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Am J Physiol Heart Circ Physiol 288: H29-H36, 2005. First published August 26, 2004; doi:10.1152/ajpheart.00623.2004
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2004 CARDIOVASCULAR AND KIDNEY INVESTIGATORS MEETING

Chronic ANG II infusion increases NO generation by rat descending vasa recta

Zhong Zhang,1 Kristie Rhinehart,1 Glen Solis,2 Janos Pittner,1 Whaseon Lee-Kwon,1 William J. Welch,2 Christopher S. Wilcox,2 and Thomas L. Pallone1

1Division of Nephrology and Department of Physiology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; and 2Division of Nephrology and Hypertension, Georgetown University, Washington, DC

Submitted 23 June 2004 ; accepted in final form 25 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng·kg–1·min–1 for 11–13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N{omega}-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca2+ response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.

medulla; kidney; microcirculation; calcium; oxidative stress

ADMINISTRATION of ANG II to rats at a rate that is too low to cause immediate hypertension induces a later hypertensive response when the infusion is sustained for several days. That "slow pressor" effect is studied as an experimental model akin to two-kidney, one-clip Goldblatt hypertension. Reactive oxygen species (ROS) favor vascular smooth muscle constriction and hypertension by a number of mechanisms. Superoxide (O2–·) prevents the full vasodilatory effects of nitric oxide (NO) from being achieved because it avidly reacts with NO to form peroxynitrite. Nonenzymatic reaction of ROS with arachidonic acid generates vasoconstrictor isoprostanes and ROS such as H2O2 and O2–· that act as signaling molecules to modify ion channel activity (7, 29, 35). It is known that ROS are generated in response to both acute (9, 36, 42) and chronic exposure of microvessels to ANG II, prompting recent investigations into the possibility that ROS play a role in the slow pressor response (2, 37, 38).

Descending vasa recta (DVR) branch from juxtamedullary glomerular efferent arterioles to supply the medulla with blood flow. They are lined by a continuous endothelium and enveloped by small contractile cells called pericytes (23). These features imply a role for DVR to regulate blood flow to the medulla. Evidence has accumulated to implicate renal medullary ROS synthesis in the generation of hypertension in animal models. Bioavailability of NO is modulated by ROS, and NO plays a role to modulate medullary perfusion, sodium excretion, and blood pressure (1, 5, 6, 21). Furthermore, it is known that ROS generation can be upregulated in vascular endothelia and smooth muscle by ANG II. To determine whether alterations of DVR function occur during exposure to ANG II, we examined the effects of chronic ANG II infusion on NO and ROS generation in rats. The results show that the contractile response of DVR to ANG II is diminished and that NO generation is increased by chronic ANG II. The capacity of DVR from chronically ANG II-infused rats to oxidize dihydroethidium (DHE) was unaltered. These results agree with a role for increased NO generation by DVR endothelia to protect the renal medulla from vasoconstrictor-induced ischemia (44, 46). The results do not support modification of DVR function to augment ROS generation as a key factor in the genesis of ANG II slow pressor hypertension.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ANG II infusion via osmotic minipump. All investigations involving animal use described herein were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. ANG II was chronically infused into Sprague-Dawley rats (Harlan) by osmotic minipump (model 2002, Alzet). Rats were weighed and then anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). To maintain body temperature, the animals were placed on a warmed surgical table. The area between the scapulae was shaved and prepared with Betadine. With the use of sterilized surgical instruments, a 0.5-cm incision was created following which a subcutaneous tunnel was formed by inserting the end of closed hemostats under the skin. The osmotic minipump was inserted and the incision closed with stainless steel clips. The rats were allowed to recover from anesthesia in a warmed cage after which they were permitted ad libium access to food and water. The rate of infusion of ANG II was either zero (controls) or 250 ng·kg–1·min–1 based on weight at the time of minipump insertion.

Isolation of DVR. Between days 11 and 13 of infusion, kidneys were harvested from the rats. Rats were preanesthetized by an intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg), and the kidneys were excised, sliced, and placed in buffer at 4°C. Individual DVR were dissected from vascular bundles of the renal outer medulla and transferred to the stage of an inverted microscope. Fluorescent microscopy and microperfusion studies were performed as previously described (22, 24, 26, 42). The solution used to dissect and microperfuse DVR contained (in mM) 140 NaCl, 10 Na acetate, 5 KCl, 1.2 MgSO4, 1.2 Na2HPO4, 1 CaCl2, 5 HEPES, 5 L-alanine, and 5 D-glucose. The pH was adjusted to 7.55 at room temperature to yield a pH of ~7.4 at 37°C.

Measurement of DVR diameter. Vasoactivity was monitored in DVR perfused in vitro on concentric pipettes (22, 24). Vessel images were recorded on videotape (Panasonic WV-BL90) and diameters analyzed during playback. DVR luminal diameter was observed with a x40 objective to yield a final magnification of x1,300. Internal diameters were measured with calipers at the point of maximal constriction. Diameter changes are expressed as percent constriction, given by the following: [1 – (D/D0)] x 100, where D and D0 are the experimental and baseline diameters, respectively.

Measurement of cytosolic Ca2+ concentration. DVR were loaded with fura 2 by incubating them with the AM ester (2 µM, Molecular Probes; Eugene, OR) for 20 min at 37°C. A photon-counting photomultiplier assembly (PMT) was employed to measure the fluorescent emission of fura 2 at 510 nm. Excitation was provided by a 75-W xenon arc lamp using a 350/380 nm wavelength combination isolated with a computer-controlled monochromator (Photon Technology International; Lawrenceville, NJ). Fluorescent emission was isolated with a 510WB40 band-pass filter (Omega Optical; Brattleboro, VT) and collected with a Nikon CF fluor x40 oil immersion objective (1.3 numerical aperture). The background-subtracted ratio of fluorescent emission (R350/380) was converted to the cytosolic Ca2+ concentration ([Ca2+]cyt) assuming a dissociation constant of 224 nM for fura 2 at 37°C. Rmax and Rmin were measured by exposing vessels to buffer containing 5 mM CaCl2 or 0 CaCl2 and 0.5 mM EGTA, respectively, along with 10 µM ionomycin (24, 26).

Measurement of ROS generation by oxidation of DHE. As previously described, we measured generation of ROS by including DHE (10 µM) in the bath (42). Within the cells, this probe is oxidized by O2–· to fluorescent products that are trapped by intercalation into DNA. This probe is generally thought to be specific for O2–·, but ethidium (ETH) may not be its sole fluorescent product (43). Single DVR were dissected and immobilized on pipettes in flowing bath. ETH was excited at 540 nm and emission isolated at 620 nm using wavelength selection from a xenon lamp by either a motor-driven monochromator (Photon Technology International) or a solenoid driven, filter-based (IonOptix) light source. ETH fluorescence at time 0 was subtracted from the record as background signal. To assess the distribution of ETH in the DVR wall, fluorescent images of some vessels were obtained with a Zeiss LSM410 laser-scanning confocal microscope and quantified in pericytes and endothelia using NIH Image J software.

Fluorescent detection of NO with DAFFM. 3-Amino-4-aminomethyl-2',7'-difluorofluorescein diacetate 2DA (DAFFM-DA) is a NO-sensitive probe that is loaded into cells and trapped by deesterification (20 min, 37°C). When covalently modified by NO, DAFFM forms a fluorescent triazolo-fluorescein analog. The fluorescent signal provides an integrated measure of intracellular NO concentration. DAFFM was excited at 480 nm, and the emission was isolated at 535 nm (27).

Alternating measurement of DAFFM and ETH fluorescence in individual vessels. In some experiments, both DAFFM and ETH were simultaneously loaded to enable alternating measurement of their emissions. To accomplish this, a fluorescent microscopy system was equipped with dual photomultipliers (PMT, Photon Technology International) (Fig. 1). Nikon B-2E/C and G-2E/C filter cube assemblies that house the excitation filter, dichroic mirror, and emission filters needed for measurement of each probe were manually moved into position at the desired time points. DAFFM and ETH emit green and red light, respectively, that was directed to the dual PMT assembly by a beam splitter. Within the dual PMT assembly, the DAFFM and ETH emissions were split to the two separate PMTs using a 565-nm dichroic mirror. Barrier filters centered at 520 and 605 nm, respectively, were also used in front of the PMTs to isolate the emissions (Fig. 1A). Examples of DAFFM and ETH tracings from the PMTs are shown in Fig. 1, B and C. Excellent separation of the signals was achieved.



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  		Fig. 1. Detection of nitric oxide (NO) and superoxide (O2–·) with 3-amino-4-aminomethyl-2',7'difluoroscein (DAFFM) and ethidium (ETH) in the same isolated descending vasa recta (DVR). A: DAFFM and ETH, when excited at 485 nm and 535 nm, respectively, emit green and red light centered at ~520 and ~620 nm. During experiments, the desired excitation cube was manually positioned to measure the probe of interest. The DAFFM and ETH emissions were directed to separate photomultiplier tubes (PMT) by inserting a 565-nm dichroic mirror in the optical path. The ~520-nm DAFFM emission was reflected to PMT1 and the ~620-nm ETH emission was transmitted to PMT2. B: record of DVR ETH fluorescence during excitation with the G-2E/C cube assembly. Fluorescence was continuously recorded except for the intermittent periods designated by the arrows. At those times, the B-2E/C cube for DAFFM excitation was moved into place to detect NO. Note that during DAFFM excitation, contaminating signal at PMT2 was insignificant. C: DAFFM fluorescence intermittently detected at the times designated by the arrows in B. Note that between DAFFM monitoring times, no contaminating signal from ETH detected by PMT1 was insignificant.

 
O2–· generation by rat tissue homogenates. Superoxide was measured by lucigenin-enhanced chemiluminescence as described (37). Briefly, NADPH (100 µM) was added to the supernatant from the renal cortex or medulla in a reaction containing lucigenin (5 µM) in assay buffer. Chemiluminescence was determined over 20 min in a luminometer (Lumat LB 9501; Berthold; Pforzheim, Germany). Basal and NADPH-stimulated O2–· production were expressed as relative luminescence units per milligram of protein. Protein was measured using the Bradford method (Bio-Rad; Hercules, CA).

Reagents. Fura 2, DAFFM, and DHE were from Molecular Probes (Eugene, OR). They were stored frozen in DMSO, thawed, and diluted for daily use. ANG II, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), and N{omega}-nitro-L-arginine methyl ester (L-NAME) were stored in water at –20°C. ANG II, Tempol, and L-NAME were from Sigma (St. Louis, MO). Reagents were diluted 1:100 or 1:1,000 on the day of the experiment, and the excess was discarded at the end of each day.

Statistics and analysis. Data in the text and figures are shown as means ± SE. Fluorescence acquisition rates were often so rapid that graphing all error bars would obscure variation of the data. In some of the figures in this study, error bars have been suppressed for clarity of presentation. The significance of differences between means was evaluated using Student's t-test (paired or unpaired, as appropriate) and ANOVA. For ANOVA, the Student-Newman-Keuls test was used to evaluate significance.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of chronic ANG II infusion on DVR contractile response. Over the time of infusion, ANG II-exposed rats gained 85 ± 6 g compared with 110 ± 4 g in controls. A similar reduction of weight gain during chronic ANG II infusion has been observed by other investigators (28). The contractile response of DVR to ANG II was diminished in chronically ANG II-infused rats, a finding that contrasts with the enhanced responses observed in renal cortical vessels from rats (10) and rabbits (37, 38). Microperfused DVR were exposed to ANG II added to the bath. After baseline diameter measurement, ANG II concentration was raised to 0.1, 1.0, and 10 nM at 10-min intervals. The contractile response of DVR from chronically ANG II-infused rats (n = 11) was significantly less than that of controls (n = 8) at all ANG II concentrations tested (Fig. 2). When the bath was stepped from 1 to 10 nM ANG II, DVR from control and chronically ANG II-infused rats showed an initial increase in constriction followed by some waning of the response. That pattern in DVR has been observed in past studies (see Figs. 2 and 11 of Refs. 24 and 26, respectively).



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  		Fig. 2. ANG II induced constriction by control and ANG II-infused DVR. DVR isolated from control or ANG II-infused rats were mounted on concentric pipettes to pressurize their lumens. Video microscopy was performed as ANG II was exchanged into the bath at 0.1, 1, and 10 nM concentration. Values on the ordinate indicated percent reduction of internal diameter. *P < 0.05 vs. control. Baseline internal diameter of vessels from control and ANG II-infused rats was similar, 14.1 ± 0.5, and 14.0 ± 0.4 µm, respectively.

 
Oxidation of DHE and NO generation in chronically ANG II-infused rats. We next tested the ability of DVR from chronically ANG II-infused rats to oxidize DHE and generate NO. DVR were suspended on pipettes and loaded with DHE and DAFFM to quantify ROS and NO, respectively, and the fluorescent emissions were alternately monitored using the method illustrated in Fig. 1. Compared with controls (n = 10), DVR from ANG II-infused rats (n = 10) tended to oxidize DHE at a slower rate (Fig. 3A), but the effect did not achieve significance (P = 0.1 at time = 50 min). DAFFM fluorescence, indicating NO generation, was significantly greater in DVR from ANG II-infused rats (Fig. 3B, P < 0.05). Vascular tone of isolated DVR is modulated by NO (33, 41), and the increase in NO generation of DVR from ANG II-infused rats might partially explain the reduced contractile response observed in Fig. 2.



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  		Fig. 3. Effect of chronic ANG II infusion on NO and O2–·. A: rate of increase in ETH fluorescence, indicating dihydroethidium (DHE) oxidation, was measured in DVR from control and ANG II-infused rats. ETH oxidation by control DVR tended to be higher than that of ANG II-treated vessels, but the effect did not reach significance during 50 min of observation. B: rate of increase in DAFFM fluorescence, indicating NO generation, was measured in DVR from control and ANG II-infused rats. NO generation by chronically ANG II exposed vessels was higher than that of controls. *P < 0.05 vs. control.

 
We examined ETH fluorescence in DVR pericytes and endothelia. White light, fluorescent, and merged images captured with a cooled CCD camera using conventional microscopy were obtained from DVR of control and chronically ANG II-infused rats (Fig. 4). In control vessels, ETH appeared to be diffusely distributed between endothelia and pericytes with some predominance of fluorescence in the latter cell type. In contrast, relative to pericyte fluorescence, ETH in endothelia of DVR from ANG II-infused rats seemed lower (similar to n = 4). In some experiments, control DVR were exposed to DHE for 30 min and then examined with confocal microscopy. Optical sections through the longitudinal axis were analyzed by setting regions of interest within the cells. Fluorescence was quantified as the average pixel intensity within the region of interest and normalized by dividing by the mean of the values from endothelial cells. As previously described (42), fluorescence was found to be highest in pericytes, 1.66 ± 0.04 times greater than that of adjacent endothelia (Fig. 5, 1.0 ± 0.08, P < 0.001).



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  		Fig. 4. ETH fluorescence in DVR. DHE was loaded for 30 min into DVR from control (left) and ANG II-infused rats (right). White light (top), fluorescent (middle), and merged (bottom) images were captured using time exposures with conventional microscopy. *Locations where pericyte cell bodies protrude from the abluminal surface of the vessels.

 


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  		Fig. 5. ETH fluorescence in DVR by confocal microscopy. DHE was loaded into control DVR for 30 min after which ETH fluorescence was examined with confocal microscopy. Top: optical images obtained by sectioning through the vessel long axis. Abluminal pericyte cell bodies are more fluorescent than the luminal endothelia. Bottom: quantification of pixel intensity in the pericytes and endothelia, normalized to the mean value for the endothelium.

 
Effect of NOS inhibition on oxidation of DHE. In a separate series of experiments, DHE oxidation and NO production by DVR from control (n = 8) and ANG II (n = 8)-infused rats were measured in the presence of the nonspecific NOS inhibitor L-NAME (100 µM). L-NAME increased the rate of DHE oxidation by DVR from ANG II-infused rats (Fig. 6A, asterisk, P < 0.05). Moreover, in the presence of L-NAME, the rate of DHE oxidation by DVR from control and ANG II-infused rats (Fig. 6A) was similar; i.e., there was no tendency (P = 0.1 in Fig. 3A) for ETH fluorescence of control DVR to exceed that of DVR from ANG II-treated rats. Recording time in the experiments shown in Fig. 6 was shorter (25 min) than those in Fig. 3 (50 min), but the rate of increase of ETH fluorescence was comparable. In DVR from control rats not exposed to ANG II, L-NAME did not significantly enhance the rate of DHE oxidation (Fig. 6B, n = 8, each group). The effect of L-NAME to eliminate reduction of DHE oxidation (Fig. 6A) in DVR from ANG II-exposed animals might be explained by its inhibition of NO; i.e., O2–· generation rate of control, and ANG II-infused vessels are identical but O2–· levels are modulated by reaction with NO. Then inhibition of NO formation by L-NAME might normalize DHE oxidation rates in ANG II-treated vessels. In parallel measurements, the efficacy of L-NAME to block NO production was verified (Fig. 6C).



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  		Fig. 6. DHE oxidation by DVR in the presence of N{omega}-nitro-L-arginine methyl ester (L-NAME). A: ETH fluorescence was measured in DVR from control and ANG II-infused rats in the presence of L-NAME (100 µM). L-NAME increased the rate of rise of ETH fluorescence suggesting that NO reduces the rate of oxidation of DHE by cellular O2–· (*P < 0.05 vs. ANG II + L-NAME). With L-NAME, ETH fluorescence was nearly identical in vehicle and ANG II-infused rats, confirming that DVR O2–· production does not increase after chronic ANG II exposure (see Fig. 4A). B: ETH fluorescence was measured in control DVR in the presence and absence of L-NAME (100 µM). L-NAME tended to increase the rate of rise of ETH fluorescence (not significant). C: rate of increase of DAFFM fluorescence, indicating NO generation, is markedly reduced by L-NAME.

 
Tempol increases bioavailable NO. O2–· reduces bioavailable NO by reacting with it to form peroxynitrite (29). Thus a reduction of O2–· by chronic ANG II infusion might account for the increase in NO found in DVR from ANG II-infused rats (Fig. 3B). To test that hypothesis, intracellular O2–· was reduced by including the cell-permeant O2–· dismutase mimetic Tempol (1 mM) in the bath and measuring bioavailable NO by DAFFM fluorescence (Fig. 7). Experiments in Fig. 7 were abbreviated to 25 min of observation compared with 50 min in Fig. 3B. As expected, Tempol increased DAFFM fluorescence compared with controls (P < 0.05, time > 16 min). Chronic ANG II exposure further increased DAFFM fluorescence to levels that exceeded that of Tempol treatment alone (P < 0.05, time > 15 min). This result supports increased NO generation rather than reduction of O2–· generation as the mechanism that underlies the reduced DHE oxidation rate in DVR from ANG II-infused rats (Figs. 3A and 6A).



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  		Fig. 7. ANG II increases NO generation during O2–· dismutation. NO generation, indicated by DAFFM fluorescence, is shown in the presence of the cell-permeant SOD mimetic Tempol. Tempol enhanced DAFFM fluorescence of DVR from control rats, implying that O2–· has a basal effect to modify DVR NO. In agreement with the results shown in Fig. 4B, chronic ANG II exposure increased NO generation above that of DVR from control rats (P < 0.05 for time > 15 min) even when O2–· was reduced through Tempol-induced dismutation.

 
Ca2+ signaling and NO generation in ACh-stimulated DVR. The above results demonstrate that basal NO generation is increased in DVR from ANG II-infused rats (Figs. 3B and 7). We also tested whether the endothelial [Ca2+]cyt and NO responses to ACh stimulation are altered by ANG II infusion. Vessels were loaded with fura 2 to measure [Ca2+]cyt. We have shown that fura 2 loads predominantly into endothelia of intact DVR with little contaminating fluorescent signal from pericytes (22–24). Exposure to ACh (10 µM) via the bath induced a brisk rise in [Ca2+]cyt. The magnitude of the response was lower in vessels from ANG II-infused rats (P < 0.05, time = 1.5 to 8.2 min, Fig. 8A). The reduction of [Ca2+]cyt response was not associated with a failure of ACh to increase NO generation. ACh-stimulated NO generation, indicated by DAFFM fluorescence, was similar in DVR from control and ANG II-infused rats. ACh did increase the rate of NO generation compared with vessels that were not exposed to ACh (Fig. 8B).



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  		Fig. 8. Effect of ACh stimulation on cytosolic Ca2+ concentration ([Ca2+]cyt) signaling and NO generation. Endothelial [Ca2+]cyt response to ACh and rates of NO generation were examined in DVR from vehicle and ANG II-infused rats. [Ca2+]cyt responses from chronic ANG II vessels were less (P < 0.05, time = 1.5–8.2 min; A), but this was not associated with a diminished response of NO generation to ACh (B).

 
O2–· generation by rat tissue homogenates. The above data (Figs. 3A and 6A) show that chronic ANG II infusion does not increase the rate of DHE oxidation by isolated DVR. Using lucigenin chemiluminescence, we also tested whether overall O2–· generation is increased when NADPH is added to cortical or medullary tissue homogenates from ANG II and vehicle infused rats (Fig. 9). ANG II infusion tended to increase O2–· production in cortical homogenates (n = 12, P = 0.02) but not medullary homogenates (n = 6, P = 0.84).



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  		Fig. 9. Effect of ANG II infusion on superoxide generation by cortical and medullary tissue homogenates. Lucigenin chemiluminescence generated by adding NADPH to renal cortical and medullary tissue homogenates is shown for vehicle and ANG II-infused rats. Data were normalized by dividing individual values by the mean of the controls for the cortex (1,318 luminescence units, left) or medulla (9,115 luminescence units, right).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Accumulating evidence favors a role for renal synthesis of ROS in rodent models of hypertension. In some of these, ANG II may play a direct role to promote ROS generation. Blood pressure elevation in the spontaneously hypertensive rat (SHR) is associated with enhanced urinary excretion of 8-isoprostaglandin F2{alpha}, a marker of oxidative stress generated through nonenzymatic conversion of arachidonic acid. Both hypertension and 8-isoprostaglandin F2{alpha} excretion are blunted by treatment of the SHR with the SOD mimetic Tempol (30, 31). Infusion of L-NAME eliminates the antihypertensive effect of Tempol, implying that reduction of NO availability through reaction with O2–· partially underlies the genesis of SHR hypertension (30). Intrarenal oxygen tension and the efficiency of renal oxygen utilization are low in the SHR. A role for chronic ANG II ANG type 1 (AT1) receptor stimulation as a mediator of oxidative stress in the SHR is supported by the finding that oxygen utilization can be normalized by treatment of the SHR with the ANG II AT1 blocker, candesartan (40).

Evidence, obtained with the slow pressor model of ANG II hypertension, also favors a role for intrarenal oxidative stress to cause salt retention and hypertension. Kawada and colleagues (11) showed that ANG II-infused mice have hypertension that is ameliorated by Tempol. Both pressure natriuresis and renal blood flow autoregulation are impaired in ANG II-infused rats (39). Associated afferent arteriolar contractile responses are enhanced in that model (10, 37, 38). Similarly, chronic alteration of intrarenal responsiveness to ANG II has been implicated in two-kidney, one-clip Goldblatt hypertension (18). Chronic ANG II infusion leads to glomerular alterations and vascular remodeling in the rat (8, 14). An increase in urinary excretion of the oxidative stress marker 8-isoprostaglandin F2{alpha}, occurs in rabbits and mice made hypertensive by infusion of ANG II (11, 37, 38).

Many enzymes are capable of transferring electrons to oxygen to generate O2–·, but it is increasingly accepted that NADPH oxidase is the dominant source in smooth muscle (9, 12, 35). Smooth muscle cells express Nox1 and Nox4 isoforms of gp91phox, the electron transporting subunit of NADPH oxidase. Nox1 may be upregulated in vascular smooth muscle in response to chronic ANG II exposure (4, 12, 32). It has been demonstrated that ANG II infusion enhances NADPH oxidase expression in the renal cortex (2), but specific vascular and tubular effects of chronic ANG II on the regulation in the medulla have not been examined. In this study we sought to verify or refute the hypothesis that chronic exposure of rats to ANG II infusion would lead to production of ROS by DVR examined ex vivo. To test this, we infused ANG II into rats via osmotic minipump (250 ng·kg–1·min–1) for 11–13 days and isolated DVR from control and ANG II-infused animals. We examined contractility (Fig. 2), the ability to oxidize DHE (Figs. 3–6), and the capacity to synthesize NO in the basal state (Figs. 3, 6, and 7) and after ACh stimulation (Fig. 8). The principal finding is that ANG II infusion led to enhancement of basal NO production, but the capacity of DVR to oxidize DHE was unaltered. Failure of oxidative stress to be generated in the rat renal medulla by ANG II infusion is supported by the overall lack of enhancement of superoxide generation, gauged by lucigenin chemilumenescence (Fig. 9).

The enhancement of NO generation by DVR from chronically ANG II-infused rats was accompanied by a parallel reduction of contractile response to acute application of ANG II (Fig. 2). This finding contrasts with results from similar experiments that examined generalized vasoactivity (25) and the ability of ANG II to contract the renal afferent arteriole. Wang et al. (37, 38) infused rabbits with ANG II at 60 and 200 ng·kg–1·min–1. The latter dose led to enhanced expression of afferent arteriolar p22phox and increased the intensity of afferent constriction in response to acute ANG II. In the same study, a role for interaction of O2–· and NO was supported by the finding that Tempol augmented ACh-induced afferent dilation (37, 38). Studies by Imig (10), using the juxtamedullary nephron preparation in rats, demonstrated similar enhanced responsiveness of afferent arterioles to ANG II.

We speculate that the mechanism that underlies the tendency toward reduction of ETH fluorescence in DVR from chronically ANG II-infused animals (Fig. 3A) is consumption of O2–· through its reaction with NO. This is supported by the results shown in Fig. 6A, where L-NAME blocked reduction of ETH fluorescence in DVR from those animals. The fluorescent images in Fig. 4 are also consistent with that possibility because endothelia are the probable source of NO in isolated DVR, and ETH fluorescence appears lower in the endothelia from ANG II-infused animals.

The mechanism underlying the difference in the cortical afferent arteriolar and medullary DVR responses to chronic ANG II infusion is uncertain, but possibilities include reduced expression or modification of ANG II receptors, alteration of receptors that respond to vasodilators, and enhanced synthesis of compensatory vasodilators. In favor of the latter, we found that chronic ANG II infusion enhanced basal production of NO by DVR (Figs. 3B and 7). That result is consistent with the results of Zou and Cowley (44, 46) who measured the spectral shift of hemoglobin to detect NO in microdialysis samples from the renal medullary interstitium. They showed that infusion of ANG II (44, 46) and other constrictors (5, 45) cause a compensatory rise in interstitial NO concentration.

Actions resulting from ANG II AT2 receptor stimulation might also underlie the disparate effects of chronic ANG II infusion to augment contraction of afferent arterioles but not DVR. AT2 receptors mediate vasodilatory processes that augment renal medullary perfusion and might therefore hypothetically abrogate vasoconstriction via ROS generation (1, 34). We have identified several ANG II effects on DVR that are modified through AT2 stimulation. In a past study (26), Pallone and colleagues showed that AT2 blockade intensifies DVR vasoconstriction and modulates endothelial [Ca2+]cyt signaling responses to ACh and bradykinin. CGP42112A, an AT2 agonist, blocks vasoconstriction and enhances the [Ca2+]cyt response to ACh (26). Recently, we examined the ability of ANG II to increase DVR ROS generation when ANG II was acutely applied to isolated vessels via the bath. A significant increase in DHE oxidation was observed when AT1 receptors were stimulated by a combined exposure to ANG II and the AT2 receptor blocker PD-123319 (42). In contrast, combined AT1 and AT2 receptor activation by ANG II did not increase DHE oxidation, implying that AT2 receptor blockade is necessary for ANG II to increase ROS production. It also seems possible, that under the conditions of chronic ANG II exposure employed in the current study, activation of AT2 receptors prevented AT1-induced upregulation of the cellular machinery responsible for ROS generation. Such AT2-mediated effects have been observed in other circumstances. Chabrashvilli et al. (2) found that concomitant infusion of ANG II and the AT2 antagonist PD-123319 accentuated the increase in expression of Nox-1 and p22phox compared with ANG II alone. ANG II stimulation has been shown to increase effects of endothelin. Activation of endothelin B receptors might also be a compensatory response stimulated by chronic ANG II exposure (28).

Although we have not found an effect of chronic ANG II to enhance ROS generation in DVR, we cannot rule out an important role for ROS generation in chronic ANG II, slow pressor hypertension. Enhanced medullary ROS generation does occur in other hypertensive models. Zou and colleagues (16, 17) showed that intramedullary infusion of the SOD inhibitor diethylthiocarbamate reduces medullary blood flo, and raises arterial blood pressure. Conversely, infusion of the SOD mimetic Tempol increases medullary blood flow and sodium excretion, an effect that is enhanced when H2O2 is eliminated with catalase (3, 16, 45). A role for renal medullary generation of ROS was reinforced by Meng et al. (19) who showed that hypertension in Dahl salt-sensitive rats is accompanied by reduced medullary expression of Cu/Zn-SOD and Mn-SOD. It is possible during ANG II exposure that medullary ROS generation occurs in nephrons but not vasa recta. Epithelial ROS production could favor hypertension by reducing bioavailable NO, leading to inhibition of NaCl reabsorption from tubular fluid (6, 13, 19, 21). The finding that renal cortical but not medullary tissue homogenates from ANG II-treated rabbits show enhanced capacity for O2–· generation favors generalized resistance of the medulla to upregulation of ROS generation during ANG II hypertension (38). Extensive study of ROS and NO generation in both vascular and nephron segments derived from the cortex and medulla would be required to more clearly delineate that issue. A study over a range of ANG II infusion rates would be needed to resolve any dose-dependent effects.


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 ABSTRACT
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Studies in our laboratory have been supported by National Institutes of Health Grants R37-DK-42495, R01-DK-68492, R01-DK-67621, and P01-HL-68686.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Pallone, Div. of Nephrology, N3W143, 22 S. Greene St., Univ. of Maryland at Baltimore, Baltimore, MD 21201-1595 (E-mail: tpallone{at}medicine.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
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