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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Acta Physiol (Oxf). 2014 Aug 25;213(1):249–258. doi: 10.1111/apha.12358

Oxidative status in the macula densa modulates tubuloglomerular feedback responsiveness in Ang II-induced hypertension

Jiangping Song 1,2, Yan Lu 2,3, En Yin Lai 4, Jin Wei 2, Lei Wang 2, Kiran Chandrashekar 3, Shaohui Wang 2, Chunyu Shen 2, Luis A Juncos 2,3, Ruisheng Liu 2,3,*
PMCID: PMC4389650  NIHMSID: NIHMS619732  PMID: 25089004

Abstract

Aim

Tubuloglomerular feedback (TGF) is an important mechanism in control of signal nephron glomerular filtration rate. The oxidative stress in the macula densa, primarily determined by the interactions between nitric oxide (NO) and superoxide (O2−), is essential in maintaining the TGF responsiveness. However few studies examining the interactions between and amount of NO and O2− generated by the macula densa during normal and hypertensive states.

Methods

In this study, we used isolated perfused juxtaglomerular apparatus to directly measure the amount and also studied the interactions between NO and O2− in macula densa in both physiological and slow pressor Angiotensin II (Ang II) induced hypertensive mice.

Results

We found that slow pressor Ang II at a dose of 600ng/kg/min for two weeks increased mean arterial pressure by 26.1±5.7 mmHg. TGF response increased from 3.4±0.2 μm in control to 5.2±0.2 μm in hypertensive mice. We first measured O2− generation by the macula densa and found it was undetectable in control mice. However, O2− generation by the macula densa increased to 21.4 ± 2.5 unit/min in Ang II-induced hypertensive mice. Then we measured NO generation and found that NO generation by the macula densa was 138.5 ± 9.3 unit/min in control mice. The NO was undetectable in the macula densa in hypertensive mice infused with Ang II.

Conclusions

Under physiological conditions, TGF response is mainly controlled by the NO generated in the macula densa; in Ang II induced hypertension, the TGF response is mainly controlled by the O2− generated by the macula densa.

Keywords: macula densa, tubuloglomerular feedback, hypertension, oxidative stress

INTRODUCTION

The macula densa plaque consists of a group of specialized epithelial cells located in the distal segment of the thick ascending limb of the nephron which serve as luminal sensors of sodium chloride (NaCl) concentration. An increase in the NaCl delivery to the macula densa promotes release of adenosine and/or ATP, leading to afferent arteriolar constriction and decrease in the single nephron glomerular filtration rate (SNGFR); this process is termed as the tubuloglomerular feedback (TGF) response (Briggs & Schnermann 1986; Ollerstam et al. 1997; Welch et al. 2000; Ren et al. 2000). TGF may help to maintain the extracellular fluid volume by preserving the delicate balance between the glomerular filtration and tubular reabsorption rate (Briggs & Schnermann 1986; Welch & Wilcox 1990; Schnermann & Briggs 1992). TGF does this by establishing a negative feedback through which any change in NaCl delivery to the macula densa induces a reciprocal change in SNGFR and tubular flow, thereby preventing acute fluctuations of flow and NaCl delivery in the distal nephron. Hence, TGF limits urinary volume and sodium excretion, thereby helping maintain the salt and water homeostasis. Several in-vivo and in-vitro models of hypertension and sodium retaining states such as cirrhosis and acute renal failure have been found to have an augmented TGF response (Welch et al. 1999; Welch et al. 2000; Persson et al. 2000; Blantz et al. 2002; Carlstrom et al. 2009a; Carlstrom et al. 2013), implying that abnormal TGF responses may have a potential role in these pathophysiological conditions.

Several factors regulate TGF responsiveness, including angiotensin II (Welch & Wilcox 1990; Ren et al. 2002c; Vallon et al. 2004), adenosine (Sun et al. 2001; Ren et al. 2002a; Li et al. 2013; Sallstrom et al. 2013), arachidonic acid metabolites (Zou et al. 1994a; Zou et al. 1994b; Imig et al. 1996; Imig 2000), ATP (Inscho et al. 1992; Nishiyama et al. 2001; Nishiyama & Navar 2002; Ren et al. 2004), atrial natriuretic factor (Huang & Cogan 1987), superoxide (O2−)(Welch et al. 2000; Ren et al. 2002b; Liu et al. 2004b) and nitric oxide (NO)(Ollerstam et al. 1997; Ichihara et al. 1998; Ichihara & Navar 1999; Welch et al. 2000; Ren et al. 2000; Liu et al. 2004a). Among these factors, oxidative stress in the macula densa (which primarily depends on the interaction between NO and O2−), is crucial in controlling TGF responses both in normal and hypertensive animals models (Welch et al. 2000; Araujo & Welch 2006; Fu et al. 2012; Zhang et al. 2014). Several studies have demonstrated that both the NO and O2− generated by the macula densa are enhanced during TGF and in turn regulate the TGF responsiveness (Liu et al. 2002; Kovács et al. 2003; Liu et al. 2004a; Liu et al. 2004b; Liu et al. 2008; Zhang et al. 2009). Additionally, O2− is vital in regulating TGF responses during pathophysiological situations such as hypertension, diabetes and high salt intake (Welch & Wilcox 1997; Welch et al. 1999; Welch et al. 2006; Zhang et al. 2014). However, despite their obvious significance, to date most of the studies have only targeted the enzymes producing either NO or O2− with antagonists or genetic approaches. There has been a lack of studies examining the interactions between and also the amount of NO and O2− being generated by the macula densa during physiological conditions and the possible alterations to these molecules during hypertension. Hence, in this study, we hypothesized that interactions between NO and O2− determines the oxidative stress in the macula densa and regulates TGF responsiveness. We directly measured the NO and O2− generation in the macula densa with fluorescent dyes in isolated perfused juxtaglomerular apparatus and also compared their levels in normal and hypertensive states.

METHODS

All procedures and experiments were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. All chemicals were purchased from Sigma (St. Louis, MO) except as indicated.

Telemetry transmitter implantation

As described previously (Zhang et al. 2014), C57BL/6 mice (18-20 g, Harlan) were anesthetized with inhaled isoflurane (Butler chemicals, UK). A small incision was made in the middle of neck and a telemetry transmitter (PA-C10, Data Sciences International) was inserted under sterile conditions. The catheter of the transmitter was placed in the left carotid artery and advanced down to the aortic arch. The body of the transmitter was placed subcutaneously in the right ventral flank of the animal. The mice were then allowed to recover for ten days.

Minipump Insertion

Osmotic minipumps (Alzet Corp) were filled with either slow-pressor dose of Angiotensin II (Ang II;600ng/kg/min) or saline (control) previously described (Zhang et al. 2014). On the day of surgery, the animals which had ten days to recover from the telemetry transmitter implantation were again anesthetized with isoflurane. Under sterile conditions, a small incision was made in the mid-scapular area on the animal's back. Using a hemostat, the subcutaneous tissue was separated and spread so as to create a small pouch in which the minipump was inserted. Following insertion, the wound was sutured and the animals were allowed to recover for a day. Measurements of the mean arterial pressure (MAP) were started on the second day after minipump implantation at 10 minute intervals. All in vitro experiments using hypertensive mice were performed at day 14 after minipump insertion.

Isolation and microperfusion of the mouse Af-Art and attached macula densa and measurement of TGF

The Afferent-arteriole (Af-Art) and attached macula densa were isolated and microperfused using previously described techniques (Liu et al. 2004b; Fu et al. 2012). Briefly, mice (18 to 20 g) were anesthetized with inhaled isoflurane. The kidneys were removed and sliced along the corticomedullary axis. These slices were then placed in ice-cold minimum essential medium (MEM; Gibco, Grand Island, NY) containing 5% bovine serum albumin and dissected under a stereo-microscope (model SMZ 1500; Nikon). A single superficial Af-Art and its intact glomerulus were microdissected together with adherent tubular segments consisting of portions of the thick ascending limb of the loop of Henle, macula densa, and early distal tubule. Using a micropipette, the microdissected complex was transferred to a temperature-regulated chamber mounted on an inverted microscope (Eclipse Ti; Nikon) with Hoffmann modulation. Both the Af-Art and the end of either the distal tubule or thick ascending limb were cannulated with an array of glass pipettes. The Af-Art was then perfused with MEM, and the intraluminal pressure was measured with a pressure pipette and maintained at 60 mm Hg throughout the experiment. The macula densa was perfused with physiologic saline consisting of (in mmol/L) 10 HEPES; 1.0 CaCO3; 0.5 K2HPO4; 4.0 KHCO3; 1.2 MgSO4; 5.5 glucose; 0.5 Na acetate; 0.5 Na lactate; and either 80 (high NaCl) or 10 NaCl (low NaCl). The pH of the solution was 7.4. The bath was exchanged continuously at a rate of 1 mL/min. Microdissection was completed within 60 minutes at 8°C, the samples were then transferred into the bath which was gradually warmed to 37°C for the rest of the experiment. After the 30-minute equilibration period, the macula densa perfusate was switched from 10 to 80 mmol/L NaCl, and luminal diameter of the Af-Art was observed for 5 minutes. The change in the diameter of the Af-Art was considered as the TGF response. The imaging system consisted of a microscope (Eclipse Ti; Nikon), digital charge-coupled device camera (CoolSnap; Photometrics), xenon light (LB-LS/30; Shutter Instruments), and optical filter changer (Lambda 10–3; Shutter Instruments). Images were displayed and analyzed with NIS-Elements imaging software (Nikon).

Measurement of O2− and NO with fluorescent dyes in isolated perfused macula densa

The macula densa with its attached glomerulus was isolated and micro-perfused using previously described techniques (Liu & Persson 2004; Liu et al. 2007; Zhang et al. 2009; Lu et al. 2010). Briefly, a single superficial intact glomerulus was microdissected together with adherent tubular segments consisting of portions of the TAL, macula densa and early distal tubule. The TAL was cannulated with an array of glass pipettes. Another pipette was used to hold and stabilize the glomerulus. The sample was arranged so that each macula densa cell could be clearly visualized on the edge of the glomerulus.

A O2− sensitive fluorescent dye, dihydroethidium, was used to detect O2− levels (Fu et al. 2010; Zhang et al. 2014). Briefly, once the TAL was perfused, macula densa cells were loaded with 10 μM dihydroethidium in 0.1% dimethyl sulfoxide (DMSO) plus 0.1% pluronic acid from the lumen for 30 minutes then washed for 20 minutes. The loaded macula densa cells were exposed to 380 and 490 nm light to excite dihydroethidium and oxyethidium, respectively. Emitted fluorescence from dihydroethidium was recorded using a 420 nm dichroic mirror with a 460/50 nm bandpass filter; for oxyethidium we used a 565 nm dichroic mirror with a 605/55 nm bandpass filter. Square-shaped regions of interest were set inside the cytoplasm of macula densa cells and their mean intensity recorded every 5 seconds for 2 minutes. We recorded and calculated the rate of the changes for oxyethidium/dihydroxyethidium as an indicator of O2− production.

A NO-sensitive fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) was used to detect NO production in macula densa (Liu et al. 2004a; Lu et al. 2010). The macula densa cells were loaded with 10 μM DAF-2 DA in 0.5% DMSO plus 0.1% pluronic acid from the tubular lumen for 30 to 40 minutes, then washed for 10 minutes. DAF-2 was excited at 490 nm with a xenon arc light, and the emitted fluorescence was recorded at wavelengths of 510 to 550 nm. Square-shaped regions of interest were set inside the cytoplasm of macula densa cells and mean intensity within the regions of interest was recorded every 5 seconds for 2 minutes. Relative changes in DAF-2 fluorescence intensity were recorded. We calculated the rate of the changes for DAF-2 intensity as an indicator of NO production. In the experiments using inhibitors, we added inhibitors in tubular lumen and bath for 15 minutes before measurement.

Statistical analysis

Data were collected as repeated measures over time under different conditions. We tested only the effects of interest, using analysis of variance (ANOVA) for repeated measures and a post-hoc Fisher LSD test or a Student's paired t-test when appropriate. The changes were considered to be significant if p< 0.05. Data are presented as mean ± SEM.

RESULTS

Hypertension induced with slow-pressor Ang II infusion in mice

C57BL/6 mice were infused with slow pressor Ang II at a dose of 600ng/kg/min for two weeks and the blood pressure was measured using telemetry. The MAP gradually increased from a basal level of 101.4±3.7 mmHg to reach a peak of 127.5±8.2 mmHg on days 10-14 of the infusion (p< 0.01; n= 8). The MAP of control mice infused with saline did not vary significantly (basal MAP changed from 99.4±4.3 to 103.7±5.6 mmHg at the end of 2 weeks of infusion, respectively; n=11, Fig 1).

Figure 1. A slow pressor dose of Angiotensin II infusion induced hypertension.

Figure 1

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Saline (control; n=11) or slow pressor dose of Angiotensin II (Ang II) at 600 ng/min/kg (n=8) was infused for 2 weeks in C57BL/6 mice. In the Ang II group the mean arterial pressure (MAP) increased from 101.4±3.7 mmHg to 127.5±8.2 mmHg after two weeks. In the control group the blood pressure did not vary significantly (MAP was 99.4±4.3 and 103.7±5.6 mmHg at basal and 2 weeks of infusion) ( * P< 0.01 vs saline-infusion and basal)

TGF response in hypertensive mice induced with slow-pressor Ang II

TGF response was measured in isolated perfused JGAs from the hypertensive mice induced with slow-pressor Ang II and compared with controls. In mice without infusion of Ang II or saline, the diameter of the Af-Art was 16.3±0.8 μm when the tubule was perfused with a 10 mmol/L NaCl. After we switched the tubular NaCl concentration to 80 mmol/L, the Af-Art constricted to 12.9±0.9 μm. In this setting, the TGF response (as indicated by the delta changes of the A-Art diameter) was 3.4±0.2 μm (n= 6; Fig 2). In the mice infused with saline, when we increased tubular NaCl concentration from 10 to 80 mmol/L in the isolated perfused JGAs, Af-Art constricted from 17.1±0.7 to 13.3±0.6 μm. TGF response was 3.8±0.3 μm. (n= 6 ; Fig 2). In the AngII infused hypertensive mice, when the tubular NaCl concentration was increased from 10 to 80 mmol/L in the isolated perfused JGAs, Af-Art constricted from 15.8±0.7 to 10.6±0.9 μm. TGF response was 5.2±0.2 μm (p< 0.01 vs. Basal and saline-infused samples; n= 6; Fig 2). These data indicate that TGF responses were enhanced in slow-pressor AngII induced hypertensive mice.

Figure 2. Slow pressor Ang II induced Tubuloglomerular Feedback (TGF) response.

Figure 2

Figure 2

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The delta changes of the diameters of the afferent arterioles (Af-Art) are taken as an indicator of the TGF response. In mice without Ang II or saline infusion (basal state), the diameter of the Af-Art was 16.3±0.8 μm when the tubule was perfused with a 10 mmol/L sodium chloride (NaCl). When the tubular NaCl concentration was switched to 80 mmol/L, the Af-Art constricted to 12.9±0.9 μm (Fig 2A). The delta changes/ TGF response was 3.4±0.2 μm (n= 6; Fig 2B). Two weeks later in the control mice, increasing the tubular NaCl concentration from 10 to 80 mmol/L in the isolated perfused JGAs, constricted the Af-Art from 17.1±0.7 to 13.3±0.6 μm (Fig 2A). TGF was 3.8±0.3 μm. (n= 6; Fig 2B) and in the Ang II treated mice, the Af-Art constricted from 15.8±0.7 to 10.6±0.9 μm (Fig 2A). TGF was 5.2±0.2 μm ( * P< 0.01 vs saline-infusion and basal; n= 6; Fig 2B).

O2− generation by the macula densa in Ang II-induced hypertensive mice

First we determined if the O2− generated by the macula densa cells increased following Ang II infusion. For this, we isolated and perfused the JGA and measured the O2− generated using dihydroethidium in mice infused with Ang II or saline for 2 weeks and compared them to basal values (Fig 3). The macula densa was perfused with an 80 mmol/L NaCl solution. O2− generation by the macula densa was undetectable in mice infused with saline and at basal conditions. However, O2− generation by the macula densa increased to 21.4 ± 2.5 unit/min in Ang II-induced hypertensive mice (p < 0.01, n =7, Fig 4). These data suggest that in control mice, the macula densa generated a greater amount of NO compared to O2− ; in hypertensive mice, O2− generation by the macula densa significantly increased.

Figure 3. Representative experiments for measurement for O2− and NO with fluorescent dyes.

Figure 3

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A. The macula densa plaque was loaded with dihydroethidium and oxyethidium was measured with Ex/Em at 565/605 nm. B. A light microscopy image for dihydroethidium loading. C. The macula densa plaque was loaded with DAF-2 DA and NO was measured with Ex/Em at 490/540 nm. D. A light microscopy image for DAF-2 DA loading. G: glomerulus arrow: macula densa plaque

Figure 4. O2− generation by the macula densa in normal and hypertensive mice.

Figure 4

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O2− was measured in the isolated perfused juxtaglomerular apparatus (JGA) using dihydroethidium in mice infused with Ang II or saline for 2 weeks and compared them to basal values. The macula densa was perfused with 80 mmol/L NaCl solution. O2− generation by the macula densa was undetectable in mice infused with saline and at basal conditions. However, O2− generation by the macula densa increased to 21.4 ± 2.5 unit/min in Ang II-induced hypertensive mice ( * p < 0.01, n =7).

Next, in order to determine the O2− generation by the macula densa cells without any interaction from NO, we repeated the above protocols in the presence of a non-selective NOS inhibitor, L-NAME (10 −4 mol/L) in tubular lumen and bath in separate experiments. The macula densa was perfused with an 80 mM NaCl solution. Under basal conditions the O2− generation by the macula densa was 12.3 ± 1.1 unit/min. In saline infused mice, it was 14.7 ± 2.0 unit/min. In the slow-pressor Ang II infused hypertensive mice, the O2− generation by the macula densa increased to 45.3 ± 3.7 unit/min (p < 0.01, n =5, Fig 5). These data indicate that there is a significant increase in O2− generation by the macula densa in Ang II-hypertensive mice.

Figure 5. O2− generation by the macula densa cells in the presence of NO inhibition.

Figure 5

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The previous experiments were repeated but this time in the presence of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (10 −4 mol/L) in tubular lumen and bath. Following perfusion of the macula densa with 80 mM NaCl solution, in basal conditions the O2− generation by the macula densa was 12.3 ± 1.1 unit/min. In saline infused mice, it was 14.7 ± 2.0 unit/min. In the slow-pressor Ang II infused hypertensive mice, the O2− generation by the macula densa increased to 45.3 ± 3.7 unit/min ( * p < 0.01, n =5).

Finally, in order to establish whether the fluorescent signals measured were actually from O2−, we repeated the above protocols in normal control mice in the presence of L-NAME (10 −4 mol/L) and a O2− scavenger, tempol (10−4 mol/L) in the bath and lumen in separate experiments. The macula densa was perfused with an 80 mmol/L NaCl solution. O2− generation was undetectable in the macula densa, indicating that fluorescent signals we measured were selective for O2− (n =5).

NO generation by the macula densa in Ang II-induced hypertensive mice

To determine whether NO generation by the macula densa increased in hypertensive mice, we measured NO using DAF-2 DA in isolated perfused JGAs in mice infused with Ang II. The macula densa was perfused with an 80 mmol/L NaCl solution. NO generation by the macula densa was 138.5 ± 9.3 unit/min in mice at basal state (without infusion of Ang II or saline). In mice infused with saline, NO generation by the macula densa was 143.9 ± 10.5 unit/min. However, the NO was undetectable in the macula densa in hypertensive mice infused with Ang II. (p < 0.01, n =7, Fig 6), indicating that in control mice, a greater amount of NO was generated compared to O2− by the macula densa; in hypertensive mice, there is more O2− generation by the macula densa than that of NO.

Figure 6. NO generation by the macula densa in normal and hypertensive mice.

Figure 6

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NO was measured in the isolated perfused juxtaglomerular apparatus (JGA) using DAF-2 in mice infused with Ang II or saline for 2 weeks and compared them to basal values. The macula densa was perfused with 80 mmol/L NaCl solution. NO generation by the macula densa was 138.5 ± 9.3 and 143.9 ± 10.5 unit/min in mice at basal conditions and infused with saline. However, NO generation by the macula densa was undetectable in Ang II-induced hypertensive mice ( * p < 0.01, n =7).

To determine the NO generated by the macula densa cells without any interaction from O2−, we repeated the above protocols in the presence of an O2− scavenger, tempol (10 −4 mol/L) in the bath and lumen in separate experiments. As before, the macula densa was perfused with an 80 mM NaCl solution. NO generation by the macula densa was 168.4 ± 12.4 units/min in mice under basal conditions (without infusion of Ang II or saline). In mice infused with saline, NO generation by the macula densa was 172.6 ± 13.8 units/min. In the Ang II hypertensive mice, NO generation by the macula densa was 215.1± 14.9 units/min (p < 0.01, n = 6, Fig 7). These data indicate that Ang II infusion enhances the NO generated by the macula densa.

Figure 7. NO generation by the macula densa cells in the presence of O2− scavenger.

Figure 7

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The previous experiments were repeated in separate JGA preparations, but this time in the presence of tempol (a O2− scavenger, 10 −4 mol/L) in the bath and lumen. When the macula densa was perfused with 80 mM NaCl solution, the NO generation by the macula densa was 168.4 ± 12.4 unit/min under basal conditions (without infusion of Ang II or saline). In mice infused with two weeks of saline, NO generation by the macula densa was 172.6 ± 13.8 unit/min. In the Ang II infused animals, NO generation by the macula densa was 215.1± 14.9 unit/min ( * p < 0.01, n = 6).

Next, in order to determine the source of the NO being generated, we repeated above protocols in the presence of tempol (10−4 mol/L) and a selective NOS1 inhibitor 7-nitroindazole (7-NI, 10−4 mol/L) in the bath and lumen in normal control mice in separate experiments. The macula densa was perfused with an 80 mmol/L NaCl solution. Under these conditions, NO generation was undetectable, indicating that the source of the macula densa NO generation was from NOS1 (n =4).

DISCUSSION

In this study we examined the interaction between NO and O2− generated by the macula densa and their role in the TGF response, under physiological and hypertensive states. The most important and novel findings are; that the interactions between NO and O2− in the macula densa regulates responsiveness of TGF; in addition these interactions vary in normotensive and hypertensive animals. In normotensive mice, there is a greater generation of NO compared to O2− by the macula densa and the NO dominates in controlling the TGF response. In contrast however, in Ang II-induced hypertension, O2− generation by the macula densa is greater than that of NO and the O2− becomes dominant in controlling the TGF response.

The slow-pressor Ang II-induced hypertension, which we have used in our present study is well recognized to be a good prototype of human hypertension and has been found to induce a gradual and sustained elevation in blood pressure over a period of time(Lever 1993; Rajagopalan et al. 1996; Reckelhoff et al. 2000). This slow-pressor Ang II induces oxidative stress, salt-sensitive hypertension, increases pro-inflammatory factors and leads to progressive renal injury(Cassis et al. 2009; Capone et al. 2011). The doses needed to cause these slow-pressor Ang II-induced changes vary based on route and species of animals. For example, previous studies have indicated that the changes induced by slow pressor doses of AngII in Sprague Dawley rats are generally stable and consistent (Chandrashekar et al. 2012). However, the changes induced in mice by such a dose are inconsistent; indeed the doses of slow pressor AngII needed to induce such changes in blood pressure and renal changes have varied considerably. The reasons are not clear, and may be due to variable release rates of the different models of minipumps or due to the varying responses of mice themselves. In mice, the slow-pressor dose fluctuates anywhere from 400-1000ng/kg/min in published reports (Daugherty et al. 2000; Brancaccio et al. 2003; Welch et al. 2006; Aragon et al. 2008; Cassis et al. 2009; Capone et al. 2011; Gao et al. 2011). In our hands, we found that Ang II at a dose of 600 ng/min/kg consistently elevated MAP by about 20 mmHg in 2 weeks (Zhang et al. 2014).

We found that TGF response was enhanced in isolated perfused JGAs from Ang II-induced hypertensive mice. Similarly, Dr. Welch's lab recently reported that slow-pressor Ang II infusion increased TGF response in vivo as measured by micropuncture (Araujo & Welch 2010). In our in vitro preparation, we have excluded all the confounding factors such as circulating Ang II and sympathetic activity. Hence, the observed enhanced TGF response should be due to the intrinsic changes in the macula densa cells and Af-Arts. It was not surprising and supported by our recent findings that both expression and activities of NOX2 and NOX4 in the macula densa were enhanced during Ang II-induced hypertension(Zhang et al. 2014). Enhanced oxidative stress in the macula densa augments TGF response (Wilcox & Welch 2000; Liu et al. 2004b).

We have reported previously that during TGF when we increased NaCl concentration in the macula densa from 10 to 80 mmol/L, both NO (Liu et al. 2002; Liu et al. 2004a; Liu & Persson 2005) and O2− (Liu et al. 2008) generation by the macula densa increased. However, their interaction at physiological and hypertensive conditions have not yet been clarified. The net effect between NO and O2− generated by the macula densa is vital in modulating the TGF responsiveness.(Wilcox et al. 1998; Welch et al. 2000; Liu et al. 2004a; Liu et al. 2004b). In our present study, we performed a series of experiments to examine the interaction between NO and O2− generated by the macula densa during TGF, by directly measuring the concentration of NO and O2− with fluorescent dyes. We first measured the amount of O2− in normal mice and could not detect any signals for O2−, implying that the O2− was perhaps being scavenged by NO. To confirm this, we repeated the experiment in the presence of a NOS inhibitor to exclude the interaction between NO and O2−.We found a constant O2− generation by the macula densa in normal mice when the NO generation was inhibited, which was similar to our previous findings(Liu et al. 2007; Zhang et al. 2009). We next measured the amount of NO generated by the macula densa in normal mice and found a constant NO generation. The NO we measured is the net result of the interaction between NO and O2− in the macula densa. It also indicated under physiological conditions, the macula densa generates greater amounts of NO compared to O2−. To further confirm this, we repeated the experiments in the presence of the O2− scavenger, tempol. We found that NO generation increased by about 20% compared with experiments without tempol.

We have previously reported that Ang II activates NOS1 in the macula densa and enhances NO generation via AT1 receptors(Liu & Persson 2004). Moreover, we have also recently published that Ang II enhances O2− via increasing activity and expression of NOX2 and/or NOX4 isoforms of NAD(P)H oxidase in the macula densa. A series of elegant studies have been conducted regarding the function of oxidative stress in the macula densa in control of TGF and renal hemodynamics in Ang II infused animals (Wilcox & Welch 2000; Welch et al. 2006; Nouri et al. 2007; Carlstrom et al. 2009b; Wang et al. 2010; Carlstrom et al. 2010; Gao et al. 2011; Lai et al. 2012). In our present study we have directly measured the generation of NO and O2− and their interaction in the macula densa in Ang II-induced hypertensive mice. We found a constant O2− generation in the macula densa in Ang II-induced hypertensive mice, indicating more O2− was generated than NO in the macula densa. In the presence of NO inhibition, O2− generated increased by 1 fold compared with experiments without NO inhibition. Also, the O2− generation increased by more than 3 folds compared with experiments without hypertension. When we measured NO generation in the macula densa in Ang II-induced hypertensive mice, we did not detect any signals for NO, indicating that the NO was being scavenged by O2− generated in the macula densa. To confirm this, we repeated the experiments in the presence of the O2− scavenger, tempol. We found a constant NO generation in the macula densa in hypertensive mice and the NO generation increased by 25%. These data indicate that in Ang II-induced hypertensive mice, generation of both NO and O2− is increased by the macula densa. However, greater amount of O2− is generated compared to NO thereby contributing to the enhanced TGF response in Ang II-induced hypertension. These data also indicate that NAD(P)H oxidase is more sensitive to Ang II stimulation than NOS1 in the macula densa and plays key role in pathophysiological conditions such as Ang II-induced hypertension. However, we need to be aware the limitations and pitfalls using fluorescent dyes, such as photobleaching, fluorophore saturation, background offsets and detector saturation to minimize the artifact(Brown 2007; Wardman 2007).

In summary, we examined the generation and interaction between NO and O2− generated by the macula densa both in physiological and hypertensive conditions. We found that under normal conditions, the generation of both NO and O2− by the macula densa increased during TGF. Under physiological conditions, NO dominates the oxidative stress in the macula densa. However, in Ang II-induced hypertension, while the macula densa increased generations of both NO and O2−, the amount of O2− generated is greater than that of NO. Hence, during Ang II chronic infusion, the O2− dominates the oxidative stress in the macula densa, which enhances TGF responsiveness and may contribute to the development of hypertension.

Acknowledgments

Sources of Funding:

This work was supported by National Institutes of Health Grants R01DK098582, R01DK099276 and American Heart Association Postdoctoral Fellowship Award 13POST1422006 (to Y. Lu).

Footnotes

Conflict of Interest: None

Reference List

  1. Aragon AC, Goens MB, Carbett E, Walker MK. Perinatal 2,3,7,8-tetrachlorodibenzo-pdioxin exposure sensitizes offspring to angiotensin II-induced hypertension. Cardiovasc.Toxicol. 2008;8:145–154. doi: 10.1007/s12012-008-9023-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Araujo M, Welch WJ. Oxidative stress and nitric oxide in kidney function. Current Opinion in Nephrology & Hypertension. 2006;15:72–77. doi: 10.1097/01.mnh.0000191912.65281.e9. [DOI] [PubMed] [Google Scholar]
  3. Araujo M, Welch WJ. Tubuloglomerular feedback is decreased in COX-1 knockout mice after chronic angiotensin II infusion. Am.J Physiol Renal Physiol. 2010;298:F1059–F1063. doi: 10.1152/ajprenal.00547.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blantz RC, Deng A, Lortie M, Munger K, Vallon V, Gabbai FB, Thomson SC. The complex role of nitric oxide in the regulation of glomerular ultrafiltration. Kidney Int. 2002;61:782–785. doi: 10.1046/j.1523-1755.2002.00220.x. [DOI] [PubMed] [Google Scholar]
  5. Brancaccio M, Fratta L, Notte A, Hirsch E, Poulet R, Guazzone S, De AM, Vecchione C, Marino G, Altruda F, Silengo L, Tarone G, Lembo G. Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat.Med. 2003;9:68–75. doi: 10.1038/nm805. [DOI] [PubMed] [Google Scholar]
  6. Briggs JP, Schnermann J. Macula densa control of renin secretion and glomerular vascular tone: evidence for common cellular mechanisms. Renal Physiol.(Basel) 1986;9:193–203. doi: 10.1159/000173084. [DOI] [PubMed] [Google Scholar]
  7. Brown CM. Fluorescence microscopy--avoiding the pitfalls. J Cell Sci. 2007;120:1703–1705. doi: 10.1242/jcs.03433. [DOI] [PubMed] [Google Scholar]
  8. Capone C, Faraco G, Park L, Cao X, Davisson RL, Iadecola C. The cerebrovascular dysfunction induced by slow pressor doses of angiotensin II precedes the development of hypertension. Am.J.Physiol Heart Circ.Physiol. 2011;300:H397–H407. doi: 10.1152/ajpheart.00679.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlstrom M, Brown RD, Sallstrom J, Larsson E, Zilmer M, Zabihi S, Eriksson UJ, Persson AE. SOD1 deficiency causes salt sensitivity and aggravates hypertension in hydronephrosis. Am.J Physiol Regul.Integr.Comp Physiol. 2009a;297:R82–R92. doi: 10.1152/ajpregu.90843.2008. [DOI] [PubMed] [Google Scholar]
  10. Carlstrom M, Brown RD, Yang T, Hezel M, Larsson E, Scheffer PG, Teerlink T, Lundberg JO, Persson AE. L-arginine or tempol supplementation improves renal and cardiovascular function in rats with reduced renal mass and chronic high salt intake. Acta Physiol (Oxf) 2013;207:732–741. doi: 10.1111/apha.12079. [DOI] [PubMed] [Google Scholar]
  11. Carlstrom M, Lai EY, Ma Z, Patzak A, Brown RD, Persson AE. Role of NOX2 in the regulation of afferent arteriole responsiveness. Am.J.Physiol Regul.Integr.Comp Physiol. 2009b;296:R72–R79. doi: 10.1152/ajpregu.90718.2008. [DOI] [PubMed] [Google Scholar]
  12. Carlstrom M, Lai EY, Ma Z, Steege A, Patzak A, Eriksson UJ, Lundberg JO, Wilcox CS, Persson AE. Superoxide dismutase 1 limits renal microvascular remodeling and attenuates arteriole and blood pressure responses to angiotensin II via modulation of nitric oxide bioavailability. Hypertension. 2010;56:907–913. doi: 10.1161/HYPERTENSIONAHA.110.159301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cassis LA, Gupte M, Thayer S, Zhang X, Charnigo R, Howatt DA, Rateri DL, Daugherty A. ANG II infusion promotes abdominal aortic aneurysms independent of increased blood pressure in hypercholesterolemic mice. Am.J.Physiol Heart Circ.Physiol. 2009;296:H1660–H1665. doi: 10.1152/ajpheart.00028.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chandrashekar K, Lopez-Ruiz A, Juncos R, Nath K, Stec DE, Vera T, Liu R, Juncos LA. The Modulatory Role of Heme Oxygenase on Subpressor Angiotensin II-Induced Hypertension and Renal Injury. Int.J Hypertens. 2012;2012:392890. doi: 10.1155/2012/392890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J.Clin.Invest. 2000;105:1605–1612. doi: 10.1172/JCI7818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fu Y, Hall JE, Lu D, Lin L, Manning RD, Jr., Cheng L, Gomez-Sanchez C, Juncos LA, Liu R. Aldosterone blunts tubuloglomerular feedback by activating macula densa mineralocorticoid receptors. Hypertension. 2012;59:599–606. doi: 10.1161/HYPERTENSIONAHA.111.173195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fu Y, Zhang R, Lu D, Liu H, Chandrashekar K, Juncos LA, Liu R. NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa. Am.J.Physiol Regul.Integr.Comp Physiol. 2010;298:R707–R712. doi: 10.1152/ajpregu.00762.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gao X, Patzak A, Sendeski M, Scheffer PG, Teerlink T, Sallstrom J, Fredholm BB, Persson AE, Carlstrom M. Adenosine A(1)-receptor deficiency diminishes afferent arteriolar and blood pressure responses during nitric oxide inhibition and angiotensin II treatment. Am.J Physiol Regul.Integr.Comp Physiol. 2011;301:R1669–R1681. doi: 10.1152/ajpregu.00268.2011. [DOI] [PubMed] [Google Scholar]
  19. Huang CL, Cogan MG. Atrial natriuretic factor inhibits maximal tubuloglomerular feedback response. American Journal of Physiology. 1987;252:F825–F828. doi: 10.1152/ajprenal.1987.252.5.F825. [DOI] [PubMed] [Google Scholar]
  20. Ichihara A, Inscho EW, Imig JD, Navar LG. Neuronal nitric oxide synthase modulates rat renal microvascular function. American Journal of Physiology. 1998;274:F516–F524. doi: 10.1152/ajprenal.1998.274.3.F516. [DOI] [PubMed] [Google Scholar]
  21. Ichihara A, Navar LG. Neuronal NOS contributes to biphasic autoregulatory response during enhanced TGF activity. American Journal of Physiology. 1999;277:F113–F120. doi: 10.1152/ajprenal.1999.277.1.F113. [DOI] [PubMed] [Google Scholar]
  22. Imig JD. Eicosanoid regulation of the renal vasculature. American Journal of Physiology-Renal Physiology. 2000;279:F965–F981. doi: 10.1152/ajprenal.2000.279.6.F965. [DOI] [PubMed] [Google Scholar]
  23. Imig JD, Zou AP, Stec DE, Harder DR, Falck JR, Roman RJ. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. American Journal of Physiology. 1996;270:R217–R227. doi: 10.1152/ajpregu.1996.270.1.R217. [DOI] [PubMed] [Google Scholar]
  24. Inscho EW, Ohishi K, Navar LG. Effects of ATP on pre- and postglomerular juxtamedullary microvasculature. American Journal of Physiology. 1992;263:F886–F893. doi: 10.1152/ajprenal.1992.263.5.F886. [DOI] [PubMed] [Google Scholar]
  25. Kovács G, Komlósi P, Fuson A, Peti-Peterdi J, Rosivall L, Bell PD. Neuronal nitric oxide synthase: its role and regulation in macula densa cells. J.Am.Soc.Nephrol. 2003;14:2475–2483. doi: 10.1097/01.asn.0000088737.05283.2b. [DOI] [PubMed] [Google Scholar]
  26. Lai EY, Solis G, Luo Z, Carlstrom M, Sandberg K, Holland S, Wellstein A, Welch WJ, Wilcox CS. p47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice. Hypertension. 2012;59:415–420. doi: 10.1161/HYPERTENSIONAHA.111.184291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lever AF. In: The fast and the slowly developing pressor effect of angiotensin II. The Renin-Angiotensin System. Ian J, Robertson S, Nicholls MG, editors. Gower Medical Publishing; London: 1993. pp. 28.1–28.9. [Google Scholar]
  28. Li L, Mizel D, Huang Y, Eisner C, Hoerl M, Thiel M, Schnermann J. Tubuloglomerular feedback and renal function in mice with targeted deletion of the type 1 equilibrative nucleoside transporter. Am.J Physiol Renal Physiol. 2013;304:F382–F389. doi: 10.1152/ajprenal.00581.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu R, Carretero OA, Ren Y, Garvin JL. Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback. Kidney Int. 2004a;67:1837–1843. doi: 10.1111/j.1523-1755.2005.00282.x. [DOI] [PubMed] [Google Scholar]
  30. Liu R, Carretero OA, Ren Y, Wang H, Garvin JL. Intracellular pH regulates superoxide production by the macula densa. Am.J.Physiol Renal Physiol. 2008;295:F851–F856. doi: 10.1152/ajprenal.90204.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu R, Garvin JL, Ren Y, Pagano PJ, Carretero OA. Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase. AJP - Renal Physiology. 2007;292:1867–1872. doi: 10.1152/ajprenal.00515.2006. [DOI] [PubMed] [Google Scholar]
  32. Liu R, Persson AE. Angiotensin II stimulates calcium and nitric oxide release from macula densa cells through AT1 receptors. Hypertension. 2004;43:649–653. doi: 10.1161/01.HYP.0000116222.57000.85. [DOI] [PubMed] [Google Scholar]
  33. Liu R, Persson AE. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration. J.Physiol. 2005;563:895–901. doi: 10.1113/jphysiol.2004.078154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu R, Pittner J, 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. 2002;13:2688–2696. doi: 10.1097/01.asn.0000033275.17169.67. [DOI] [PubMed] [Google Scholar]
  35. Liu R, Ren Y, Garvin JL, Carretero OA. Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole. Kidney Int. 2004b;66:268–274. doi: 10.1111/j.1523-1755.2004.00727.x. [DOI] [PubMed] [Google Scholar]
  36. Lu D, Fu Y, Lopez-Ruiz A, Zhang R, Juncos R, Liu H, Manning RD, Jr., Juncos LA, Liu R. Salt-sensitive splice variant of nNOS expressed in the macula densa cells. Am.J.Physiol Renal Physiol. 2010;298:F1465–F1471. doi: 10.1152/ajprenal.00650.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nishiyama A, Majid DSA, Walker M, III, Miyatake A, Navar LG. Renal interstitial ATP responses to changes in arterial pressure during alterations in tubuloglomerular feedback activity. Hypertension. 2001;37:753–759. doi: 10.1161/01.hyp.37.2.753. [DOI] [PubMed] [Google Scholar]
  38. Nishiyama A, Navar LG. ATP mediates tubuloglomerular feedback. Am J Physiol Regul.Integr.Comp Physiol. 2002;283:R273–R275. doi: 10.1152/ajpregu.00071.2002. [DOI] [PubMed] [Google Scholar]
  39. Nouri P, Gill P, Li M, Wilcox CS, Welch WJ. p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats. Am J Physiol Heart Circ Physiol. 2007;292:H1685–H1689. doi: 10.1152/ajpheart.00976.2006. [DOI] [PubMed] [Google Scholar]
  40. Ollerstam A, Pittner J, Persson AEG, Thorup C. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J.Clin.Invest. 1997;99:2212–2218. doi: 10.1172/JCI119394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Persson AE, Gutierrez A, Pittner J, Ring A, Ollerstam A, Brown R, Liu R, Thorup C. Renal NO production and the development of hypertension. Acta Physiol.Scand. 2000;168:169–174. doi: 10.1046/j.1365-201x.2000.00669.x. [DOI] [PubMed] [Google Scholar]
  42. Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman BA, Griendling KK, 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. 1996;97:1916–1923. doi: 10.1172/JCI118623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reckelhoff JF, Zhang H, Srivastava K, Roberts LJ, II, Morrow JD, Romero JC. Subpressor doses of angiotensin II increase plasma F2-isoprostanes in rats. Hypertension. 2000;35(Suppl):476–479. doi: 10.1161/01.hyp.35.1.476. [DOI] [PubMed] [Google Scholar]
  44. Ren Y, Arima S, Carretero OA, Ito S. Possible role of adenosine in macula densa control of glomerular hemodynamics. Kidney Int. 2002a;61:169–176. doi: 10.1046/j.1523-1755.2002.00093.x. [DOI] [PubMed] [Google Scholar]
  45. Ren Y, Carretero OA, Garvin JL. Mechanism by which superoxide potentiates tubuloglomerular feedback. Hypertension. 2002b;39:624–628. doi: 10.1161/hy0202.103299. [DOI] [PubMed] [Google Scholar]
  46. Ren Y, Garvin JL, Carretero OA. Role of macula densa nitric oxide and cGMP in the regulation of tubuloglomerular feedback. Kidney Int. 2000;58:2053–2060. doi: 10.1111/j.1523-1755.2000.00377.x. [DOI] [PubMed] [Google Scholar]
  47. Ren Y, Garvin JL, Carretero OA. Vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles. Hypertension. 2002c;39:799–802. doi: 10.1161/hy0302.104673. [DOI] [PubMed] [Google Scholar]
  48. Ren Y, Garvin JL, Liu R, Carretero OA. Role of macula densa adenosine triphosphate (ATP) in tubuloglomerular feedback. Kidney Int. 2004;66:1479–1485. doi: 10.1111/j.1523-1755.2004.00911.x. [DOI] [PubMed] [Google Scholar]
  49. Sallstrom J, Eriksson T, Fredholm BB, Persson AE, Palm F. Inhibition of sodium-linked glucose reabsorption normalizes diabetes-induced glomerular hyperfiltration in conscious adenosine A -receptor deficient mice. Acta Physiol (Oxf) 2013 doi: 10.1111/apha.12152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schnermann J, Briggs JP. Function of the juxtaglomerular apparatus. Control of glomerular hemodynamics and renin secretion. In: Seldin DW, Giebisch G, editors. The Kidney: Physiology and Pathology. Raven Press; New York: 1992. pp. 1249–1289. [Google Scholar]
  51. Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, Schnermann J. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proceedings of the National Academy of Sciences. 2001;98:9983–9988. doi: 10.1073/pnas.171317998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vallon V, Richter K, Huang DY, Rieg T, Schnermann J. Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice. Pflugers Arch. 2004;448:214–221. doi: 10.1007/s00424-004-1239-8. [DOI] [PubMed] [Google Scholar]
  53. Wang D, Luo Z, Wang X, Jose PA, Falck JR, Welch WJ, Aslam S, Teerlink T, Wilcox CS. Impaired endothelial function and microvascular asymmetrical dimethylarginine in angiotensin II-infused rats: effects of tempol. Hypertension. 2010;56:950–955. doi: 10.1161/HYPERTENSIONAHA.110.157115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wardman P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic.Biol Med. 2007;43:995–1022. doi: 10.1016/j.freeradbiomed.2007.06.026. [DOI] [PubMed] [Google Scholar]
  55. Welch WJ, Chabrashvili T, Solis G, Chen Y, Gill PS, Aslam S, Wang X, Ji H, Sandberg K, Jose P, Wilcox CS. Role of extracellular superoxide dismutase in the mouse angiotensin slow pressor response. Hypertension. 2006;48:934–941. doi: 10.1161/01.HYP.0000242928.57344.92. [DOI] [PubMed] [Google Scholar]
  56. Welch WJ, Tojo A, Lee J-U, Kang DG, Schnackenberg CG, Wilcox CS. Nitric oxide synthase in the JGA of the SHR: expression and role in tubuloglomerular feedback. American Journal of Physiology. 1999;277:F130–F138. doi: 10.1152/ajprenal.1999.277.1.F130. [DOI] [PubMed] [Google Scholar]
  57. Welch WJ, Tojo A, Wilcox CS. Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR. American Journal of Physiology-Renal Physiology. 2000;278:F769–F776. doi: 10.1152/ajprenal.2000.278.5.F769. [DOI] [PubMed] [Google Scholar]
  58. Welch WJ, Wilcox CS. Feedback responses during sequential inhibition of angiotensin and thromboxane. American Journal of Physiology. 1990;258:F457–F466. doi: 10.1152/ajprenal.1990.258.3.F457. [DOI] [PubMed] [Google Scholar]
  59. Welch WJ, Wilcox CS. Role of nitric oxide in tubuloglomerular feedback: effects of dietary salt. Clin.Exp.Pharmacol.Physiol. 1997;24:582–586. doi: 10.1111/j.1440-1681.1997.tb02095.x. [DOI] [PubMed] [Google Scholar]
  60. Wilcox CS, Deng X, Welch WJ. NO generation and action during changes in salt intake: roles of nNOS and macula densa. American Journal of Physiology. 1998;274:R1588–R1593. doi: 10.1152/ajpregu.1998.274.6.R1588. [DOI] [PubMed] [Google Scholar]
  61. Wilcox CS, Welch WJ. Interaction between nitric oxide and oxygen radicals in regulation of tubuloglomerular feedback. Acta Physiologica Scandinavica. 2000;168:119–124. doi: 10.1046/j.1365-201x.2000.00668.x. [DOI] [PubMed] [Google Scholar]
  62. Zhang J, Chandrashekar K, Lu Y, Duan Y, Qu P, Wei J, Juncos LA, Liu R. Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice. Am.J Physiol Renal Physiol. 2014;306:F344–F350. doi: 10.1152/ajprenal.00515.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhang R, Harding P, Garvin JL, Juncos R, Peterson E, Juncos LA, Liu R. Isoforms and Functions of NADPH Oxidase at the Macula Densa. Hypertension. 2009;53:556–563. doi: 10.1161/HYPERTENSIONAHA.108.124594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zou AP, Imig JD, Kaldunski M, Ortiz de Montellano PR, Sui Z, Roman RJ. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. American Journal of Physiology. 1994a;266:F275–F282. doi: 10.1152/ajprenal.1994.266.2.F275. [DOI] [PubMed] [Google Scholar]
  65. Zou A-P, Imig JD, Ortiz de Montellano PR, Sui Z, Falck JR, Roman RJ. Effect of P-450 omega-hydroxylase metabolites of arachidonic acid on tubuloglomerular feedback. American Journal of Physiology. 1994b;266:F934–F941. doi: 10.1152/ajprenal.1994.266.6.F934. [DOI] [PubMed] [Google Scholar]

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