(Hypertension. 1995;26:925-931.)
© 1995 American Heart Association, Inc.
Articles |
Endothelin in the Kidney in Malignant Phase Hypertension
From the Centre for Genome Research, University of Edinburgh (C.E.W., M.M.V., J.J.M.); Institute of Molecular Medicine, John Radcliffe Hospital, Oxford (J.D.F.); and Department of Renal Medicine, Royal Infirmary of Edinburgh (A.D.C.) (UK).
Correspondence to Dr Caroline Whitworth, Centre for Genome Research, University of Edinburgh, West Mains Road, Edinburgh, UK.
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Abstract A role for endothelin in malignant phase hypertension has been suggested on the basis of reported increases of circulating plasma immunoreactive endothelins in animal models. Recently, a hypertensive rat model that exhibits a genetically determined tendency for developing spontaneous onset malignant hypertension has been described. Expression of the three genes endothelin-1, endothelin-2, and endothelin-3 was quantified in the kidney by specific RNase protection assays in rats with established malignant hypertension, in rats with benign hypertension with and without a genetic susceptibility to malignant hypertension, and in normotensive Sprague-Dawley rats. Endothelin-1 mRNA levels were significantly elevated in the group with malignant hypertension compared with the other three groups. For determination of whether endothelin-1–mediated effects were crucial in the transition from benign to malignant phase hypertension, an oral nonspecific combined endothelin-A and endothelin-B receptor antagonist (bosentan) was given to hypertensive rats susceptible to malignant hypertension. No hypotensive effects were observed, and no significant difference in the incidence of malignant hypertension was observed between treated and control groups. In conclusion, although increased endothelin-1 mRNA expression was found in kidney tissue from rats developing malignant hypertension, blockade of endothelin-1–mediated effects did not prevent the transition from benign phase hypertension. Hence, increased renal endothelin-1 expression in this model of malignant hypertension does not appear to have a causative role and may simply reflect cellular damage and ischemia.
Key Words: endothelin • malignant hypertension • kidney • animals, transgenic
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The transition from BH to MH involves a pressure-induced natriuresis and diuresis, an accelerating rise in BP, myointimal proliferation, and fibrinoid necrosis particularly affecting afferent arterioles and interlobular arteries in the kidney.1 In addition, there is good evidence that activation of the renal renin-angiotensin system contributes to the vicious circle of increasing BP and vascular damage.2 3 4 5 The factors that initiate the transition from BH to MH remain to be identified. We have recently described a rat model of human MH in which spontaneous transition to the malignant phase arises in transgenic TGR(mREN-2)27 heterozygotes. Male HanRen2/Edin- rats, derived from crossing homozygote TGR(mREN-2)27 rats6 with Edinburgh SD (EdinSD) rats, show a 73.5% incidence (95% confidence limits, 65.7% to 81.3%) of MH, and HanRen2/Lew- rats, derived from a cross with the Lewis strain, develop BH but not MH despite attaining similar BP levels.7 8 Results from an analytic cross designed to segregate EdinSD genes suggested that a genetically determined factor, acting in addition to but independent of BP, was important in initiating this transition.8
Endothelin may be a candidate for this role. Identification of this endothelial cell–derived peptide by Yanagisawa et al9 was followed by recognition of both its potent vasoconstrictor activity, particularly in the renal vasculature, and promitogenic effects.9 10 11 12 The reported effects of exogenous ET-1 given to rats and dogs include systemic hypertension,13 renal vascular constriction,14 increased renal vascular resistance,15 glomerular hypertension, decreased renal plasma flow and glomerular filtration rate, but increased filtration fraction. Both natriuretic effects16 and salt and water retention have been reported to occur in response to intravenous endothelin, although such effects may depend on dose and species.17 18 19 20 In humans, arterial and microvascular vasoconstriction and venoconstriction have been reported,21 22 and it has been postulated that ET-1–mediated vasoconstriction may be normally attenuated by endothelium-derived relaxing factors forming a local endothelial autoregulatory system.23 24
In essential hypertension both unchanged and increased basal plasma endothelin levels have been reported.25 26 Some animal models of induced MH have been shown to have elevated plasma ET-1 levels and exhibit hypotensive responses to ETA receptor blockade,27 28 suggesting a role for ET-1 in this condition.
We therefore asked the following questions: (1) Is the spontaneous transition to MH from BH in the susceptible heterozygote cross HanRen2/Edin- associated with increased renal endothelin gene expression? and (2) Is activation of the endothelin system responsible for inducing the accelerated rise in BP leading to afferent vascular damage, renal renin-angiotensin system activation, and progression to renal failure and death? Since it seemed likely that endothelins act as local autocrine and paracrine factors rather than as circulating hormones and that the activity of the endothelin system is modulated at the mRNA level, we used specific RNase protection assays to quantify renal expression of the three endothelin isoforms. We found that ET-1 mRNA levels but not ET-3 mRNA levels were increased in the kidneys of rats with MH. On administration of the combined nonpeptidic ETA and ETB receptor antagonist bosentan29 to HanRen2/Edin- rats from 25 days (prehypertensive stage), it was observed that nonselective endothelin receptor blockade did not lower BP, nor did it significantly prevent transition to MH. This therefore suggested that although increased renal ET-1 gene expression occurred in MH, ET-1–mediated effects were not involved in the normal transition to and progression of MH.
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Methods |
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Endothelin mRNA Expression in MH
Animals
Male heterozygotes, hemizygous for the mouse Ren-2 transgene, were bred as previously described.7 Hypertensive HanRen2/Edin- rats (incidence of MH phenotype in males, 73.5%), HanRen2/Lew- rats (incidence of MH phenotype, 0%), and nontransgenic, normotensive EdinSD rats were maintained in controlled conditions with a 12-hour light/dark cycle, temperature of 18° to 20°C, humidity of 45% to 65%, and normal sodium (0.32%) diet (SDS). All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 and followed the ethical guidelines of the institute.
MH was identified on the basis of the previously described clinical appearances of weight loss and apathy, agitation, or seizures and was confirmed by the presence of fibrinoid necrosis of renal arterioles on histopathologic examination of 3-µm kidney sections fixed in 4% formal saline and stained with hematoxylin and eosin as described previously.8 Four groups of 8- to 10-week-old rats were studied: (1) HanRen2/Edin- rats with MH (n=11), (2) HanRen2/Edin- rats with BH and no signs of MH (n=6), (3) HanRen2/Lew- rats with BH (n=4), and (4) normotensive EdinSD rats (n=4). Rats were briefly anesthetized with 2% halothane anesthesia in oxygen and killed by cervical dislocation. Kidneys were quickly dissected out, snap-frozen in liquid nitrogen, and stored at -70°C before RNA extraction.
RNase Protection Assay
Total RNA was extracted from whole kidney by the guanidine
isothiocyanate/phenol method.30 Solutions were treated
with diethylpyrocarbonate (0.01%, Sigma Chemical Co) when appropriate.
Briefly, whole-kidney tissue was homogenized (Polytron
homogenizer, Janke and Kunkel GmbH) in RNAzol B
(Biogenesis Ltd) on ice according to manufacturers' instructions and
quantified by spectrophotometry. RNA was then reprecipitated in 1/10th
vol of 0.3 mol/L sodium acetate and 2 vol absolute ethanol and stored
at -70°C before assay. The quality of extracted RNA was
assessed on a 1% agarose gel stained with ethidium bromide.
RNase protection assays for rat ET-1, ET-2, and ET-3 were performed as
previously described.31 In brief, uniformly labeled
antisense RNA transcripts were generated by in vitro transcription with
SP6 polymerase (Amersham International) and [
-32P]GTP
(410 Ci/[mmol/L], Amersham). In each case the riboprobe template used
contained genomic sequence that included a part of exon 2 of the gene
of the relevant endothelin, which is the region coding for the mature
peptide. For mRNA analysis, precipitated total RNA from coded
samples was dissolved in hybridization buffer (80% formamide, 40
mmol/L
piperazine-N,N,'-bis[2-ethanesulfonic
acid], 400 mmol/L NaCl, 1 mmol/L EDTA [pH 8]), and RNA concentration
was determined by absorbance measurement at 260 nm with a DU-62
spectrophotometer (Beckman Instruments Inc). One microgram of RNA
extracted from the human cell line K562 containing abundant
-globin mRNA was added to 30 µg of rat kidney RNA in a final
volume of 50 µL. RNA was denatured at 90°C for 10 minutes.
Hybridization was performed overnight at 60°C with
2.5x105 cpm of the appropriate endothelin probe and
2.5x105 cpm of a probe specific for human -globin.
A comparison of the recovery of -globin mRNA from individual
samples allowed a correction to be made for any variation in the
efficiency of processing and gel loading. After hybridization, RNase
digestion was carried out at 37°C for 30 minutes by the addition of
350 µL of solution containing 40 µg/mL RNase A (Boehringer
Mannheim UK), 10 mmol/L Tris (pH 7.5), 5 mmol/L EDTA, and 300 mmol/L
NaCl. This reaction was terminated by the addition of 60 µL of
proteinase K (1 mg/mL) with 3% sodium dodecyl sulfate and
further incubation for 30 minutes. Phenol-chloroform and then
chloroform extractions were performed and the RNA fragments
precipitated with 2.5 vol of absolute ethanol. Precipitated RNA was
dissolved in 5 µL of 80% formamide running buffer, and the reaction
mix was electrophoresed on a denaturing 8% polyacrylamide gel.
After electrophoresis the gels were dried and subjected to
autoradiography at -70°C, after which the
autoradiographs were aligned with their corresponding gels, and the
protected endothelin and -globin mRNA bands were excised. These
were then counted with the use of a flat-bed liquid scintillation
counter (1205 Beta Plate, Pharmacia-Wallac OY). Results were expressed
as counts per minute (mean±SD) after (1) subtraction of background
(counts per minute derived from counting a sample containing no RNA),
(2) correction for recovery of -globin mRNA in individual
samples (which did not vary over a range of more than 15% on any gel),
and (3) correction for the counts per minute obtained from three
external standards run on each gel. These external standards,
containing 15, 30, and 60 µg from a pool of RNA derived from the
kidneys of normal rats, were required because the counts per minute
obtained from any particular gel depended not only on the samples
themselves but also on the activity of the particular batch of probes
used, which was made freshly each week. Statistical analysis
was initially performed with the Kruskal-Wallis one-way ANOVA,
corrected for ties, to look for differences within the four groups.
Subsequently, a Mann-Whitney U test was used to test for
significant differences between individual groups. The probability
value for statistical significance was taken to be less than .05.
Effects of an Endothelin Antagonist on Transition
to MH
Animals
Male HanRen2/Edin- rats were housed as described above in
groups of three or four and fed a 0.32% sodium diet (Harlan-Olac) from
weaning at 25 days. They were randomly assigned to a treatment group
(n=17), given bosentan at a dose of 100 mg/kg per day, or a control
group (n=17), given the same diet without bosentan added. The drug was
thoroughly mixed with powdered foodstuff in aliquots of 1.75 g/kg.
Correct dose was verified by periodic weighing of both rats and food
intake. Both groups had tap water ad libitum to drink. Survival at 100
days of age was compared between treated and untreated groups, because
it has been previously demonstrated in a study of 117 male rats that
development of MH occurred in 73.5% of HanRen2/Edin- rats (95%
confidence limits, 65.7% to 81.3%) by 100 days of age. MH was defined
as present on the development of clear signs of the syndrome as
previously described, with light microscopic examination (Leitz
Laborlux S) of tissues fixed in 4% formal saline, wax embedded,
sectioned at 3 µm thickness, and stained with hematoxylin and eosin,
to confirm MH by the presence of fibrinoid necrosis and myointimal
proliferation.8
BP Measurement
For determination of the effect of bosentan on BP in conscious
transgenic Ren-2 rats with established hypertension, an additional
group of 11 male rats (body weight, 340 to 435 g) had transmitters
(TA11PA-C40) implanted under anesthesia for telemetric
recording of continuous arterial pressure (Data
Sciences International) as previously described.32 33 At
least 2 weeks after surgery rats were administered by gavage single
doses of 100 mg/kg per day bosentan dissolved in 5% gum arabic
(Fisons). Three rats were given further single doses of up to 175 mg/kg
a week later to ensure that any increase in dose would not affect BP.
MBP was recorded for 24 hours before and 48 hours after dosing in
conscious unrestrained rats, and comparisons were made between MBP at
equivalent times of the day to correct for circadian rhythm
effects.34 35
During the study of long-term bosentan-treated versus untreated groups, SBP and heart rate were measured weekly in anesthetized rats with an indirect tail-cuff plethysmographic method (IITC Life Sciences Inc). Brief anesthesia was achieved with 2% halothane in oxygen (Acoma Vaporizer F, International Market Supply) before measurement. Five SBP readings were obtained at each time point, and a mean was recorded for the individual rat. Body weight was measured weekly. Statistical comparison between groups was performed by Student's t test, with a value of P<.05 taken to be significant.
Effects of Exogenous Endothelin
We examined the effect of exogenous endothelin in treated and
control rats to show that treatment with bosentan given mixed in food
had effectively blocked endothelin receptors. On completion of the
study at 105 days of age, random surviving bosentan-treated (n=3)
and untreated (n=4) rats were anesthetized with
ketamine HCl (Vetalar, 12 mg/100 g body wt) and xylazine
(Rompun, 0.3 mg/100 g body wt). The internal jugular vein was
cannulated for intravenous administration. The carotid
artery was cannulated (PE-20) and the catheter connected to a pressure
transducer (model CK-590, Gould) and chart recorder to obtain
continuous direct arterial pressure measurements. Minimal
arterial flushes with 0.9% NaCl/0.1% bovine serum
albumin with 10 U/mL heparin were used if required, up to a
maximum of 1.5 mL in total. Porcine big endothelin (Sigma) was
administered at a dose of 0.3 nmol/kg in 0.9% NaCl/0.1% bovine serum
albumin at a final volume of 0.5 mL/kg after a stable baseline
BP had been achieved. The BP response was recorded over the
following 60 minutes.
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Results |
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In kidney tissue ET-1 mRNA and ET-3 mRNA were detected. ET-2 mRNA was not found in kidney tissue from any group of rats. RNase protection assays (Fig 1) on RNA from whole kidney showed protected bands for ET-1 and ET-3 and also human
-globin
(as a loading control). Excised bands were then counted and results
depicted as counts per minute after correction for efficiency of
processing, gel loading, and background activity (Fig 2).
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ET-1 mRNA levels differed significantly between the four groups
(Kruskal-Wallis one-way ANOVA, 
2=11.757,
df=3, P=.008), with a mean (±SD) of 81.8±24.7
cpm in HanRen2/Edin- rats with clinical signs and histopathologic
evidence of MH compared with 49.5±19.1 cpm in age-matched
HanRen2/Edin- rats with BH, 37.2±15.7 cpm in HanRen2/Lew-
rats with BH, and 36.2±6.8 cpm in normotensive EdinSD rats. On
comparison of the four groups, ET-1 mRNA levels in HanRen2/Edin-
rats with MH were significantly higher than those found in kidney
tissue from either HanRen2/Edin- rats with BH (Mann-Whitney
U test, Z=-2.3, P=.020) or from
HanRen2/Lew- rats (Z=-2.49, P=.013) or EdinSD
rats (Z=-2.3, P=.019).
On examination for differences in ET-3 mRNA levels between the four
groups, there was a small difference that did not reach statistical
significance (Kruskal-Wallis one-way ANOVA,
2=7.810, df=3, P=.0501).
Lower levels were observed to occur in kidney from HanRen2/Edin-
rats with MH (9.15±5.7 cpm) compared with the control groups
(HanRen2/Edin- rats with BH and a genetic susceptibility to MH,
16.2±7.5 cpm; HanRen2/Lew- rats with BH, 12.9±1.1 cpm; and
normotensive EdinSD rats, 20.9±10.0 cpm).
Administration of the nonspecific endothelin receptor antagonist bosentan (100 to 175 mg/kg) by gavage to transgenic Ren-2 rats with established hypertension had no significant effect on MBP either within the first 12 hours or from 12 to 24 hours after a single dose in conscious rats (Fig 3). Likewise, a repeat dose given 1 week later did not lower BP. Although the rats used in this experiment to investigate the effects of short-term oral administration of bosentan were older than those that received the endothelin antagonist on a long-term basis, no hypotensive response was elicited from any of them.
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HanRen2/Edin- rats were therefore randomly assigned at weaning to either long-term oral treatment with bosentan or no treatment. No effect on MBP (measured by tail-cuff plethysmography in anesthetized rats), heart rate, or body weight was observed up to 15 weeks of age (Fig 4). There was no statistically significant difference in the survival curves between the two groups, with 9 of 17 (53%) control rats developing MH and 11 of 17 (65%) treated rats (Fig 5) with the pathological features of MH observed in affected cases.
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) and untreated controls (
)
(n=17 per group at the start of the study) were followed with weekly
measurements; results are expressed as group means and SD. SBP and
heart rate were determined for each individual rat at each time point
from the mean of five measurements. Treated rats received bosentan
mixed with powdered food at a dose of 100 mg/kg per day; controls
received the same diet without the drug.
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To verify that both the dose of endothelin antagonist used and the method of administration had achieved satisfactory blockade of endothelin receptors, we gave three treated survivors and four untreated survivors, while still on their assigned diet, intravenous porcine big endothelin (0.3 nmol/kg). The maximal increase in mean BP occurring during the subsequent 60-minute period in untreated rats ranged from +24.5% to +42.4%, but in the bosentan-treated rats an increase in MBP of only 0% to +5.6% was observed, indicating that at the dose used adequate blockade of endothelin receptor–mediated vasopressor effects had been achieved (Fig 6).
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The identification of a genetic susceptibility to MH in transgenic Ren-2 rats led to the question of what factor or factors additional to high BP might be responsible for initiating the transition to MH, with an accelerating rise in arterial pressure, development of myointimal proliferation and fibrinoid necrosis principally of small renal arteries and afferent arterioles, activation of the renal renin-angiotensin system, and decline in renal function. Susceptibility appeared to be conferred by crossing transgenic Hannover SD rats (HanRen2/HanRen2) with EdinSD rats (Edin-/Edin-), resulting in 73.5% (65.7% to 81.3%) of HanRen2/Edin- males developing MH by 100 days of age; whereas a cross with the Lewis strain, obtaining HanRen2/Lew- heterozygotes, resulted in hypertension (MBP, 180 mm Hg) of a similar degree but no transition to MH.8 This observation has obvious and important clinical parallels in that it remains unknown why the transition to MH occurs in some hypertensive individuals but not in others who have the same level of arterial pressure.36
A role for endothelin had been suggested on the basis of its potent vasoconstrictor effects, particularly on the kidney, and promitogenic effects. Induction of endothelin expression within vascular endothelial cells has been shown to occur in response to low levels of shear stress and to mediators such as thrombin and angiotensin II,9 which may be involved in the process of MH. Elevated plasma immunoreactive ET-1 has been reported in two experimental rat models of MH, either resulting from deoxycorticosterone acetate–salt administration to SHR or long-term administration of caffeine to two-kidney, one clip rats.27 SHR have been reported to exhibit both normal and elevated plasma endothelin levels,27 37 and the ETA-specific antagonist BQ-123 has been shown to have both antihypertensive effects and no effect in SHR.28 37 However, in stroke-prone SHR (20 to 29 weeks old), elevated plasma ET-1 levels when compared with levels of normotensive Wistar-Kyoto controls and a hypotensive response to intravenous BQ-123 led the authors to suggest a role for ET-1 in the maintenance of high BP in MH.28
The short plasma half-life, very low circulating plasma levels, and little evidence of storage of endothelin peptides have strongly supported an autocrine or paracrine role, which makes any interpretation of plasma endothelin levels difficult.24 Measurement of tissue endothelin mRNA levels was therefore thought to be a better reflection of activity of the endothelin system.
In the present investigation the kidney was specifically studied in view of its likely involvement in MH, with an initial natriuretic response, developing renal impairment, afferent vascular damage, and activation of the renal renin-angiotensin system. We used specific RNase protection assays that could clearly differentiate among ET-1, ET-2, and ET-3 mRNA. The significant finding was of a twofold increase in ET-1 mRNA expression in kidneys taken from rats with MH. Interestingly, there was a suggestion of a reciprocal relationship, which has previously been observed,31 between expression of ET-1 and ET-3, with the lowest levels of ET-3 occurring in MH kidney. However, the differences in ET-3 mRNA among groups did not reach statistical significance. ET-2 was not detected in rat kidney, as previously reported.31
Previous investigators have demonstrated by Northern blot analysis an increase in arterial vessel wall ET-1 mRNA expression in the deoxycorticosterone acetate–salt hypertensive rat compared with uninephrectomized normotensive controls.38 This was associated with elevated immunoreactive ET-1 peptide in both aortic and mesenteric arterial endothelial cell layers demonstrated by immunohistochemistry.39 Our findings have shown that increased renal expression of ET-1 mRNA is associated with the development of MH, but it was important to identify an active role before suggesting that endothelin peptides may be causative in MH, because an increase in ET-1 in such a condition may simply be an epiphenomenon reflecting endothelial cell damage.40
To examine this possibility we studied the effect of pharmacological manipulation of the endothelin system on the development of MH. ETA receptors preferentially bind ET-1 compared with the other isoforms, have a high efficacy, and mediate vasoconstrictor effects although the relative contribution of the ETA and ETB receptors to vasoconstriction may depend on the vascular bed studied. ETB receptors show equal affinity for all three isoforms and have been linked to the formation of nitric oxide and prostacyclin.24 It may be that the ratio of ET-1 to ET-3, highest in MH, is relevant in reflecting an imbalance between local vasoconstrictor and vasodilator effects.
In the present study we examined in the MH model the effect of the nonspecific endothelin antagonist bosentan, which blocks both ETA- and ETB-mediated vasoconstrictor effects.29 To specifically answer the question as to whether endothelin receptor blockade would prevent the onset of MH, it was important to exclude any antihypertensive effects of the antagonist. Oral administration of a dose (100 mg/kg per day) that has previously been demonstrated to effectively block both ETA and ETB receptors29 had no BP-lowering effect either on short-term administration to rats that had BP monitored in a conscious state on a continuous basis by telemetry or on long-term administration. This clearly demonstrated that BP in transgenic Ren-2 rats was not mediated in the BH state by either receptor. Second, it was confirmed at the end of the 11-week period of oral administration of the antagonist that complete blockade of any hypertensive effect resulting from exogenous administration of porcine big endothelin (0.3 nmol/kg) was present, suggesting that adequate receptor blockade had been achieved.
Long-term administration of the endothelin receptor antagonist did not significantly alter survival at 100 days of age, basal SBP, heart rate, or body weight gain. Although untreated HanRen2/Edin- rats did develop MH slightly earlier than those in the bosentan-treated group, the age at onset for both groups did not differ significantly from the age range (median age, 59 days; range, 46 to 102 days) in which a larger group (86 of 117) of untreated rats developed the syndrome.8
The absence of a hypotensive response to bosentan in Ren-2 rats is in contrast to the findings reported for some other rat models of hypertension. In the deoxycorticosterone acetate–salt hypertensive rat, in which both increased immunoreactive ET-1 peptide in acid extracts of thoracic aorta and mesenteric vasculature and increased ET-1 gene expression were seen,38 39 3 weeks of treatment with bosentan (100 mg/kg per day) resulted in a small but statistically significant attenuation in the rise in SBP measured by tail-cuff plethysmography.41 In the present study the effects of bosentan on SBP were examined essentially in the BH state when rats were clinically healthy and when no significant differences in renal ET-1 or ET-3 mRNA levels were demonstrated between transgenic rats with BH and normotensive control SD rats. It is therefore not possible to draw any conclusions from this study about the contribution of ET-1 to the maintenance of systemic BP during MH at a time when increased renal ET-1 mRNA levels were found. Previous studies have demonstrated that the transition to MH in this model is relatively acute, leading to a terminal phase of accelerating BP, weight loss, and renal failure of only a few days' duration.8
In summary, the present study has clearly demonstrated increased ET-1 mRNA expression in the kidney on transition to MH in the hypertensive transgenic HanRen2/Edin- cross, but in an age-matched susceptible group of healthy HanRen2/Edin- rats that might be considered to be potentially premalignant, there were no significant differences in ET-1 mRNA levels relative to the nonsusceptible cross HanRen2/Lew- with equivalent BP or to normotensive SD rats. The cell type within kidney tissue responsible for the increase in ET-1 expression has not been identified in this study. However, with the use of the nonselective endothelin receptor antagonist bosentan at a dose that was able to block the hypertensive response to a relatively large dose of exogenous big endothelin, no significant reduction in the incidence of MH was seen. This has suggested that in this model the pathophysiological effects of endothelin are not involved in either initiating the transition from BH to MH or in the progression of the natural course of MH but that increased renal ET-1 mRNA expression may occur in response to the onset of MH, perhaps as a consequence of renal vascular endothelial cell damage.
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Acknowledgments |
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This study was funded by the BBSRC and CEC Concerted Action "Transgeneur." Thanks to Dr Martine Clozel, Hoffmann–La Roche, Basel, Switzerland, for kindly giving us bosentan and to Kay Yeates for technical assistance with the endothelin RNase protection assays. Thank you to Louise Anderson and Gillian Brooker for all their help.
Received February 24, 1995; first decision March 27, 1995; accepted August 24, 1995.
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