Spontaneously hypertensive rats (SHRs) have normal glomerular capillary pressure even though renal perfusion pressure is higher, suggesting that preglomerular vessels exhibit abnormally high resistance. This may be due to increased superoxide (O2−) production, which contributes to the vasoconstriction in hypertension. We tested the hypothesis that the myogenic response of the afferent arteriole (Af-Art) is exaggerated in SHRs because of increased levels of reactive oxygen species (ROS). Single Af-Arts were microdissected from kidneys of SHRs and Wistar-Kyoto (WKY) rats and microperfused in vitro. When perfusion pressure in the Af-Art was increased stepwise from 60 to 140 mmHg, the luminal diameter decreased by 8.4 ± 2.9% in WKY Af-Arts but fell by 29.3 ± 5.6% in SHR Af-Arts. To test whether ROS production is enhanced during myogenic response in SHRs, we measured chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) florescence before and after increasing intraluminal pressure from 60 to 140 mmHg. Pressure-induced increases in ROS were fourfold greater in SHR Af-Arts compared with WKY Af-Arts (SHR, 48.0 ± 2.2%; and WKY, 12.2 ± 0.3%). To test whether O2− contributes to the myogenic response in SHRs, either the membrane-permeant O2− scavenger Tempol or the nox2-based NADPH oxidase (NOX2) inhibitor gp91ds-tat were added to the Af-Art lumen and bath and the myogenic response was tested before and after treatment. Both Tempol (10−4 M) and gp91ds-tat (10−5 M) significantly attenuated the pressure-induced constriction in SHR Af-Arts but not in WKY Af-Arts. We conclude that 1) pressure-induced constriction is exaggerated in SHR Af-Arts, 2) NOX2-derived O2− may contribute to the enhanced myogenic response, and 3) O2− exerts little influence on the myogenic response under normotensive conditions.
renal blood flow is highly regulated, with at least three mechanisms contributing to its fine tuning: myogenic response, tubuloglomerular feedback, and a third mechanism, possibly the recently described connecting tubule glomerular feedback (38, 42). In the myogenic response, the afferent arteriole (Af-Art) responds to changes in perfusion pressure per se with increased pressure causing constriction. Because of its very short activation delay, the myogenic response is particularly well suited for isolating the glomerular capillary pressure from the rapidly changing renal perfusion pressure (32).
Spontaneously hypertensive rats (SHRs), a model of genetic hypertension, exhibit remarkably little renal injury compared with other hypertensive models (35). In SHRs, the renal blood flow autoregulatory curve is “shifted to the right,” so that autoregulation is essentially preserved within its usual blood pressure ranges (23). By contrast, models with impaired autoregulation, such as 5/6 nephrectomized rats or Dahl salt-sensitive rats, have greater renal damage, as do stroke-prone SHRs when they are taken above their autoregulatory perfusion pressure range by salt supplementation (33). This evidence points to the autoregulation of renal blood flow playing a key role not only in regulating glomerular filtration and distal sodium delivery but also in protecting against renal injury (34). Thus SHRs are an ideal model to study renal autoregulation in hypertension. We have previously shown that isolated, perfused Af-Arts from SHRs display enhanced vasoconstriction in response to increased perfusion pressure compared with Wistar-Kyoto (WKY) rats (22); however, the mechanism underlying this phenomenon has not been elucidated.
Numerous studies in humans (18) and animals (3, 19) suggest that an increased production of superoxide (O2−) contributes significantly to the functional alterations of arteries in hypertension. Vascular O2− generation is increased in hypertensive rats chronically infused with angiotensin II (ANG II), and the O2− scavenger Tempol prevents ANG II-induced increases in peripheral vascular resistance and arterial pressure (53). Studies have also shown that the development of hypertension seen in SHRs is associated with a greater production of O2− in vascular tissues (50, 51). Therefore, oxidative stress clearly plays an important role in developing and maintaining high blood pressure. However, we know of no direct evidence that O2− production is induced by increased transmural pressure in hypertensive animals during myogenic response. In the present study, we hypothesized that in Af-Arts from SHRs, the myogenic response is exaggerated because of the activation of NADPH oxidase (NOX) and the generation of O2−. To test this hypothesis, we microdissected and perfused a single Af-Art from a rat kidney. This preparation is ideal to study the role of autacoids in the control of Af-Art tone because it allows us to control perfusion pressure in the Af-Art as well as to obtain real-time images in the absence of systemic hemodynamic, hormonal, or neural influences.
We used methods similar to those previously described to isolate the Af-Art with glomerulus intact (21, 37). Male SHRs and age-matched WKY and Sprague-Dawley rats (Charles River, Wilmington, MA) were used at 5 and 12 wk of age, corresponding to the prehypertensive and early hypertensive stages of SHRs. Both kidneys were removed under ketamine-xylazine anesthesia and sliced longitudinally along the corticomedullary axis. This protocol was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee and adhered to the American Physiological Society's “Guiding Principles in the Care and Use of Animals,” including the provision to comply with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The kidney slices were placed in ice-cold minimum essential medium (Gibco) containing 5% BSA (Intergen), and a single superficial Af-Art with glomerulus intact was microdissected under a stereomicroscope. With the use of a micropipette, the arteriole was transferred to a temperature-controlled chamber mounted on an inverted microscope (IMT-2; Olympus) with Hoffmann modulation and cannulated with an array of glass pipettes. Af-Arts were perfused from the proximal end in an orthograde direction. Intraluminal pressure was measured by Landis' technique using a fine pipette introduced into the arteriole through the perfusion pipette (29). Microdissection and cannulation were completed within 90 min at 8°C, after which the bath was gradually warmed to 37°C for the rest of the experiment. Once the temperature was stable, a 30-min equilibration period was allowed before any measurements were taken. Images of the Af-Art were displayed at magnifications up to ×1,980, recorded at 5-s intervals with a video camera, and measured with a computer equipped with Metavue image analysis system (MDS Analytical Technologies, Toronto, Canada). For the purpose of standardizing our measurements, each data point resulted from averaging three individual measurements taken at the site of maximum constriction and ±5 μm around it.
Response of the Af-Art to increased intraluminal pressure.
To examine the time course of the myogenic response in Af-Arts of 12-wk-old SHRs and WKY rats, we monitored the luminal diameter of Af-Arts for 5 min after raising intraluminal pressure in 20-mmHg increments from 60 to 140 mmHg. To examine whether the increased myogenic response seen in SHRs was secondary to hypertension, we also tested Af-Arts from 5-wk-old SHRs and WKY and Sprague-Dawley rats.
Direct measurement of reactive oxygen species production induced by increased luminal pressure.
To examine whether raising intraluminal pressure enhances reactive oxygen species (ROS) production more in SHRs than in WKY rats, we measured ROS production directly by using chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) fluorescence (47). Af-Arts were perfused at 60 mmHg, loaded with 5 μM CM-H2DCFDA for 20 min, and washed for 15 min at 37°C. They were then excited at 490 nm, and the emitted fluorescence was recorded at 540 nm. Fluorescence intensity was measured once every 10 s for 5 min before and 5 min after increasing intraluminal pressure to 140 mmHg. The rate of increase in fluorescence intensity was calculated for each period.
Effect of scavenging O2− on myogenic response.
To confirm that the myogenic response is reproducible, we obtained two consecutive pressure-response curves in Af-Arts from SHRs and WKY rats in the absence of any treatment. We found that myogenic response is reproducible in both strains. Subsequent experiments were performed in a similar fashion, with the first curve serving as the control. To examine whether O2− plays a role in myogenic response in SHR and WKY Af-Arts, we measured the Af-Art diameter before and after treating them with the O2− scavenger Tempol, (10−4 M) for 30 min, both in the lumen and bath.
Effect of gp91ds-tat, a selective inhibitor of the nox2-based NADPH oxidase, on myogenic response.
To test whether nox2-based NOX (NOX2)-derived O2− is necessary for the enhanced myogenic response in SHR Af-Arts, we used gp91ds-tat. Gp91ds-tat is a cell-permeant chimeric peptide that selectively interacts with p47phox, inhibiting NOX2 assembly and activity (8). We measured the Af-Art diameter before and after treating them with gp91ds-tat (10−5 M) for 30 min, both in the lumen and bath. In a separate control experiment, we used scrambled gp91ds-tat.
Myogenic response was defined as the percent change in Af-Art diameter when switching intraluminal pressure from 60 to 80, 100, 120, and 140 mmHg (baseline Af-Art diameters were similar between SHRs and WKY rats at both 5 and 12 wk of age). Experiments comparing curves obtained from different groups of animals were analyzed using repeated-measures ANOVA, followed by pairwise comparisons using the pooled variance from the ANOVA. Experiments comparing two curves obtained sequentially in the same preparation (i.e., control and treated) were analyzed using a paired t-test. Data are expressed as means ± SE. When multiple comparisons were performed, Hochberg's adjustment was used to determine significance.
Response of the Af-Art to Increase Intraluminal Pressure
As expected, Af-Arts exhibited constriction in response to increasing perfusion pressure. Diameters of WKY Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 18.1 ± 0.9, 18.2 ± 0.8, 17.8 ± 0.8, 16.7 ± 1.1, and 16.4 ± 0.9 μm, respectively (n = 10). Corresponding diameters in Af-Arts from SHRs were 15.9 ± 0.6, 16.3 ± 0.8, 15.2 ± 0.7, 12.6 ± 0.9, and 11.6 ± 0.9 μm (n = 9; Fig. 1). Thus the maximum decrease in diameter was 8.4 ± 2.9% in WKY Af-Arts and 29.3 ± 5.6% in SHR Af-Arts (P < 0.01, WKY vs. SHR).
To test whether this enhanced myogenic response of SHRs was due to hypertension, we studied Af-Arts from 5-wk-old WKY rats and age-matched prehypertensive SHRs and found that pressure-induced constriction was stronger in young SHRs. Diameters of WKY Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 13.3 ± 0.8, 13.4 ± 1, 12.7 ± 0.9, 11.7 ± 0.9, and 11.2 ± 1.1 μm, respectively (n = 5). Corresponding diameters in Af-Arts from SHRs were 12.8 ± 0.5, 13.4 ± 0.4, 12.4 ± 0.6, 9.2 ± 0.8, and 7.4 ± 0.6 μm (n = 8; Fig. 2). When the pressure was increased to 140 mmHg, the diameter decreased by 16.3 ± 3.4% in WKY Af-Arts but fell by 42.7 ± 3.6% in SHR Af-Arts (P < 0.001). This suggests that the exaggerated myogenic response observed in SHRs is not secondary to hypertension.
To eliminate the possibility that the observed difference between SHR and WKY Af-Art myogenic response was due to a specific defect in myogenic response in WKY rats, we tested Af-Arts from Sprague-Dawley rats. As shown in Fig. 2, the myogenic response curve in Sprague-Dawley rats was no different from that in WKY rats. Thus our data show that Af-Art myogenic response is enhanced in SHRs.
Direct Measurement of ROS Production Induced by Increased Luminal Pressure
To determine whether increasing intraluminal pressure enhances ROS production, we subjected isolated Af-Arts to increased luminal pressure and studied ROS production by measuring the rate of increase in CM-H2DCFDA fluorescence intensity (Fig. 3). We found that pressure-induced increases in ROS were fourfold greater in SHR Af-Arts (n = 5) compared with WKY Af-Arts (n = 4) when arteriolar pressure was increased from 60 to 140 mmHg. This shows that pressure-induced ROS production is enhanced in SHRs.
Effect of Scavenging O2− on Myogenic Response
To test whether O2− contributes to myogenic response in hypertension, we used the membrane-permeant O2− scavenger Tempol. Fig. 4A illustrates a representative experiment in an SHR Af-Art. As summarized in Fig. 4B, the diameters of SHR Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 16.9 ± 0.9, 17.2 ± 1.1, 17.2 ± 0.9, 13.6 ± 1.3, and 11.7 ± 1.6 μm, respectively, in the control period, whereas after the addition of Tempol (10−4 M), they were 16.9 ± 1.1, 17.2 ± 1.1, 17.0 ± 1.1, 16.3 ± 1.1, and 16.0 ± 1.2 μm (n = 8; the last two values bearing significance at P < 0.01, with vs. without Tempol). SHR Af-Arts significantly constricted when perfused at 120 and 140 mmHg, and this effect was blunted by scavenging O2− with Tempol, although Tempol did not alter the basal diameter. In contrast, adding Tempol to WKY Af-Arts had no effect on pressure-induced constriction (Fig. 5). The diameters of WKY Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 19.6 ± 1.3, 19.8 ± 1.3, 19.2 ± 1.6, 17.5 ± 1.7, and 16.9 ± 1.8 μm, respectively, in the control period, whereas after the addition of Tempol (10−4 M), they were 18.1 ± 1.7, 17.9 ± 1.7, 17.8 ± 1.8, 16.6 ± 2.1, and 15.8 ± 2.0 μm (n = 8). These data suggest that O2− is involved in the exaggerated myogenic response in SHRs but may not play an important role under normotensive conditions.
Effect of Inhibiting NOX2 on Myogenic Response
We next tested whether NOX2-derived O2− contributes to the exaggerated myogenic response in SHRs. Gp91ds-tat, a blocker of gp91phox-p47phox interactions, was used to selectively inhibit NOX2. In SHR Af-Arts, gp91ds-tat at 10−5 M did not alter basal diameter but significantly attenuated pressure-induced constriction. Diameters of SHR Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 18.4 ± 0.4, 18.4 ± 0.7, 17.5 ± 1.0, 12.2 ± 1.1, and 9.9 ± 0.9 μm, respectively, in the control period, whereas after the addition of gp91ds-tat (10−5 M), they were 17.6 ± 0.6, 18.0 ± 0.5, 17.0 ± 0.8, 15.1 ± 0.8, and 14.3 ± 1.0 μm (n = 5; the last two values bearing significance at P < 0.05 and P < 0.01, respectively, with vs. without gp91ds-tat; Fig. 6). However, the addition of scrambled gp91ds-tat to the Af-Art lumen and bath had no effect on pressure-induced constriction. Af-Art diameters were 15.7 ± 1.1, 17.2 ± 1.0, 14.9 ± 0.9, 12.4 ± 0.8, and 11.6 ± 0.9 μm in the control period and 16.2 ± 1.6, 15.7 ± 1.4, 13.5 ± 1.7, 11.1 ± 1.5, and 11.0 ± 2.0 μm after treatment with scrambled gp91ds-tat (n = 4). These data suggest that NOX2-derived O2− contributes to the enhanced myogenic response in SHR Af-Arts. In contrast, the addition of gp91ds-tat to WKY Af-Arts had no effect on pressure-induced constriction. Diameters of WKY Af-Arts perfused at 60, 80, 100, 120, and 140 mmHg were 18.8 ± 1.4, 19.1 ± 1.5, 17.9 ± 2.0, 17.3 ± 1.9, and 16.6 ± 1.7 μm, respectively, in the control period, whereas after the addition of gp91ds-tat (10−5 M), they were 19.3 ± 2.0, 19.6 ± 1.8, 19.2 ± 1.8, 17.5 ± 2.0, and 17 ± 1.5 μm (n = 3). These data suggest that NOX2-derived O2− does not play an important role in the myogenic response under normotensive conditions.
In the present study we found that isolated, perfused Af-Arts from SHRs displayed an enhanced myogenic response compared with those from age-matched WKY or Sprague-Dawley controls, confirming our previous observations with this preparation (22) and the results of Hayashi et al. (17) in in vitro perfused hydronephrotic kidneys. This increase was accompanied by heightened ROS production. Scavenging O2− with Tempol or blocking O2− production by inhibiting NOX2 activation attenuated the exaggerated myogenic response.
When the Af-Art of hypertensive versus normotensive rats was compared, there was some concern about the magnitude of the intravascular pressure that should be applied. For this reason, we compared the myogenic response at various pressures to the same perfusion pressure in both strains. Increased myogenic response has been previously reported in arterioles of the cremaster muscle of SHRs and in the renal interlobular arteries of stroke-prone SHRs (13, 17, 20). Because these observations were made after hypertension was already established, it was unclear whether the enhanced myogenic response was secondary to hypertension. We addressed this issue by studying the myogenic response in young SHRs, before hypertension is fully developed, and found a similar enhancement of the myogenic response, suggesting that such enhancement is not secondary to hypertension itself. However, young SHRs, commonly referred to as prehypertensive, actually have a slightly higher blood pressure than age-matched WKY rats as early as at birth (4).
It must also be noted that the enhanced myogenic response is not due to hypertrophy in SHR Af-Arts, since the media-to-lumen ratio of SHR Af-Arts is no different from that of WKY Af-Arts (44, 45). Furthermore, we observed that the enhancement of myogenic response in SHRs can be completely reversed acutely, pointing to a functional rather than structural difference between strains.
It could be argued that our findings reflect an impairment in the myogenic response in WKY rats, rather than an enhancement in SHRs. To exclude this possibility, we also included a Sprague-Dawley control group. Myogenic response was essentially identical in WKY and Sprague-Dawley rats.
Previous studies show that increased ROS production plays an important role in several types of hypertension, including genetic hypertension (49), renovascular hypertension (19), and angiotensin-induced hypertension (28), and contributes to functional changes in arteries in humans (18) and animals (10, 15). We first studied the production of ROS induced by raising intraluminal pressure in SHR and WKY Af-Arts. SHR Af-Arts displayed a fourfold greater increase in ROS production as measured by CM-H2DCFDA fluorescence, demonstrating that high-ROS generation is associated with the enhancement of the myogenic response in SHR Af-Arts.
H2DCF-DA is a commonly used fluorescent dye with good sensitivity for measurements of ROS. It enters the cell and once cleaved cannot escape, therefore measuring intracellular ROS. However, the use of this dye is not free of weaknesses, including that DCF itself can generate ROS, that the generation of the fluorescent DCF requires peroxidase activity, and most relevant, that it is not specific for a given ROS but reflects general oxidative stress (11). Several ROS can directly or indirectly cause H2DCF oxidation, including singlet oxygen, hydroxyl radical, O2−, and H2O2; hence, CM-H2DCFDA is a marker of generalized oxidative stress. Based on previous studies showing that the development of hypertension seen in SHRs is associated with a greater production of O2− in vascular tissues (50, 51), we chose to test the effect of scavenging O2− on the myogenic response in SHRs. Our results showed that scavenging O2− with Tempol inhibited the myogenic response in SHRs, suggesting that O2− mediates most of the myogenic response in this strain. Note that Tempol did not completely abolish the myogenic response; in fact, in the absence of myogenic response, the perfusion pressure would passively distend the Af-Art, causing an increase in its diameter (see dashed line in Fig. 1). This suggests that in SHRs, when perfusion pressure is high, O2− acts as a modulator which enhances the myogenic response mechanism and that scavenging O2− in SHR reveals an underlying myogenic response of similar magnitude to that of normotensive rats. In addition, because Tempol dismutates O2− into H2O2, it is possible that H2O2 contributed to the inhibition of the myogenic response. Depending on the concentrations employed and the blood vessel studied, H2O2 can act as a vasoconstrictor or a vasodilator (1). In the Af-Art, extracellularly applied H2O2 can block ANG II-induced increases in intracellular calcium (14). To further define the role of O2− in the myogenic response in SHR, we inhibited the synthesis of O2− with gp91ds-tat, a specific NOX2 inhibitor.
NOXs are the main source of O2− in vascular tissue (16). Three NOXs are expressed in the kidney cortex, NOX1, 2, and 4, but only NOX2 shows an increased expression in SHR kidneys compared with WKY controls (6). Recently, it was shown that NOX2 is necessary for the full vasoconstrictor effect of agonists such as ANG II and adenosine on the Af-Art (5). Our studies demonstrate that the enhanced myogenic response seen in SHR Af-Arts can be attenuated gp91ds-tat, suggesting that NOX2-derived O2− exacerbates the myogenic response in SHRs. Our results are consistent with those of Keller et al. (25) who showed that NOX2-derived O2− potentiates the myogenic response in resistance arteries isolated from the hamster gracilis muscle.
Our results are in agreement with studies showing that ROS may lead to enhanced renal vascular tone, increased sensitivity to vasoconstrictors, impaired endothelium-dependent vasodilation, and enhanced tubuloglomerular feedback (40). However, they are in disagreement with Sharma et al. (43) who found that an excess of TGF-β impairs Af-Art autoregulation through ROS production (43). Such disagreement may depend on the animal model or the stimulus for ROS production (i.e., TGF-β vs. increasing luminal pressure). It must be noted that very high O2− levels could be damaging to the vascular tissue, thereby impairing its function (2).
In our gp91ds-tat experiments in SHRs, there is some residual vasoconstriction not blocked by this peptidic inhibitor, as opposed to the complete blockade observed with Tempol treatment. There are three possible explanations for this discrepancy. First, there may be an additional source of O2−, not inhibited by gp91ds-tat. Of note, NOX1 and NOX4 are also expressed in vascular smooth muscle cells, and NOX1 has been shown to mediate ANG II-induced O2− production (30). Second, it is likely that the myogenic response is due to two or more vasoconstrictors, only one of which is O2−. Tempol scavenges O2− and in the process produces H2O2, which, acting as a vasodilator, may counteract the effect of the other vasoconstrictor(s), whereas gp91ds-tat decreases both O2− and H2O2. And third, the permeation of the cells by gp91ds-tat, a peptide, may be a limitation.
While the mechanism by which O2− enhances the myogenic response in SHRs remains unknown and even the exact mechanism of the myogenic response itself remains to be completely elucidated (9), several steps in the myogenic response may be sensitive to O2−. The myogenic response is initiated by mechanosensitive cation channels that cause sodium entry and depolarization (46). Mechanosensitive cation channels can be stimulated by O2− and peroxinitrite, at least in ventricular myocytes (12). Depolarization is followed by the opening of voltage-dependent, L-type calcium channels, and the enhanced activity of these channels has been proposed to mediate the increased myogenic response in SHRs (39). Of note, NOX-derived O2− has recently been shown to increase the open probability of L-type calcium channels, at least in cardiomyocytes (52). In addition, O2− could also act by activating protein kinase C (PKC) (27). PKC is known to potentiate the myogenic response either by facilitating the opening of mechanosensitive cation channels as shown in cerebral arteries (31) or by modulating the delayed rectifier potassium channels as shown in Af-Arts (26). Therefore, we speculate that O2− could enhance the myogenic response in SHRs by affecting PKC, mechanosensitive channels, or L-type calcium channels.
Unlike in the SHRs, O2− does not seem to participate in the myogenic response in the WKY rat, since neither Tempol nor gp91ds-tat inhibited the myogenic response in this normotensive strain. These results are in agreement with those of Ozawa et al. (36) who showed that the myogenic response was resistant to Tempol in hydronephrotic kidneys from Sprague-Dawley rats. Our results are also consistent with in vivo studies showing that Tempol reduces blood pressure and renal vascular resistance in SHRs but not in WKY rats (41, 48).
We conclude that myogenic response is enhanced in Af-Arts from both prehypertensive and hypertensive SHRs. This enhancement is accompanied by increased ROS production. Scavenging O2− or inhibiting NOX2 can normalize the myogenic response, suggesting that increased O2− production plays a role in the control of Af-Art vascular tone in hypertension. However, this role is likely complex. On the one hand, O2− and the consequent increase in Af-Art resistance can contribute to the development of hypertension. On the other hand, after hypertension is established, O2− could provide protection from glomerular barotrauma and injury by enhancing myogenic response and Af-Art autoregulation, at least in SHRs.
Our study may be relevant to patients with impaired renal blood flow autoregulation, such as those with diabetes and chronic kidney disease. These patients are at a high risk for barotrauma-induced renal injury when blood pressure values are even slightly elevated. In fact, current guidelines recognize this risk and recommend a lower blood pressure goal for such patients (7).
This work was supported by American Heart Association Grant D20607 and National Heart, Lung, and Blood Institute Grant HL-28982.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Present address of R. Liu: Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS.
Present address of P. J. Pagano: Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA.
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