Bosentan Prevents Preglomerular Alterations During Angiotensin II Hypertension
Abstract The present study was performed to characterize structurofunctional alterations of preglomerular vessels during chronic angiotensin II (Ang II)–induced hypertension (Ang II group: 400 ng · kg−1 · min−1, 10 days) and to assess the role of endothelin-1 in rats receiving Ang II and the mixed receptor antagonist bosentan (Ang II+B group: 30 mg · kg−1 · d−1, 10 days). Systolic blood pressure rose by 56±3 and 54±6 mm Hg in Ang II and Ang II+B rats, respectively. Albuminuria increased similarly in both Ang II–treated groups, reflecting glomerular barrier dysfunction. Preglomerular vessels were isolated after HCl maceration and comprised arcuate arteries and their branches, interlobular arteries (ILA), and afferent arterioles (AA). In the Ang II group, focal vascular lesions affected 36±6%, 20±5%, and 4±1% of arcuate arterial branches, ILA, and AA, respectively. They were characterized by 74% increased media thickness and accumulation of Sudan black–positive (SB+) lipid droplets, and media cell proliferation was documented through immunohistochemistry. The occurrence of SB+ lesions was strikingly reduced with bosentan. Autoregulatory responses (AR) were assessed along ILA and AA with the use of blood-perfused juxtamedullary nephron preparations. AR were elicited by raising blood perfusion pressure from 60 to 160 mm Hg and quantified through videomicroscopy as pressure-induced constrictions. AR were inhibited in Ang II–treated rats along ILA and AA; Ang II–induced AR changes were prevented by bosentan. Maximal relaxation induced by Mn2+ revealed equal basal tone in Ang II–treated, Ang II+B–treated, and control vessels. Chronic Ang II–induced hypertension is therefore associated with the development of SB+ lesions and selective impairment of AR in juxtamedullary nephrons. Endothelin-1 likely mediates the structurofunctional alterations of preglomerular vasculature during Ang II hypertension.
The cardiovascular system undergoes major adaptations and alterations during the course of systemic hypertension produced by Ang II administration (for a recent review, see 1 . Although kidneys play a key role in the control of blood pressure2 and the development of other Ang II–dependent hypertensive models,3,4 few studies have focused on the structurofunctional alterations of renal vasculature during chronic Ang II–induced hypertension.
An array of glomerular and vascular injuries, classically associated with hypertension, was reported by Zou et al5 in uninephrectomized rats receiving low-dose (200 ng · kg−1 · min−1) Ang II infusions for 13 days. In rats treated during the same period of time with twofold higher doses of Ang II, Johnson et al6 carried out detailed immunohistochemical characterizations and found focal inflammatoproliferative vascular lesions, segmental glomerular hyalinosis with upregulation of glomerular α-SM actin, and interstitial injury. In L-NAME hypertensive rats, with the use of vasculatures isolated through a new maceration-dissection technique,7,8 we recently found focal lesions along ArcB and ILA. These lesions were characterized by proliferation of media cells, macrophage invasion, immunoreactive vascular cell adhesion molecule-1, and low-density lipoprotein.8 A salient feature of these lesions was the accumulation within the hypertrophied vessel wall of SB+ lipid droplets.8 These characteristics were suggestive of an early atherosclerotic process.9 Furthermore, development of vascular SB+ lesions could be prevented selectively by the mixed ETA/ETB receptor antagonist bosentan.8 To our knowledge, the occurrence and ET-1 dependency of preglomerular SB+ lesions have not been assessed in Ang II hypertensive rats, although Ang II promotes vascular lipid deposition10 and vascular ET production.11
In functional terms, pressure-induced vasomotor responses of preglomerular vessels constitute the effector limb of renal autoregulation.12–15 The latter plays a key functional role because it maintains glomerular filtration and protects glomeruli against barotrauma and sclerosis.16–18 Impairment of AR was repeatedly documented at whole-kidney or single arteriolar levels in several hypertensive rat models.3,16,19–22 AR were recently explored in uninephrectomized, Ang II–treated rats23 but remain unknown in intact, Ang II–treated animals.
The present study was undertaken in rats receiving Ang II for 10 days with the following aims. First, we wanted to document the existence and assess the segmental distribution of preglomerular SB+ lesions. This was achieved through microscopic examination of large, intact segments of the preglomerular vasculature isolated with the use of our maceration-dissection technique.7,8 Second, to assess autoregulatory pressure-induced vasomotor responses along the preglomerular vasculature in normotensive and Ang II hypertensive rats, we used our in vitro blood-perfused JMN preparation and videomicroscopy.13,14,24 This preparation permits direct visualization of the successive segments of the preglomerular vasculature while preserving tubulovascular relationships.24 Intrinsic renal autoregulatory mechanisms, including myogenic responses and TGF mechanism, are effective in vitro, and preglomerular vessels develop strong basal tone without the addition of exogenous constrictors.13,14,21,22,24,25 Third, we wanted to assess the influence of treatment with bosentan on the renal structurofunctional alterations induced by Ang II hypertension.
The present experiments in animals complied with French law and the guiding principles for experimental procedures as set forth in the Declaration of Helsinki. Studies were conducted in adult (weight, 230 to 350 g) male Sprague-Dawley rats (Iffa Credo) that were allowed free access to standard rat chow and tap water. SBP was assessed with tail-cuff manometry (Narco BioSystems). One group (Ang II; n=10) received a continuous Ang II infusion (400 ng · kg−1 · min−1) for 10 days via subcutaneous osmotic minipumps (Alzet model 2002, Alza Corp) containing Ang II (Sigma Chemical) dissolved in distilled water. One group (Ang II+B; n=10) received simultaneous Ang II (400 ng · kg−1 · min−1) via minipumps and bosentan (30 mg · kg−1 · d−1; Hoffman-LaRoche) via gavage for 10 days. In previous studies, this treatment inhibited peak pressor responses to exogenous ET-1.8 In Ang II and Ang II+B groups, SBP was measured before and at the end of treatment. Control animals were body weight–matched rats (n=10) and SBP was measured only on the day they were killed. Control rats were not subjected to sham-gavage. Pilot studies showed that gavage per se had no impact on either SBP or albuminuria over a period of 10 days.
Albuminuria was taken as an index of glomerular injury26,27 and was determined on 24-hour urine collections through the use of laser immunonephelometry, as described previously.8 Measurements were performed before and after Ang II and Ang II+B treatment and on the day of experiment only in control rats.
Isolation of Preglomerular Vasculature
Preglomerular vasculatures were separated from tubules after HCl maceration, as described recently.7,8 Isolated vasculatures comprised the first divisions of the renal artery into mArcA, lateral or terminal ArcB, ILA, and AA. Vessels were permeabilized with 1% Triton X-100 (Sigma) and immersion-fixed in 10% buffered formalin (Accustain; Sigma). Vessels were rinsed in distilled water and stained with Sudan black B (Sigma) as previously described8 or processed for immunohistochemistry as indicated below.
Quantification of Sudanophilic Lesions
Portions of Sudan black–stained vasculatures were observed under a stereoscope or compound microscope (Laborlux D-FS; Ernst Leitz) equipped with a 25× long-working distance objective (NPL Fluotar L 25/0.35; Ernst Leitz). To avoid dimensional distortion of vascular samples, fragments of coverslips were used as spacers between slides and coverslips. Sudanophilic (SB+) lesions were quantified along ArcB, ILA, and AA. For a given type of preglomerular vessel, relative frequency of damaged vessels was calculated as the number of vascular segments with at least one SB+ lesion divided by the total number of observed vascular segments. Using a videomicroscopic system,13,14 which gave a final magnification of 1350× on the video screen, we assessed vessel wall thickness (precision of ±0.5 μm) along SB+ lesions and adjacent “control” segments. Injured wall thickness was expressed as percent of nearby control value. Values of >100% denoted relative hypertrophy of vessel wall.
As described previously,8 detection of cell proliferation was performed on isolated vessels using the avidin-biotin immunoperoxidase method and PC10 (1:1000; DAKO A/S), a mouse monoclonal primary antibody directed against PCNA/cyclin.28
Videometric Assessment of Renal Microvascular Function
Experiments were carried out using in vitro blood-perfused JMN preparations.24 Microsurgery, perfusion procedures, and videomicroscopic techniques have been previously described in detail.13,14,24 Kidneys were obtained from control, Ang II–treated, and Ang II+B–treated rats. For each kidney donor, two older male rats (weight, 500 to 600 g) of the same strain were used as blood donors. All animals were anesthetized with pentobarbital sodium (50 mg/kg IP). The right kidney of the donor was processed through the HCl maceration procedure. Renal preparations (left kidney) were placed on the stage of the compound microscope, epi-illuminated with a fiberoptic light pipe, and continuously superfused with warm (37°C) KBR buffer, pH 7.4, containing 1% dialyzed bovine serum albumin (Fraction V; Sigma) and 5 mmol/L HEPES buffer. Preparations were perfused with an isotonic KBR buffer containing 6% dialyzed bovine serum albumin, HEPES buffer, amino acids,13,24 and washed erythrocytes (hematocrit of ≈30%), which was placed in a glass vial that was pressurized and oxygenated with a 93% O2/7% CO2 gas mixture. The nonpulsatile PP was continuously measured at the tip of the perfusion cannula in the distal renal artery; it could be adjusted stepwise by changing the gas pressure inside the blood vial. Preparations were left to stabilize for 30 minutes at a PP of 60 mm Hg.
Images of superficial vessels were displayed, printed, and recorded with the video system previously mentioned in the text, allowing measurements within ±0.5 μm. Vascular sites were identified along superficial ILA and superficial AA. These vessels were selected because they represent the major effector sites of autoregulation.13–15,25 Criteria for site selection were (1) clear optical definition of vessel walls and (2) the presence of an anatomic landmark that allowed repeated, unambiguous identification of the site. Measurement sites were also selected along the JAA segment (10 to 70 μm from glomerulus), its midportion (MAA150, 150 to 170 μm from glomerulus; MAA300, 300 to 320 μm from glomerulus), and EAA (10 to 100 μm from branching site). Due to accumulation of collagen strands in the juxtaglomerular area, diameter measurements were less frequently possible along JAA than along EAA. The former vascular site was previously shown to be a major effector of glomerular autoregulation and TGF mechanism.13,14 After equilibration, pressure-diameter curves were generated by imposing PP steps of 20 mm Hg from 60 to 160 mm Hg. Luminal diameters were measured at steady state (ie, 3 to 5 min after PP change). All selected vascular sites were followed throughout the experiment, yielding paired diameter values. Luminal diameters were normalized to their respective initial baseline value at 60 mm Hg. During previous experiments,13,14,22 we found that vascular diameters are maximal at a PP of 60 mm Hg, at which TGF and myogenic mechanisms are relaxed. After completion of pressure-diameter curves, PP was brought back to 60 mm Hg. We rejected vascular sites with a diameter that did not return to within 20% of the value previously obtained at 60 mm Hg during the autoregulation period. Keeping PP at 60 mm Hg, vessels were maximally relaxed by topical application of the nonspecific Ca2+ blocker Mn2+ (10 mmol/L MnCl2; Sigma).29 Basal vascular tone was quantified as the fractional dilation induced by Mn2+ at a PP of 60 mm Hg. Passive wall thickness–to–lumen diameter ratios were calculated by playback of videotape recordings as [(Do−Di)/Di]×100, where Do and Di are the outside and inside (luminal) diameters, respectively. Media thickness was calculated as (Do−Di)/2. Measurements were made only when outside diameters could be clearly delineated. Under these “passive” conditions, we considered differences in wall-to-lumen ratios to reflect structural differences rather than differences in vascular tone.
Between-group comparisons were performed with analysis of variance. Between-group comparisons of pressure-diameter curves were carried out using two-factor (ie, group and PP) analyses of variance. When significant interaction was found, pairwise comparisons were performed by one-way variance analysis, followed by Fisher’s protected least significant difference test. Where appropriate, within-group analyses were carried out with paired Student’s t test, or variance analysis for repeated measures, followed by Fisher’s protected least significant difference test for pairwise multiple comparisons. In all analyses, a value of P<.05 was considered to be significant. Values are given as mean±SEM.
Systemic Blood Pressure and Body Weight
Baseline mean SBP was similar in control, Ang II–treated, and Ang II+B–treated groups and averaged 126±1 (n=10), 121±2 (n=10), and 126±2 (n=10) mm Hg, respectively. Ang II treatment significantly increased SBP to a mean value of 177±4 (n=10) and 181±7 (n=10) mm Hg in Ang II and Ang II+B groups, respectively. Of importance, bosentan did not prevent Ang II–induced hypertension. Mean body weights were not different in control, Ang II–treated, and Ang II+B–treated groups and averaged 310±11 (n=10), 292±13 (n=10), and 304±13 (n=10) g, respectively.
Baseline albumin excretion was similar in control, Ang II–treated, and Ang II+B–treated groups and averaged 111±17 (n=8), 170±42 (n=6), and 202±52 (n=6) μg/24 h, respectively. This parameter significantly and strikingly increased 50- to 150-fold after 10 days of Ang II treatment, indicating alteration of glomerular barrier function. Due to its variability in Ang II–treated rats, final albumin excretion was not significantly different in Ang II and Ang II+B groups and averaged 26.4±10.8 (n=6) and 11.1±4.8 (n=6) mg/24 h, respectively.
Characteristics of Vascular Lesions
Fig 1A⇓ illustrates light microscopic appearance of preglomerular vascular lesions encountered in the Ang II–treated group after staining with Sudan black. As was the case in our previous studies with isolated preglomerular vasculatures,8 focal vascular lesions could be easily detected before staining because lipid deposits are highly light-scattering under low-angle epi-illumination. Lesions were never found at the level of main ArcA but were present along ArcB, ILA, and AA. Focal sudanophilia was always associated with hypertrophy of the vessel wall; more precisely, wall thickness was increased by 74±5% relative to adjacent control segments in 36 SB+ lesions pooled from three Ang II–treated rats. Moreover, wall thickening was always associated with proliferation of media cells as revealed by positive staining for PCNA/cyclin (see Fig 3B⇓ and 3C⇓). Scattered, PCNA-positive nuclei (ie, SM cells and, less frequently, endothelial cells) were also found along vascular segments devoid of lesions. SB+ lesions had similar characteristics in the Ang II+B group but appeared with lesser frequency.
Quantification of Vascular Lesions
Relative frequencies of SB+ vessels were assessed in 9 Ang II–treated and 9 Ang II+B–treated rats. None of the control rats had SB+ vessels. The total number of ArcB, ILA, and AA observed per rat (n=18) averaged 27±2, 129±10, and 227±14, respectively. As shown in Fig 2⇓, frequency of SB+ vessels declined from 36.3±5.8% (n=9) to 3.7±1.2% (n=9) from ArcB to AA, respectively, in Ang II rats. Treatment with bosentan significantly prevented the development of SB+ lesions along ArcB, ILA, and AA (Fig 2⇓).
Videometric Assessment of Renal Microvascular Function
Renal microvascular function was explored in 8 control, 8 Ang II–treated, and 5 Ang II+B–treated rats. These animals had similar mean body weights of 305±11, 282±13, and 327±20 g, respectively. As shown in Table 1⇓, baseline luminal diameters tended to be larger in Ang II–treated groups; however, this trend reached statistical significance in ILA only (Table 1⇓). Of importance, no lesions could be detected by focal wall thickening along any of the explored preglomerular vessels.
Pressure-diameter curves of the various segments of the preglomerular vasculature in control and Ang II–treated rats are given in Fig 3A⇓ through 3D. Vasoconstrictions elicited by raising PP from 60 to 160 mm Hg averaged 4.9±0.6 (JAA, n=16), 6.0±1.1 (MAA150, n=22), 5.1±1.6 (MAA300, n=11), 6.8±0.9 (EAA, n=29), and 9.7±1.1 (ILA, n=21) μm. Pressure-induced responses were significantly blunted in the Ang II–treated group compared with the control group, and no significant PP-induced constrictions were observed along JAA, EAA, or ILA (Fig 3A⇓, 3C⇓, and 3D⇓). In contrast, pressure-diameter curves obtained in MAA150 were not significantly different in control and Ang II–treated groups (Fig 3B⇓). A similar pattern of pressure-induced responses was obtained along MAA300 (data not shown). Along MAA300, increasing PP from 60 to 160 mm Hg induced similar mean fractional constrictions of 18.4±6.6% (n=11), 16.0±6.9% (n=11), and 22.6±2.4% (n=8) in control, Ang II–treated, and Ang II+B–treated groups, respectively. Bosentan prevented attenuation of preglomerular pressure-responsiveness; similar pressure-diameter curves were obtained at all sites in control and Ang II+B–treated groups (Fig 3A⇓ through 3D).
As shown in Fig 4⇓, the ability of preglomerular vessels to generate basal tone was not affected by Ang II treatment, as reflected by similar Mn2+-induced relative dilation. Even higher basal tone was found at the level of EAA in Ang II–treated compared with control rats (Fig 4⇓). Likewise, MAA300 sites developed similar basal tone of 18.6±5.6% (n=11), 16.4±3.0% (n=11), and 20.4±4.1% (n=8) in control, Ang II–treated, and Ang II+B–treated groups, respectively. Therefore, attenuation of pressure-induced AR in AA and ILA (Ang II group; Fig 3A⇑, 3C⇑, and 3D⇑) did not result from a nonselective impairment of vascular contractility.
Segmental values for mean media thickness are given in Table 2⇓. In all groups, media thickness increased with vessel caliber (ie, from JAA to ILA). Wall thickness was increased in Ang II–treated animals relative to control animals; this is consistent with our previously mentioned observation of scattered PCNA-positive nuclei. Ang II– and Ang II+B–treated groups had similar wall thicknesses (Table 2⇓). Table 2⇓ also provides a summary of segmental values of wall thickness–to–luminal diameter ratios obtained in control and Ang II–treated groups. In all groups, ratios decreased with increased vessel caliber (ie, from JAA to ILA). Changes in media thickness were offset by concomitant differences in passive lumen diameters, and no between-group differences in wall-to-lumen ratios were detected, with the exception of MAA150 sites of Ang II–treated rats, which had larger ratios than those of control animals. These results suggest an absence of major structural alterations in Ang II–treated vessels compared with control vessels.
The present study documents, for the first time, that sudanophilia is a hallmark of the focal and proliferative vascular lesions that develop along preglomerular vessels during chronic Ang II–induced hypertension. These SB+ lesions mainly affected ArcB and, with decreasing frequency, ILA and AA. Our present study relied on the microscopic observation of preglomerular vasculatures isolated through a HCl maceration technique,7,8 which preserved vascular lipid deposits, allowed their easy recognition after Sudan black staining, and permitted determination of their segmental distribution. The fact that proliferation of media cells and sudanophilia characterized vascular lesions during Ang II–induced hypertension extends our previous observations in L-NAME hypertensive rats,8 in which preglomerular SB+ lesions developed within a similar time frame. In contrast, SB+ lesions were never observed along AA in the L-NAME model,8 suggesting that segmental distribution of lesions is model specific. In rats receiving 400 ng · kg−1 · min−1 of Ang II for 2 weeks (ie, in a hypertensive model similar to the presently used one), Johnson et al6 documented media cell proliferation and macrophage accumulation in preglomerular arteries and arterioles. Interestingly, accumulation of vascular lipids was not reported in that study,6 as they may have been extracted by organic solvents during paraffin histology. Nevertheless, the latter study6 and ours concur to indicate that in Ang II hypertensive rats, preglomerular lesions possess key characteristics of an early atherosclerotic process.9
In the present study, chronic Ang II–induced hypertension was associated with a 100-fold increase in mean albumin excretion. This result likely reflects the occurrence of glomerular injury during Ang II–induced hypertension. In fact, at a dose similar to the presently used one (ie, 350 ng · kg−1 · min−1), Ang II was shown to impair glomerular permselectivity within 30 minutes in anesthetized rats.27 This effect occurred in the presence of increased systemic and glomerular capillary pressures and decreased glomerular blood flow.27 Lapinski et al26 confirmed such rapid and deleterious effect of Ang II on glomerular barrier function using isolated perfused rat kidneys. In addition, they demonstrated direct deleterious Ang II effects26 because they occurred when renal PP was held constant and could be prevented by an Ang II type I receptor antagonist. On a chronic basis, Johnson et al6 demonstrated dramatic upregulation of intraglomerular α-SM actin after 1 week of Ang II infusion. This observation6 is in keeping with the present one because α-SM actin expression is a good index of glomerular injury and sclerosis.30 Along the same lines, we previously noted good parallelism between albuminuria and glomerular expression of α-SM actin in L-NAME hypertensive rats.8
One important finding of the present study is that bosentan, a mixed ETA/ETB receptor blocker, at a dose that did not affect the final hypertensive effect of 10 days of Ang II treatment prevented development of SB+ lesions along ArcB, ILA, and AA. Therefore, our results suggest that ET-1 specifically mediated the development of SB+ lesions during chronic Ang II hypertension. However, because blood pressure was not continuously monitored, our present study does not allow us to rule out possible differences in terms of amplitude or frequencies in blood pressure fluctuations between Ang II– and Ang II+B–treated rats that may have influenced the development of SB+ lesions. Nevertheless, the present findings extends our previous observations in L-NAME hypertensive rats.8 Consistent with the atherosclerosis-like nature of SB+ lesions, it was recently shown that ET promotes the early inflammatory phase of atherosclerosis in hamsters that have been fed cholesterol.31 The lack of pressure-lowering effect of the presently used dose of bosentan permitted, within the previously stated limits, the avoidance of confounding systemic influences but does not allow us to reach a conclusion regarding the involvement or lack of involvement of ET-1 in Ang II–induced hypertension. In fact, we could prevent the hypertensive effects of a lower Ang II dosage (200 ng · kg−1 · min−1, 10 days)32 by using the same dose of bosentan. Similar pressure-lowering effect was obtained by d’Uscio et al33 using a specific ETA receptor antagonist, thus suggesting that hypertensive Ang II effects involved activation of ETA receptors. Both ET-1 and Ang II were shown to be progression growth factors in cultured vascular SM cells34 but to require competence growth factors such as platelet-derived growth factor to exert their mitogenic effects. In this contention, it is interesting to note that platelet-derived growth factor B-chain mRNA was indeed reported to increase in the areas of vascular injury during Ang II hypertension.6
In the present study, the beneficial vascular effects of bosentan were not paralleled by improvement of albuminuria, suggesting maintained glomerular injury that may reflect direct, deleterious effects of Ang II.26 Similarly disparate vascular and glomerular responses to bosentan treatment were previously noted in L-NAME hypertensive rats.8 Although we cannot exclude that the presently used dose of bosentan was not sufficient to prevent glomerular alteration, our results indicate that vascular and glomerular injuries occurring during Ang II hypertension are controlled by different cell mechanisms.
Use of the JMN preparation and videomicroscopy13,14,24 allowed us to assess pressure-induced AR along ILA and AA, the major effectors of autoregulation.25 One incentive to explore AR was that they buffer the effects of steady changes in blood pressure and filter those of blood pressure fluctuations in the low-frequency range (ie, ≈40 to 200 mHz)35 and their impairment promotes glomerular injury/sclerosis.16–18 A second incentive was to seek a functional correlate to the pathological processes leading to focal SB+ lesions.
Consistent with previous studies performed with blood-perfused JMN preparations,13–15,22 control preglomerular vessels exhibited significant pressure-induced constrictions, with the highest responses obtained in JAA. AR were impaired in Ang II–treated compared with control rats at the level of JAA, EAA, and ILA. In contrast, pressure responsiveness of MAA sites was well preserved after 10 days of Ang II treatment. Such a differential pattern of responses cannot be attributed to intergroup differences in baseline diameters or to differences in vascular structure because passive wall thickness–to–lumen diameter ratios were unaffected by Ang II treatment. Furthermore, segmental loss of pressure responsiveness was not due to nonspecific impairment of SM cell contractility because all vascular sites of Ang II–treated rats developed a similar, if not higher, basal tone compared with controls. Therefore, our results demonstrate specific and segment-dependent impairment of preglomerular pressure responsiveness in Ang II–treated rats with preserved SM cell contractility. The maintained contractility currently observed in vitro is consistent with the increases in renal vascular resistances observed during chronic Ang II treatment in vivo,1,36 where nervous and systemic constrictor influences are fully expressed.1,23,37
The current functional findings are not unique to the present model of hypertension in that impairment of AR was found at whole or zonal kidney and single arteriolar levels in various other hypertensive rat models associated with glomerular injury.3,16,19–22 More specifically, with the use of blood-perfused JMN preparations, diminished pressure-responsiveness with maintained basal tone was found in the nonclipped kidney of two-kidney, one clip hypertensive rats21 and in L-NAME hypertensive rats.22 In apparent contrast to the present findings, Ichihara et al23 reported maintained pressure responsiveness of AA using blood-perfused JMN preparations from uninephrectomized, Ang II–treated rats (200 ng · kg−1 · min−1, 13 days), a model associated with significant vascular and glomerular alterations.5 However, an examination of rat body weights and baseline AA diameters given in the former study23 suggests that diameter measurements were performed at sites presently defined as MAA, along which pressure responsiveness was currently preserved.
Our present results demonstrate impairment of AR at the level of JAA and ILA. Because the AR of these vascular segments mainly reflect the activity of TGF and myogenic mechanisms, respectively,12,14 both mechanisms were therefore impaired by chronic Ang II treatment. To our knowledge, no data are available regarding whole-kidney autoregulation during chronic Ang II hypertension. In the nonclipped kidney of two-kidney, one clip hypertensive rats, impaired autoregulation was found at whole-kidney level3 and in blood-perfused JMN preparation.21 However, because we cannot exclude the fact that Ang II may differentially affect the various nephron populations, we cannot extrapolate the present findings to the entire nephron population. In any case, should the current functional impairment be limited to the JMN population, it would still affect medullary function and sodium and water handling and may contribute to the salt sensitivity of Ang II hypertension.38
An important mechanistic finding of the present study was that bosentan prevented both impairment of vascular pressure responsiveness and development of SB+ lesions. Our study therefore suggests that ET-1 is a common mediator for both structural and functional alterations of preglomerular vessels during Ang II hypertension. No lesions were present in the set of functionally studied vessels, and we did not assess whether functional events preceded lesion formation. Therefore, a causal relationship between autoregulation and lesion formation cannot be inferred from the present study. Nevertheless, our results are in keeping with the concept that vascular ET-1 expression is a crucial determinant of vascular pathology during hypertension.39–41
In summary, focal and proliferative SB+ lesions develop along ArcB, ILA, and AA during chronic Ang II–induced hypertension in rats. In addition to structural alterations, pressure-induced vasomotor responses of ILA and AA were impaired in JMN, and glomerular barrier function was altered at whole-kidney level. Bosentan prevented structurofunctional alterations of the vasculature without affecting development of Ang II–induced hypertension or glomerular injury. Our results therefore suggest that ET-1 selectively mediates vascular pathological changes during Ang II hypertension.
Selected Abbreviations and Acronyms
|AA||=||afferent arteriole, arterioles|
|Ang II||=||angiotensin II|
|ArcB||=||arcuate arterial branch, branches|
|EAA||=||early afferent arteriole, arterioles|
|ETA||=||ET-1 type A receptor|
|ETB||=||ET-1 type B receptor|
|ILA||=||interlobular artery, arteries|
|JAA||=||juxtaglomerular afferent arteriole, arterioles|
|JMN||=||juxtamedullary nephron, nephrons|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|MAA150, MAA300||=||midafferent arteriolar sites|
|mArcA||=||main arcuate artery, arteries|
|PCNA||=||proliferating cell nuclear antigen|
|PP||=||blood perfusion pressure|
|SBP||=||systolic blood pressure|
|SB+||=||Sudan black–positive, sudanophilic|
|SM||=||smooth muscle, muscles|
This work was supported by a grant from Institut National de la Santé et de la Recherche Médicale (Dr Casellas) (INSERM CRE 930404). During the course of these studies, Dr Bouriquet was supported by a Research Fellowship from the Société de Néphrologie. We acknowledge Annie Artuso for her expert technical assistance. Micrographs were printed by Patrick Schuman (INSERM, Service d’Iconographie, Montpellier, France).
Presented in part at the 29th Annual Meeting of the American Society of Nephrology in New Orleans, La, November 1996.
This work has appeared in abstract form (J Am Soc Nephrol. 1996;7:1531).
- Received May 9, 1997.
- Revision received June 12, 1997.
- Accepted July 10, 1997.
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