Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation*

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT1 receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT1 and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT1 antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT2 antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT1 receptors and PKCα-dependent NADPH oxidase.

decreases renal blood flow by constricting renal vessels (4), reduces glomerular filtration rate by enhancing tubuloglomerular feedback (5) and also promotes salt reabsorption along the nephron (6,7). Excessive O 2 . generation within the kidneys contributes to the development of hypertension (8), renal damage (9,10), and atherosclerosis (11,12). Thus clarifying the mechanisms that regulate O 2 . production within the kidney may help us understand the etiology and pathophysiology of many diseases and develop new targets for treatment. O 2 . can be generated by several types of cells within the kidney (13,14); however, it is primarily produced by the medullary thick ascending limb of the loop of Henle (TAL) 2 (14). In the TAL, O 2 . production can be stimulated by several factors, including Ang II (15,16). Ang II can activate two types of receptors: AT 1 and AT 2 . Activation of AT 1 is associated with the salt-retaining and pro-hypertensive actions of Ang II (17,18). In the TAL, Ang II acutely stimulates O 2 . production (15), but neither the receptor nor the signaling cascade involved has been identified. O 2 . can be produced by NADPH oxidase, xanthine oxidase, and the mitochondria (19). In the absence of Ang II stimulation, NADPH oxidase appears to be the main source in the renal medulla (20), particularly the TAL (14,21,22); however, the source of O 2 . in the TAL during Ang II stimulation is still unknown. In many types of cells, including TAL cells, activation of protein kinase C (PKC) has been shown to stimulate O 2 . production in response to different stimuli, including Ang II (23,24,25). The PKC family of serine/threonine kinases is composed of many isoforms, some of which are expressed in the TAL, including PKC␣, -␤, -␦, -⑀, and - (26,27 the day of the experiment, animals were anesthetized with ketamine (100 mg/kg body weight, intraperitoneally) and xylazine (20 mg/kg body weight, intraperitoneally). All protocols were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee. Medullary TAL Suspensions-TAL suspensions were obtained from rats weighing 150 -220 g as described previously (28). This procedure yields a suspension of TALs that is Ͼ90% pure (29), so that contamination by other types of cells in our preparation was minimal or absent.
Measurement PKC Reporter-PKC activity was measured using a fluorescence resonance energy transfer (FRET)-based PKC reporter, CKAR (generously provided by Dr. Alexandra C. Newton, Howard Hughes Medical Institute, University of California-San Diego) (30). This probe consists of the consensus sequence for PKC phosphorylation with cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP) at either end. Under basal conditions CFP emission excites YFP due to its close proximity (FRET signal). Upon PKC activation, CKAR becomes phosphorylated and the probe "opens," resulting in an increased CFP signal and decreased FRET. Thus an increase in the CFP/YFP ratio indicates heightened PKC activity. The probe was subcloned into the adenoviral shuttle vector pVQ Ad5CMV K-NpA, which contains the CMV promoter, and sent to ViraQuest (North Liberty, IA) for viral production.
Dominant Negative PKC Isoforms-The HA (hemagglutinin)-tagged dominant negative PKC␣ and PKC␤1 plasmids (dn-PKC␣ and dn-PKC␤1) were kindly provided by Dr. Jae-Won Soh, Biomedical Research Center for Signal Transduction Networks, Incheon, Korea. The dominant negatives consist of kinase-dead PKC isoforms, generated by single amino acid substitution within the kinase domain. Sequences encoding for both proteins were subcloned into a shuttle vector containing the CMV promoter. Plasmids were sent to ViraQuest for adenoviral production.
In Vivo Gene Delivery of CKAR and dn-PKC Isoforms-TALs were transduced in vivo with recombinant replication-deficient adenoviruses expressing the dn-PKC␣, dn-PKC␤1, or CKAR sequence as we reported previously (31,32). Briefly, kidneys of a 95-to 105-g rat were exposed via a flank incision, and the renal artery and vein were clamped. Four 20-l virus injections (1 ϫ 10 12 particles/ml) were made along the longitudinal axis at a flow rate of 20 l/min. The renal vessels were unclamped; kidneys were returned to the abdominal cavity, the muscle incision was sutured, and the skin was clipped. Because we previously found that maximum expression occurred 3-5 days after injection of the adenovirus (32,33), all experiments were performed within these time points. Expression of the dominant negatives was confirmed by Western blots.
Expression of dn-PKC␣ and -␤-Western blots were performed as routinely done in our laboratory (28,29). Briefly, 40 g of TAL suspension homogenates was loaded onto an 8% polyacrylamide gel, and electrophoresis was performed for 2 h at 92 mV. After an overnight transfer, the polyvinylidene difluoride membrane was blocked in a buffer containing 20 mM Tris, 137 mM NaCl, 5% nonfat dried milk, and 0.1% Tween 20 (TBS-T) and 5% milk for 1 h at room temperature and then incubated with either a 1:1,000 dilution of a mouse monoclonal anti-HA antibody (Abgent, San Diego, CA), 1:1,000 dilution of a mouse anti-PKC␣ antibody (BD Biosciences, San Jose, CA) or a 1:250 dilution of a mouse anti-PKC␤ antibody (BD Biosciences) for 1 h at room temperature. The membrane was washed using TBS-T and incubated for another hour with a 1:1,000 dilution of the appropriate IgG conjugated to horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature. The reaction products were detected using a chemiluminescence kit (Amersham Biosciences) and by exposure to Fuji RX film.
Measurements of PKC Activity by FRET-On the day of the experiment, TAL suspensions were obtained from CKARtransduced kidneys as indicated above and 1/5 of the suspension was seeded in a temperature-controlled chamber and warmed to 37°C. The flow rate of the bath was 0.3 ml/min. During the 30-min equilibration period, images were acquired (100ϫ oil objective, numerical aperture: 1.3) by alternately exciting CFP (442 nm) and YFP (514 nm) and monitoring YFP emission at 540 nm to determine expression of the FRET sensor and highlight regions of interest. During the control period, CFP/YFP emission ratios were measured by exciting CFP at 442 nm once a minute for 5 min and simultaneously monitoring CFP and YFP emissions at 440 -480 (CFP) and 540 -545 nm (YFP). At the end of the control period, Ang II was added to the bath and CFP/YFP monitored once every minute for 15 min. The averages corresponding to the 5-min control period and the last 5 min of the experimental period were compared. To

Angiotensin Stimulates TAL O 2 .
21324 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 28 • JULY 9, 2010 confirm that the YFP signal was due to FRET, control experiments were performed by photobleaching CFP and measuring the decrease in YFP emission. Images were acquired using the same settings (laser intensity, detector gain and offset, resolution, and exposure time). Statistics-All statistical analyses were performed by the Biostatistics Department of Henry Ford Hospital. Results are expressed as mean Ϯ S.E. Data were analyzed using Student's t-tests. A version designed for unequal standard deviations was used when necessary. Some comparisons were studied using contrast statements. When multiple testing was involved, Hochberg's method was used.  (Fig. 2A). In a different set of experiments, apocynin alone significantly reduced basal O 2 . production by 80%

RESULTS
(p Ͻ 0.005; n ϭ 5). To make sure the effect of apocynin was due to specific inhibition of NADPH oxidase, we performed experiments using tubules isolated from p47 phoxϪ/Ϫ mice. In the presence of Ang II, O 2 . production was 1.45 Ϯ 0.12 nmol/ min/mg in wild-type controls but undetectable in TALs from p47 phoxϪ/Ϫ (0.00 Ϯ 0.32 nmol/min/mg) (p Ͻ 0.02; n ϭ 4 for each group) (Fig. 2B). These data indicate that NADPH oxidase is the primary source of O 2 . in the TAL under both basal and Ang II-stimulated conditions. To test whether Ang II directly enhances PKC activity in the rat TAL, we measured the effect of Ang II on PKC activity using FRET. In tubules expressing CKAR and incubated with vehicle (0.005% acetic acid) the CFP/YFP ratio was 0.02 Ϯ 0.03 arbitrary unit. Upon adding Ang II (1 nM) to the same tubules,  . production by rat thick ascending limbs. Rat thick ascending limbs were incubated with 1 nM angiotensin II for 10 min in the presence or absence of apocynin and superoxide production measured. n ϭ 5 per group. Bottom, effect of 1 nM angiotensin II for 10 min on O 2 . production by thick ascending limbs isolated from wild-type and p47 phox knock-out mice (p47 phoxϪ/Ϫ ). n ϭ 4. Ang II, angiotensin II.

JOURNAL OF BIOLOGICAL CHEMISTRY 21325
CFP/YFP increased to 0.13 Ϯ 0.02 arbitrary unit (p Ͻ 0.03; n ϭ 6) (Fig. 3). These data suggested that Ang II activates PKC activity in the TAL.
To determine whether activation of PKC is required for the stimulatory effect of Ang II on O 2 . production, we used a general PKC inhibitor, GF109203X. production by activating PKC␣ and/or PKC␤1.
To clarify the PKC isoform(s) involved, we transduced rat TALs in vivo so that they expressed either control DNA, dominant negative PKC␣ (dn-PKC␣) or dominant negative PKC␤1 (dn-PKC␤1). Expression of the dominant negatives was maximal 3-5 days after adenoviral injection as assessed by Western blots. The dominant negatives are HA-tagged, kinase-dead mutants generated by a single point mutation within the kinase domain. Thus, their expression can be monitored by the presence of HA and also by an increase in total PKC␣ or -␤ (because the antibodies used for Western blot also recognize the mutants). We found Ͼ500% increase of HA expression compared with the non-injected kidney (n ϭ 4 for dn-PKC␣ and n ϭ 3 for dn-PKC␤1). In addition, total PKC␣ increased by 250% in dn-PKC␣-injected versus non-injected kidney (p Ͻ 004; n ϭ 4) and PKC␤ by 293% dn-PKC␤1-injected versus noninjected kidney (p Ͻ 0.08; n ϭ 3). All experiments were performed 3-5 days after adenoviral transduction. In control rat TALs incubated with vehicle (0.005% acetic acid), O 2 . production was 1.42 Ϯ 0.12 nmol/min/mg, and with 1 nM Ang II it rose to 3.58 Ϯ 0.51 nmol/min/mg of protein (p Ͻ 0.011, n ϭ 5) (Fig.  6 (Fig. 6). Neither dn-PKC isoform had any effect on basal O 2 . (vehicle-treated suspensions; black bars in Fig. 6). These data indicated that PKC␣ mediates the stimulatory effect of Ang II on O 2 . production by TALs.

DISCUSSION
We hypothesized that Ang II acts on the AT 1 receptor to stimulate O 2 . production by the TAL, and that this process involves stimulation of PKC␣, which in turn activates NADPH oxidase. We found that: 1) Ang II stimulated rat TAL O 2 . production, and this process was halted by blocking AT 1 but not AT 2 ; 2) the NADPH oxidase inhibitor apocynin blocked the   . production by rat thick ascending limbs. n ϭ 6. . production was reduced in rat TALs expressing dn-PKC␣ but intact in tubules expressing dn-PKC␤1. We found that AT 1 mediated the stimulatory effect of Ang II on O 2 . production in the rat TAL but AT 2 did not, consistent with several studies conducted with other tissues. Fu et al. (33) recently reported that, in freshly isolated macula densa cells, Ang II stimulated O 2 . production, and this effect was blocked by the AT 1 antagonist losartan. Jaimes et al. (13) found that Ang II stimulated O 2 . production in cultured mesangial cells, and this effect was blocked by an AT 1 antagonist. Plumb et al. (35) reported that Ang II stimulated O 2 . production in human platelets, and this effect was blunted by an AT 1 receptor antagonist.
Although we recently reported that Ang II acts on AT 2 receptors to activate other signaling events in the TAL (34), in the present study the AT 2 antagonist PD123319 had no effect on Ang II-stimulated O 2 . production, suggesting that AT 2 receptors do not play a role in AT 1 -stimulated O 2 . production in the TAL. Thus it seems likely that activation of each receptor subtype leads to stimulation of independent signaling pathways. We also questioned whether NADPH oxidase is the source of O 2 . in Ang II-stimulated TALs. NADPH oxidase is an enzymatic complex that comprises five components: p40 phox , p47 phox , p67 phox , p22 phox , and NOX (35). Under basal conditions p40 phox , p47 phox and p67 phox are located in the cytosol as a complex. To make sure the effect of apocynin was specifically due to inhibition of NADPH oxidase, we tested TALs isolated from p47 phoxϪ/Ϫ mice and found that they had low basal levels of O 2 . , which were not stimulated by Ang II, indicating that: 1) p47 phox is required for Ang II-stimulated O 2 . production in TALs and 2) it maintains basal TAL levels of O 2 . . We were unable to uncover any compensatory mechanism that enables O 2 . to be generated under both basal and stimulated conditions in these mice. We recognize that the results obtained in mice cannot necessarily be extrapolated to rats. In fact, the degree of Ang II-stimulated O 2 . production was lower in mice compared with rats. However, the p47 phoxϪ/Ϫ mice were used as a tool to investigate the involvement of NADPH oxidase so that we did not rely only on pharmacological inhibition. These findings are consistent with data from Li et al. (14) showing that in unstimulated TALs NADPH oxidase is the major source of O 2 . production. In addition, a recent report from our laboratory demonstrated that luminal flow stimulated TAL O 2 . production via activation of NADPH oxidase (37 The PKC protein family is composed of at least eight members, of which five have been shown to be expressed in the TAL: PKC␣, -␤, -␦, -⑀, and - (26,27). To find out which isoform(s) might be involved in Ang II-induced O 2 . production, we used Gö6976, which inhibits both PKC␣ and -␤. We found that Gö6976 completely blocked Ang II-induced O 2 . production.
Because we know of no pharmacological inhibitor specific enough to target only PKC␣ or -␤1, we used adenoviral-mediated transduction of dn-PKC␣ or -␤. We found that Ang II stimulated O 2 . production both in controls and in dn-PKC␤1-  . production, although the exact signaling pathway remains unknown. The AT 1 receptors are coupled to G q and G i proteins. Activation of G q enhances diacylglycerol production and stimulates intracellular Ca 2ϩ (39,40), either of which is capable of activating the classic PKC isoforms ␣ and ␤ (41). In other cells, O 2 . stimulation by AT 1 activation has been attributed to increased diacylglycerol generation and subsequent activation of PKC. In addition, AT 1 -stimulated O 2 .
production is mediated by increases in intracellular Ca 2ϩ (42). Because PKC is stimulated by diacylglycerol, and both NADPH oxidase and PKC are sensitive to increases in intracellular Ca 2ϩ (42), both of these pathways could mediate AT 1 -dependent activation of PKC␣ in the TAL. In addition, Ang II has been reported to activate the small GTPase Rac (43), whose trafficking and translocation to the plasma membrane play an important role in activation of NADPH oxidase (44). In the TAL, Rac mediates NaCl-induced O 2 . production (45). Thus Rac could also participate in both Ang II-induced NADPH oxidase activation and O 2 . production in the TAL.
In this study we report that activation of PKC␣ is required for Ang II to stimulate O 2 . production in the TAL. However, we have shown previously that O 2 . activates PKC␣ in this segment (26). According to our data, PKC␣ also enhances O 2 . production via NADPH oxidase assembly with the p47 phox subunit. Therefore it is possible that Ang II initiates a cycle whereby small increases in Ang II increase O 2 . production, which in turn overstimulates PKC␣ and ultimately heightens oxidative stress. In summary, in TALs Ang II acts on the AT 1 receptor to activate PKC␣, which in turn stimulates NADPH oxidase and enhances O 2 . production. This could be an important regulatory mechanism whereby Ang II modulates O 2 . levels in the renal medulla under physiological conditions. In addition, defects in the Ang II/PKC/NADPH/O 2 . pathway in the TAL could play a role in the development of hypertension, renal damage, and atherosclerosis.