev Physiol 65:501-529,2003.
113. Schnermann J, Ploth DW and Hermle M. Activation of tubulo-glomerular feedback by chloride transport. Pflugers Arch 362:229-240,1976.
114. Scurry MT, Shear L and Barry KG. The renal tubular handling of aldosterone and its acid-labile conjugate. J Clin Invest 47:242-248,1968.
115. Sedeek M, Hebert RL, Kennedy CR, Burns KD and Touyz RM. Molecular mechanisms of hypertension:role of Nox family NADPH oxidases. Curr Opin Nephrol Hypertens 18: 122-127,2009.
116. Selemidis S, Sobey CG, Wingler K, Schmidt HH and Drummond GR. NADPH oxidases in the vasculature:molecular features, roles in disease and pharmacological inhibition. Pharmacol Ther 120:254-291,2008.
117. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y and Griendling KK. Angiotensin Ⅱ stimulation of NAD(P)H oxidase activity:upstream mediators. Circ Res 91: 406-413,2002.
118. Shiomi T, Tsutsui H, Matsusaka H, Murakami K, Hayashidani S, Ikeuchi M, Wen J, Kubota T, Utsumi H and Takeshita A. Overexpression of glutathione peroxidase prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation 109: 544-549,2004.
119. Sia YT, Lapointe N, Parker TG, Tsoporis JN, Deschepper CF, Calderone A, Pourdjabbar A, Jasmin JF, Sarrazin JF, Liu P, Adam A, Butany J and Rouleau JL. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat. Circulation 105:2549-2555,2002.
120. Sirker A, Zhang M, Murdoch C and Shah AM. Involvement of NADPH oxidases in cardiac remodelling and heart failure. Am JNephrol 27:649-660,2007.
121. Sirker A, Zhang M, Murdoch C and Shah AM. Involvement of NADPH oxidases in cardiac remodelling and heart failure. Am JNephrol 27:649-660,2007.
122. Siu YP, Leung KT, Tong MK and Kwan TH. Use of allopurinol in slowing the progression of renal disease through its ability to lower serum uric acid level. Am J Kidney Dis 47:51-59,2006.
123. Sun Y, Zhang J, Lu L, Chen SS, Quinn MT and Weber KT. Aldosterone-induced inflammation in the rat heart:role of oxidative stress. Am J Pathol 161:1773-1781,2002.
124. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 79:215-262,1999.
125. Tabet F, Savoia C, Schiffrin EL and Touyz RM. Differential calcium regulation by hydrogen peroxide and superoxide in vascular smooth muscle cells from spontaneously hypertensive rats. J Cardiovasc Pharmacol 44:200-208,2004.
126. Touyz RM. Recent advances in intracellular signalling in hypertension. Curr Opin Nephrol Hypertens 12:165-174,2003.
127. Touyz RM and Briones AM. Reactive oxygen species and vascular biology:implications in human hypertension. Hypertens Res 34:5-14,2011.
128. Touyz RM, Tabet F and Schiffrin EL. Redox-dependent signalling by angiotensin Ⅱ and vascular remodelling in hypertension. Clin Exp Pharmacol Physiol 30:860-866,2003.
129. Traynor TR, Smart A, Briggs JP and Schnermann J. Inhibition of macula densa-stimulated renin secretion by pharmacological blockade of cyclooxygenase-2. Am J Physiol 277:F706-F710,1999.
130. Valko M, Morris H and Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 12:1161-1208,2005.
131. Virdis A, Colucci R, Versari D, Ghisu N, Fornai M, Antonioli L, Duranti E, Daghini E, Giannarelli C, Blandizzi C, Taddei S and Del TM. Atorvastatin prevents endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats:role of cyclooxygenase 2-derived contracting prostanoids. Hypertension 53:1008-1016,2009.
132. Virdis A, Neves MF, Amiri F, Touyz RM and Schiffrin EL. Role of NAD(P)H oxidase on vascular alterations in angiotensin Ⅱ-infused mice. J Hypertens 22:535-542,2004.
133. Wei XF, Zhou QG, Hou FF, Liu BY and Liang M. Advanced oxidation protein products induce mesangial cell perturbation through PKC-dependent activation of NADPH oxidase. Am J Physiol Renal Physiol 296:F427-F437,2009.
134. Welch WJ and Wilcox CS. What is brain nitric oxide synthase doing in the kidney? Curr Opin Nephrol Hypertens 11:109-115,2002.
135. White CN, Figtree GA, Liu CC, Garcia A, Hamilton EJ, Chia KK and Rasmussen HH. Angiotensin Ⅱ inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase. Am J Physiol Cell Physiol 296:C693-C700,2009.
136. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney:a critical link to hypertension? Am JPhysiol Regul Integr Comp Physiol 289:R913-R935,2005.
137. Wojcik M, Burzynska-Pedziwiatr I and Wozniak LA. A review of natural and synthetic antioxidants important for health and longevity. Curr Med Chem 17:3262-3288,2010.
138. Wray DW, Uberoi A, Lawrenson L, Bailey DM and Richardson RS. Oral antioxidants and cardiovascular health in the exercise-trained and untrained elderly:a radically different outcome. Clin Sci (Lond) 116:433-441,2009.
139. Yang G, Merrill DC, Thompson MW, Robillard JE and Sigmund CD. Functional expression of the human angiotensinogen gene in transgenic mice. J Biol Chem 269:32497-32502,1994.
140. Yang G and Sigmund CD. Developmental expression of human angiotensinogen in transgenic mice. Am J Physiol 274:F932-F939,1998.
141. Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H, Spring K, Briggs JP and Schnermann J. Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line. J Biol Chem 275:37922-37929,2000.
142. Yoshida K. Pursuing enigmas on ischemic heart disease and sudden cardiac death. Leg Med (Tokyo) 11:51-58,2009.
143. Yoshimoto T, Fukai N, Sato R, Sugiyama T, Ozawa N, Shichiri M and Hirata Y. Antioxidant effect of adrenomedullin on angiotensin Ⅱ-induced reactive oxygen species generation in vascular smooth muscle cells. Endocrinology 145:3331-3337,2004.
144. Zalba G, Beaumont FJ, San JG, Fortuno A, Fortuno MA, Etayo JC and Diez J. Vascular NADH/NADPH oxidase is involved in enhanced superoxide production in spontaneously hypertensive rats. Hypertension 35:1055-1061,2000.
145. Zhang R, Harding P, Garvin JL, Juncos R, Peterson E, Juncos LA and Liu R. Isoforms and functions of NAD(P)H oxidase at the macula densa. Hypertension 53:556-563,2009.
146. Zhu X, Manning RD, Jr., Lu D, Gomez-Sanchez CE, Fu Y, Juncos LA and Liu R. Aldosterone stimulates superoxide production in the macula densa cells. Am J Physiol Renal Physiol 2011.
1. Araujo M and Welch WJ. Cyclooxygenase 2 inhibition suppresses tubuloglomerular feedback:roles of thromboxane receptors and nitric oxide. Am J Physiol Renal Physiol 296:F790-F794,2009.
2. Arima S, Kohagura K, Xu HL, Sugawara A, Abe T, Satoh F, Takeuchi K and Ito S. Nongenomic vascular action of aldosterone in the glomerular microcirculation. J Am Soc Nephrol 14:2255-2263,2003.
3. Beswick RA, Dorrance AM, Leite R and Webb RC. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension 38:1107-1111,2001.
4. Blantz RC and Pelayo JC. A functional role for the tubuloglomerular feedback mechanism. Kidney Int 25:739-746,1984.
5. DeMarco VG, Habibi J, Whaley-Connell AT, Schneider RI, Heller RL, Bosanquet JP, Hayden MR, Delcour K, Cooper SA, Andresen BT, Sowers JR and Dellsperger KC. Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat. Am J Physiol Heart Circ Physiol 294:H2659-H2668,2008.
6. Deng A, Wead LM and Blantz RC. Temporal adaptation of tubuloglomerular feedback:effects of COX-2. Kidney Int 66:2348-2353,2004.
7. El JA, Valente AJ, Lechleiter JD, Gamez MJ, Pearson DW, Nauseef WM and Clark RA. Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med 44:868-881,2008.
8. Ergul A, Johansen JS, Stremhaug C, Harris AK, Hutchinson J, Tawfik A, Rahimi A, Rhim E, Wells B, Caldwell RW and Anstadt MP. Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes:role of endothelin-1. J Pharmacol Exp Ther 313:70-77,2004.
9. Fu Y, Katsuya T, Matsuo A, Yamamoto K, Akasaka H, Takami Y, Iwashima Y, Sugimoto K, Ishikawa K, Ohishi M, Rakugi H and Ogihara T. Relationship of bradykinin B2 receptor gene polymorphism with essential hypertension and left ventricular hypertrophy. Hypertens Res 27:933-938,2004.
10. Fu Y, Zhang R, Lu D, Liu H, Chandrashekar K, Juncos LA and Liu R. NOX2 is the primary source of angiotensin 11-induced superoxide in the macula densa. Am J Physiol Regul Integr Comp Physiol 298:R707-R712,2010.
11. Gao WD, Liu Y and Marban E. Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium. Circulation 94:2597-2604,1996.
12. Gekle M, Golenhofen N, Oberleithner H and Silbernagl S. Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance. Proc Natl Acad Sci U S A 93: 10500-10504,1996.
13. Gomez-Sanchez CE, de Rodriguez AF, Romero DG, Estess J, Warden MP, Gomez-Sanchez MT and Gomez-Sanchez EP. Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 147: 1343-1348,2006.
14. Harvey BJ and Higgins M. Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells. Kidney Int 57:1395-1403,2000.
15. He H, Podymow T, Zimpelmann J and Burns KD. NO inhibits Na+-K+-2Cl-cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1). Am JPhysiol Renal Physiol 284:F1235-F1244,2003.
16. Heitzmann D, Derand R, Jungbauer S, Bandulik S, Sterner C, Schweda F, EI WA, Lalli E, Guy N, Mengual R, Reichold M, Tegtmeier I, Bendahhou S, Gomez-Sanchez CE, Aller MI, Wisden W, Weber A, Lesage F, Warth R and Barhanin J. Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis. EMBO J 21:179-187,2008.
17. Heumuller S, Wind S, Barbosa-Sicard E, Schmidt HH, Busse R, Schroder K and Brandes RP. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 51:211-217,2008.
18. Heylen E, Huang A, Sun D and Kaley G. Nitric oxide-mediated dilation of arterioles to intraluminal administration of aldosterone. J Cardiovasc Pharmacol 54:535-542, 2009.
19. Hirono Y, Yoshimoto T, Suzuki N, Sugiyama T, Sakurada M, Takai S, Kobayashi N, Shichiri M and Hirata Y. Angiotensin Ⅱ receptor type 1-mediated vascular oxidative stress and proinflammatory gene expression in aldosterone-induced hypertension:the possible role of local renin-angiotensin system. Endocrinology 148: 1688-1696,2007.
20. Hollenberg NK, Stevanovic R, Agarwal A, Lansang MC, Price DA, Laffel LM, Williams GH and Fisher ND. Plasma aldosterone concentration in the patient with diabetes mellitus. Kidney Int 65:1435-1439,2004.
21. Iwashima F, Yoshimoto T, Minami I, Sakurada M, Hirono Y and Hirata Y. Aldosterone induces superoxide generation via Racl activation in endothelial cells. Endocrinology 149:1009-1014,2008.
22. Lassegue B and Griendling KK. Reactive oxygen species in hypertension; An update. Am J Hypertens 17:852-860,2004.
23. Li Q, Zhang Y, Marden JJ, Banfi B and Engelhardt JF. Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury. Biochem J 411:531-541,2008.
24. Liu R, Carretero OA, Ren Y, Wang H and Garvin JL. Intracellular pH regulates superoxide production by the macula densa. Am J Physiol Renal Physiol 295:F851-F856,2008.
25. Liu R, Garvin JL, Ren Y, Pagano PJ and Carretero OA. Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase. Am J Physiol Renal Physiol 292:1867-1872,2007.
26. Liu R and Persson AE. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration. J Physiol 563:895-901,2005.
27. Liu R, Pittner J and Persson AE. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration. JAm Soc Nephrol 13:2688-2696,2002.
28. Liu R, Ren Y, Garvin JL and Carretero OA. Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole. Kidney Int 66:268-274,2004.
29. Lombes M, Farman N, Oblin ME, Baulieu EE, Bonvalet JP, Erlanger BF and Gasc JM. Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldosterone. Proc Natl Acad Sci USA 87:1086-1088,1990.
30. Losel R, Schultz A and Wehling M. A quick glance at rapid aldosterone action. Mol Cell Endocrinol 217:137-141,2004.
31. Lu D, Fu Y, Lopez-Ruiz A, Zhang R, Juncos R, Liu H, Manning RD, Jr., Juncos LA and Liu R. Salt-sensitive splice variant of nNOS expressed in the macula densa cells. Am JPhysiol Renal Physiol 298:F1465-F1471,2010.
32. Manning RD, Jr., Tian N and Meng S. Oxidative stress and antioxidant treatment in hypertension and the associated renal damage. Am J Nephrol 25:311-317,2005.
33. Meyer JW and Schmitt ME. A central role for the endothelial NADPH oxidase in atherosclerosis. FEBS Lett 472:1-4,2000.
34. Paliege A, Pasumarthy A, Mizel D, Yang T, Schnermann J and Bachmann S. Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 290:R694-R700,2006.
35. Rebsamen MC, Perrier E, Gerber-Wicht C, Benitah JP and Lang U. Direct and indirect effects of aldosterone on cyclooxygenase-2 and interleukin-6 expression in rat cardiac cells in culture and after myocardial infarction. Endocrinology 145:3135-3142, 2004.
36. Rudolph AE, Rocha R and McMahon EG. Aldosterone target organ protection by eplerenone. Mol Cell Endocrinol 217':229-238,2004.
37. Schiffrin EL. Effects of aldosterone on the vasculature. Hypertension 47:312-318, 2006.
38. Schnermann J and Levine DZ. Paracrine factors in tubuloglomerular feedback: adenosine, ATP, and nitric oxide. Annu Rev Physiol 65:501-529,2003.
39. Schnermann J, Ploth DW and Hermle M. Activation of tubulo-glomerular feedback by chloride transport. PflugersA rch 362:229-240,1976.
40. Scurry MT, Shear L and Barry KG. The renal tubular handling of aldosterone and its acid-labile conjugate. JClin Invest 47:242-248,1968.
41. Sun Y, Zhang J, Lu L, Chen SS, Quinn MT and Weber KT. Aldosterone-induced inflammation in the rat heart. Role of oxidative stress. Am J Pathol 161:1773-1781. 2002.
42. Traynor TR, Smart A, Briggs JP and Schnermann J. Inhibition of macula densa-stimulated renin secretion by pharmalogical blockade of cycloooxygenase-2. Am J Physiol 277:F706-F710,1999.
43. Welch WJ and Wilcox CS. Tubuloglomerular feedback and macula densa-derived NO. In:Nitric Oxide and the Kidney. Physiology and Pathophysiology., edited by Goligorsky MS and Gross SS. New York:Chapman & Hall,1997, p.216-232.
44. Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H, Spring K, Briggs JP and Schnermann J. Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line. J Biol Chem 275:37922-37929,2000.
45. Yoshimoto T, Fukai N, Sato R, Sugiyama T, Ozawa N, Shichiri M and Hirata Y. Antioxidant effect of adrenomedullin on angiotensin Ⅱ-induced reactive oxygen species generation in vascular smooth muscle cells. Endocrinology 145:3331-3337, 2004.
46. Zhang R, Harding P, Garvin JL, Juncos R, Peterson E, Juncos LA and Liu R. Isoforms and Functions of NADPH Oxidase at the Macula Densa. Hypertension 53: 556-563,2009.
1. Al-Dujaili EA and Edwards CR. The development and application of a direct radioimmunoassay for plasma aldosterone using 1251-labeled ligand--comparison of three methods. J Clin Endocrinol Metab 46:105-113,1978.
2. Alzamora R, Brown LR and Harvey BJ. Direct binding and activation of protein kinase C isoforms by aldosterone and 17beta-estradiol. Mol Endocrinol 21'.2637-2650, 2007.
3. Araujo M and Welch WJ. Cyclooxygenase 2 inhibition suppresses tubuloglomerular feedback:roles of thromboxane receptors and nitric oxide. Am J Physiol Renal Physiol 296:F790-F794,2009.
4. Arima S, Kohagura K, Xu HL, Sugawara A, Abe T, Satoh F, Takeuchi K and Ito S. Nongenomic vascular action of aldosterone in the glomerular microcirculation. J Am Soc Nephrol 14:2255-2263,2003.
5. Berl T, Katz FH, Henrich WL, de TA and Schrier RW. Role of aldosterone in the control of sodium excretion in patients with advanced chronic renal failure. Kidney Int 14:228-235,1978.
6. Beswick RA, Dorrance AM, Leite R and Webb RC. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension 38:1107-1111,2001.
7. Blantz RC and Pelayo JC. A functional role for the tubuloglomerular feedback mechanism. Kidney Int 25:739-746,1984.
8. Deng A, Wead LM and Blantz RC. Temporal adaptation of tubuloglomerular feedback: effects of COX-2. Kidney Int 66:2348-2353,2004.
9. Fu Y, Katsuya T, Matsuo A, Yamamoto K, Akasaka H, Takami Y, Iwashima Y, Sugimoto K, Ishikawa K, Ohishi M, Rakugi H and Ogihara T. Relationship of bradykinin B2 receptor gene polymorphism with essential hypertension and left ventricular hypertrophy. Hypertens Res 27:933-938,2004.
10. Fu Y, Zhang R, Lu D, Liu H, Chandrashekar K, Juncos LA and Liu R. NOX2 is the primary source of angiotensin Ⅱ-induced superoxide in the macula densa. Am J Physiol Regul Integr Comp Physiol 298:R707-R712,2010.
11. Gekle M, Golenhofen N, Oberleithner H and Silbernagl S. Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance. Proc Natl Acad Sci U S A 93:10500-10504, 1996.
12. Good DW. Nongenomic actions of aldosterone on the renal tubule. Hypertension 49: 728-739,2007.
13. Greene EL, Kren S and Hostetter TH. Role of aldosterone in the remnant kidney model in the rat. J Clin Invest 98:1063-1068,1996.
14. Harvey BJ and Higgins M. Nongenomic effects of aldosterone on Ca2+in M-1 cortical collecting duct cells. Kidney Int 57:1395-1403,2000.
15. Hirono Y, Yoshimoto T, Suzuki N, Sugiyama T, Sakurada M, Takai S, Kobayashi N, Shichiri M and Hirata Y. Angiotensin Ⅱ receptor type 1-mediated vascular oxidative stress and proinflammatory gene expression in aldosterone-induced hypertension:the possible role of local renin-angiotensin system. Endocrinology 148:1688-1696,2007.
16. Iwashima F, Yoshimoto T, Minami I, Sakurada M, Hirono Y and Hirata Y. Aldosterone induces superoxide generation via Rac1 activation in endothelial cells. Endocrinology 149:1009-1014,2008.
17. Le MC, Ouvrard-Pascaud A, Capurro C, Cluzeaud F, Fay M, Jaisser F, Farman N and Blot-Chabaud M. Early nongenomic events in aldosterone action in renal collecting duct cells:PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response. JAm Soc Nephrol 15:1145-1160,2004.
18. Liu R, Carretero OA, Ren Y and Garvin JL. Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback. Kidney Int 67:1837-1843, 2004.
19. Liu R and Persson AE. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration. J Physiol 563:895-901,2005.
20. Liu R, Pittner J and Persson AE. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration. J Am Soc Nephrol 13:2688-2696,2002.
21. Losel R, Schultz A and Wehling M. A quick glance at rapid aldosterone action. Mol Cell Endocrinol 217:137-141,2004.
22. Lu D, Fu Y, Lopez-Ruiz A, Zhang R, Juncos R, Liu H, Manning RD, Jr., Juncos LA and Liu R. Salt-sensitive splice variant of nNOS expressed in the macula densa cells. Am J Physiol Renal Physiol 298:F1465-F1471,2010.
23. Mihailidou AS, Bundgaard H, Mardini M, Hansen PS, Kjeldsen K and Rasmussen HH. Hyperaidosteronemia in rabbits inhibits the cardiac sarcolemmal Na(+)-K(+) pump. Circ Res 86:37-42,2000.
24. Paliege A, Pasumarthy A, Mizel D, Yang T, Schnermann J and Bachmann S. Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 290: R694-R700,2006.
25. Rebsamen MC, Perrier E, Gerber-Wicht C, Benitah JP and Lang U. Direct and indirect effects of aldosterone on cyclooxygenase-2 and interleukin-6 expression in rat cardiac cells in culture and after myocardial infarction. Endocrinology 145:3135-3142, 2004.
26. Rudolph AE, Rocha R and McMahon EG Aldosterone target organ protection by eplerenone. Mol Cell Endocrinol 217:229-238,2004.
27. Schiffrin EL. Effects of aldosterone on the vasculature. Hypertension 47:312-318,2006.
28. Schnermann J, Ploth DW and Hermle M. Activation of tubulo-glomerular feedback by chloride transport. PflugersArch 362:229-240,1976.
29. Sun Y, Zhang J, Lu L, Chen SS, Quinn MT and Weber KT. Aldosterone-induced inflammation in the rat heart. Role of oxidative stress. Am J Pathol 161:1773-1781, 2002.
30. Traynor TR, Smart A, Briggs JP and Scbnermann J. Inhibition of macula densa-stimulated renin secretion by pharmalogical blockade of cycloooxygenase-2. Am J Physiol 277:F706-F710,1999.
31. Welch WJ and Wilcox CS. Tubuloglomerular feedback and macula densa-derived NO. In:Nitric Oxide and the Kidney. Physiology and Pathophysiology., edited by Goligorsky MS and Gross SS. New York:Chapman & Hall,1997, p.216-232.
32. Zhang R, Harding P, Garvin JL, Juncos R, Peterson E, Juncos LA and Liu R. Isoforms and Functions of NADPH Oxidase at the Macula Densa. Hypertension 53: 556-563,2009.
33. Zhu X, Manning RD, Jr., Lu D, Gomez-Sanchez CE, Fu Y, Juncos LA and Liu R. Aldosterone stimulates superoxide production in the macula densa cells. Am JPhysiol Renal Physiol 2011.