AJH

1998;11:989 –997

Effects of Losartan and Captopril on Endothelin-1 Production in Blood Vessels and Glomeruli of Rats With Reduced Renal Mass Richard Larivie`re, Marcel Lebel, Iris Kingma, John H. Grose, and Daniel Boucher

Recently, we have reported that endothelin-1 (ET1) production is increased in blood vessels and glomeruli of rats with chronic renal failure. This study was design to investigate the role of angiotensin II (Ang II) in endogenous ET-1 production in rats with reduced renal mass. One week after subtotal (5/6) nephrectomy, uremic rats were divided into three groups, and received either no treatment, the Ang II subtype 1 receptor (AT1) antagonist losartan (10 mg/kg/day), or the angiotensin-converting enzyme inhibitor (ACE-I) captopril (30 mg/kg/day) for 6 weeks. Shamoperated rats were used as controls and received no treatment. The levels of immunoreactive ET-1 (ir-ET-1) in plasma and urine, as well as in vascular and renal tissues, were determined by radioimmunoassay (RIA) after extraction. In uremic rats, losartan and captopril completely prevented the increase in systolic blood pressure. At week 6, plasma ir-ET-1 was similar in the different groups of uremic rats and in the controls. However, ir-ET1 concentration in the mesenteric arterial bed, the thoracic aorta, preglomerular arteries, and glomeruli, as well as urinary ir-ET-1 excretion were significantly greater in uremic-untreated rats

compared to controls (P < .01). Treatment of uremic rats with losartan or captopril reduced irET-1 concentration in the thoracic aorta and preglomerular arteries (P < .05), but ir-ET-1 concentration in the mesenteric arterial bed was unchanged. Although both drugs completely prevented the increase in proteinuria, losartan but not captopril significantly reduced ir-ET-1 concentration in glomeruli (P < .05) and normalized urinary ir-ET-1 excretion. This indicates that increased ET-1 production in blood vessels and glomeruli of uremic rats is modulated, at least in part, by Ang II through the AT1 receptor. The beneficial effects of the AT1 antagonist losartan could be attributable to the attenuation of Ang IIinduced ET-1 production in this rat remnant kidney model of chronic renal failure, whereas those of the ACE-I captopril are not related to changes in ET-1 production in glomeruli. Am J Hypertens 1998;11:989 –997 © 1998 American Journal of Hypertension, Ltd.

hronic renal failure is characterized by increased filtration in residual nephrons due to glomerulosclerosis and systemic and glomerular capillary hypertension.1,2 Recently, a role for endothelin-1 (ET-1), a powerful

C

vasoconstrictor peptide of endothelial origin,3 has been proposed in these abnormalities in many diseases with renal failure. In hemodialysis patients, elevated plasma ET-1 levels have been reported,4,5 which correlated with the increase in blood pressure.5 In

Received March 13, 1997. Accepted March 9, 1998. From the Research Centre and Division of Nephrology, CHUQ, L’Hoˆtel-Dieu de Que´bec Hospital (RL, ML, IK, JHG, DB), and Departments of Pharmacology (RL), Medicine (ML, IK, JHG), and Pathology (DB), Laval University, Que´bec, Que´bec, Canada. This study was supported by a Losartan Medical School Grant

from Merck Frosst Canada Inc., and by a grant from the Fondation des Maladies du Coeur du Que´bec to RL. IK and RL hold research scholarships from the Fonds de la Recherche en Sante´ du Que´bec. Address correspondence and reprint requests to Richard Larivie`re, PhD, Centre de Recherche, CHUQ, L’Hoˆtel-Dieu de Que´bec, 11 coˆte du Palais, Que´bec, Que´bec G1R 2J6, Canada.

© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.

KEY WORDS:

Endothelin-1, angiotensin II, losartan, captopril, blood vessels, glomeruli, hypertension, chronic renal failure.

0895-7061/98/$19.00 PII S0895-7061(98)00088-0

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addition, increased urinary ET-1 concentration in rats with reduced renal mass correlated positively with heightened blood pressure, proteinuria, and glomerular sclerosis.6,7 We have reported recently that immunoreactive ET-1 (ir-ET-1) concentration is augmented in blood vessels and glomeruli of rats with chronic renal failure.8 Increased local ET-1 production may significantly affect the systemic and renal hemodynamics3,9 and promote vascular smooth muscle and the glomerular mesangial cell growth as well as the accumulation of extracellular matrix,10,11 leading to the aggravation of hypertension and the progression of renal insufficiency. Recent studies have documented a role for angiotensin II (Ang II) in stimulating ET-1 generation and release in vascular endothelial cells and in glomerular mesangial cells in vitro.12,13 In normal rats, Ang II infusion enhances urinary ET-1 excretion, indicating in vivo stimulation of renal ET-1 production.14 Moreover, ET-1 has been shown to mediate in part the pressor response to Ang II in vitro and in vivo,15,16 and the effects of Ang II on glomerular mesangial cell proliferation and extracellular matrix protein expression.11,17 On the other hand, it is well established that treatment of hypertension in chronic renal failure with either angiotensin-converting enzyme inhibitors (ACE-I) or with Ang II subtype 1 receptor (AT1) antagonists significantly reduces proteinuria and attenuates glomerulosclerosis.18,19 Similar protective effects have been recently reported in uremic rats treated with an ET subtype A receptor (ETA) antagonist.20 These studies strongly suggest that Ang II may be an important modulator of endogenous ET-1 production. Therefore, the present study was designed to examine whether Ang II modulates ET-1 production in chronic renal failure. The effects of losartan, an AT1 receptor antagonist, and the ACE-I captopril on ir-ET-1 concentration in blood vessels and glomeruli as well as on urinary ir-ET-1 excretion were investigated in rats with reduced renal mass.

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METHODS

rats were divided into three groups with similar systolic blood pressure. One group received no treatment and the other groups were given either losartan (DuP 753; DuPont Merck Pharmaceutical Co., Wilmington, DE) at a dose of 10 mg/kg/day or captopril (BristolMyers Squibb Co., Syracuse, NY) at a dose of 30 mg/kg/day, in drinking water for the following 6 to 7 weeks. We chose doses of losartan and captopril previously reported to be the minimum required for each to obtain maximum systolic blood pressure reduction in rats with reduced renal mass.19,21 Sham-operated rats receiving tap water served as controls. Systolic blood pressure was measured before the initiation of treatment and again after every 2 weeks, by the tailcuff method using an IITC blood pressure system fitted with a model 29 pulse sensor (IITC Life Science, Woodland Hills, CA) after warming and with slight restraint. The blood pressure readings were analyzed with a computerized acquisition system (model MP100, Biopac System, Goleta, CA). On average, three separate blood pressure measurements were recorded. Before treatment and at week 6, the rats were placed in metabolic cages, and 24-h urine samples were collected and stored at 220°C for the assessment of protein, sodium, and ir-ET-1. At the end of the experiment, the rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg) and blood was collected by abdominal aortic puncture. Blood samples were used for the measurement of hematocrit, plasma renin activity, and ir-ET-1, as well as serum creatinine and urea. The thoracic aorta segment, from the first to the eighth caudal ribs, and complete mesenteric vascular bed, from the 1st cranial artery to the intestinal border, were removed and cleaned of blood and adipose tissue. They were then quickly frozen on dry ice and stored at 280°C for ir-ET-1 measurements. The heart was removed, cleaned of blood, and weighed. The remnant kidney of uremic rats and the kidneys of control animals were removed and immersed in 0.9% saline for fresh preparation of preglomerular arteries and glomeruli.

Animal Experiments Animal experiments, approved by the Animal Care Committee of Laval University, were performed on 250 g male Sprague-Dawley rats (Charles River Laboratories, St.-Constant, Que´bec, Canada) allowed free access to standard laboratory rat chow and tap water. All animals were housed under controlled humidity, temperature, and 12-h light– dark cycles. Renal mass was reduced (5/6 nephrectomy) according to the method of Anderson et al18 by ligating two to three branches of the left renal artery, followed 1 week later by right nephrectomy under intraperitoneal pentobarbital anesthesia (Somnotol, 50 mg/kg; MTC Pharmaceuticals, Cambridge, Ontario, Canada). One week later the nephrectomized

Preparation of Preglomerular Arteries and Glomeruli Preglomerular arteries and glomeruli were prepared according to the methods described by De Leon and Garcia22,23 with slight modifications. To obtain a sufficient amount of tissue for assessment of ir-ET-1 concentration, kidneys were harvested from two animals per group. They were dissected longitudinally and the papilla removed. Kidney halves were homogenized by passage through a 0.4-mm stainless steel grid. Preglomerular interlobar, arcuate, and interlobular arteries as well as afferent arterioles retained on the grid were washed with 0.9% saline solution, minced with scissors, and transferred onto a 150-mm mesh nylon sieve (Nitex, B & SH Thompson Co., Mon-

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tre´al, Que´bec, Canada). The tissues were pressed against the nylon sieve with a spatula, to detach the remaining tubules, connective tissues and glomeruli, and rinsed with saline solution. The renal arteries were transferred to a centrifugation tube, whereas the filtrate was combined with the first homogenate. Glomeruli contained in the homogenate were isolated by filtration through 150, 50, and 100 mm mesh nylon sieves and washed with saline solution. Glomeruli retained on the last sieve were transferred to a centrifugation tube and an aliquot was used to determine the number of glomeruli under a light microscope. Preparations from the different groups of rats contained ,5% contamination with connective tissue and tubules. Renal arteries and glomeruli were collected by centrifugation at 3000 g for 15 min at 4°C. Supernatants were removed and pellets were frozen quickly and stored at 280°C for the assessment of tissue irET-1 content. Measurement of ir-ET-1 in Tissue, Plasma, and Urine One thoracic aorta segment, mesenteric arterial bed, or preparation of either preglomerular arteries or glomeruli was used per extraction tube and assayed individually. Frozen tissues were weighed and homogenized twice with a Tissue-Tearor (Biospec Products, Bartlesville, OK) for 15 sec in 2 mL of ice-cold extraction solution containing 1 N HCl, 1% acetic acid, 1% trifluoroacetic acid (TFA), and 1% NaCl.8 The homogenate was centrifuged at 3000 g for 30 min at 4°C. The supernatant was then collected and 100 mL of 125I-ET-1 (;1000 cpm) (DuPont NEN, Boston, MA) was added before to extraction on a C18 Sep-Pak column (Waters, Milford, MA). Similarly, plasma and urine samples (2 mL) were acidified with 0.2% TFA, and 100 mL of 125 I-ET-1 (;1000 cpm) was added before extraction on a C18 Sep-Pak column.5 The Sep-Pak columns were activated with 4 mL 60% acetonitrile and 0.1% TFA, then rinsed twice with 10 mL 0.1% TFA. After the samples were loaded, the columns were washed twice with 10 mL of 0.1% TFA. The ir-ET-1 fraction was eluted with 3 mL of 60% acetonitrile and 0.1% TFA and counted in a gamma counter. Radiolabeled ET-1 recovered was 90% to 95% for tissues and 75% to 85% for plasma and urine. The extracted samples were dried overnight in a Speed Vac (Savant Instruments Inc., Farmingdale, NY), and were reconstituted in 500 mL RIA buffer containing 100 mmol/L sodium phosphate, pH 7.4, 50 mmol/L NaCl, 0.1% bovine serum albumin, 0.1% Triton X-100, and 0.01% sodium azide. Aliquots of 100 mL and 200 mL of extracted samples or 200 mL standard ET-1 (Peninsula Laboratories, Belmont, CA) at concentrations ranging from 0.5 to 128 pg were added to 100 mL of diluted antiserum. The final reaction volume in each tube was adjusted to 300 mL with RIA buffer. Previously prepared rabbit anti-

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serum specific for ET-1 with about 30.5%, 62%, and 33.9% cross-reactivity with big ET-1, ET-2, and ET-3, respectively, was diluted at a final concentration of 1:10,000, which gave approximately 35% to 40% binding. After 24 h of incubation at 4°C, 100 mL of 125I-ET-1 (15,000 cpm) in RIA buffer was added, and the tubes were incubated for an additional 24 h at 4°C. Bound and free radioactivities were separated by the addition of 100 mL 10-fold diluted goat antirabbit IgG serum (Immunocorp, Montre´al, Que´bec, Canada) and 100 mL 25-fold diluted normal rabbit serum (Immunocorp). After a 2-h incubation period at room temperature, 0.5 mL of RIA buffer was added, and the tubes were centrifuged at 2500 g for 20 min at 4°C. The supernatant was then discarded and the pellet was counted in a gamma counter. Ir-ET-1 concentrations were corrected for losses in the extraction and purification steps. Other Biochemical Analysis Serum was obtained from 1-mL blood samples incubated for 1 h at room temperature and centrifuged for 2 min in a bench top microcentrifuge. Serum creatinine and urea as well as urinary sodium and protein were measured with an autoanalyzer system (Ilab 1800, Lexington, MA). Hematocrit was assessed in Pre-Cal microhematocrit heparinized tubes (Becton Dickinson Co., Parsippany, NJ) after 2 min of centrifugation in a bench top microcentrifuge. Plasma renin activity was measured using a RIA kit for angiotensin I purchased from DuPont NEN. Analysis of Data The results are expressed as means 6 SEM. Mean values were compared by ANOVA followed by Student-Newman-Keuls’ test for multiple comparisons. Differences were considered significant at a value of P , .05. RESULTS Changes in Systolic Blood Pressure One week after renal mass ablation, systolic blood pressure was significantly higher in uremic rats compared to the controls (155 6 5 and 91 6 3 mm Hg, respectively; P , .01). Systolic blood pressure increased progressively with time in untreated uremic rats, reaching 235 6 19 mm Hg at the end of the experiment (Table 1). In contrast, at week 2, systolic blood pressure was slightly reduced in losartan-treated and captopriltreated uremic rats, and was not different from that in control rats at weeks 4 and 6 (Figure 1). Although the decrease of systolic blood pressure by losartan was slightly less than with captopril, the difference was not statistically significant (Figure 1 and Table 1). The differences in systolic blood pressure reduction may be attributable to the fact that three captopril-treated rats died during the study and were not included in

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TABLE 1. SYSTEMIC PARAMETERS IN CONTROL, UREMIC-UNTREATED, LOSARTAN-TREATED, AND CAPTOPRIL-TREATED RATS

Group

n

Body Weight (g)

Control Uremic Uremic 1 losartan Uremic 1 captopril

10 8 9 7

454 6 16 439 6 12 407 6 18 394 6 12

Systolic Blood Pressure (mm Hg)

Serum Creatinine (mmol/L)

Serum Urea (mmol/L)

Hematocrit (%)

Plasma Renin Activity (ng Ang I/mL/h)

121 6 3 235 6 19† 155 6 9 126 6 12

43 6 1† 86 6 6 75 6 8 85 6 7

5.2 6 0.2† 17.0 6 1.6* 12.8 6 1.7 12.7 6 1.2

43.3 6 0.7† 34.0 6 1.1 35.4 6 1.2 36.4 6 1.4

39 6 5 3 6 1‡ 20 6 10 28 6 16

Values are means 6 SEM. Ang I, angiotensin I; n, number of rats studied. * P , .05 v captopril-treated uremic rats; † P , .01 v other groups of rats; ‡ P , .01 v control rats.

the data analysis, whereas only one losartan-treated rat died (Figure 1). Systemic and Renal Parameters Table 1 presents data on systemic parameters measured at week 6. Body weight was similar in the four groups. However, serum creatinine and urea were significantly higher, whereas hematocrit and plasma renin activity were lower in uremic rats compared to the controls (P , .01). Neither losartan nor captopril affected serum creatinine and hematocrit. In contrast, serum urea was

FIGURE 1. Changes in systolic blood pressure in control, uremic-untreated, losartan-treated (10 mg/kg/day) and captopriltreated (30 mg/kg/day) rats. Nx represents the time of completion of renal mass ablation, time 0 indicates the following week before the initiation of treatments, and times 1 to 6 indicate weeks of treatment. *P , .01 v control rats; **P , .01 v other groups of rats; 1P , .05 v control rats. Values in parentheses represent the number remaining from the initial 10 rats, in different groups of uremic animals at each time interval.

reduced in treated uremic rats but remained significantly higher than in the controls. Treatment of uremic rats with losartan or captopril significantly increased plasma renin activity. One week after renal mass ablation, proteinuria and urinary volume were significantly higher in uremic rats compared to the controls, whereas natriuresis was similar (Figure 2). At the end of the experimental protocol, however, all renal parameters were higher in uremic rats. Treatment with losartan or captopril completely prevented the rise in proteinuria in uremic rats (Figure 2B). Plasma, Urine, and Tissue ir-ET-1 Concentration Wet weight of the mesenteric arterial bed, the thoracic aorta and preglomerular arteries, as well as the heart wet weight to body weight ratio were significantly greater in uremic rats compared to control animals (P , .01; Table 2), suggesting cardiovascular tissue hypertrophy. Treatment of uremic rats with losartan or captopril did not change mesenteric arterial bed wet weight. In contrast, thoracic aorta wet weight was significantly reduced in treated uremic rats (P , .01), but was similar to that in the controls. Although renal preglomerular artery wet weight only tended to be lower in losartan-treated uremic rats (Table 2), it was slightly but significantly reduced in captopril-treated uremic animals (P , .05). Similarly, the heart wet weight to body weight ratio was lower in captopriltreated uremic rats (P , .05). As expected, the number of glomeruli obtained from the remnant kidneys of rats with reduced renal mass was much smaller than that from the kidneys of control animals (29,062 6 1640 and 6000 6 1068 glomeruli per kidney, respectively, P , .01). The number of glomeruli obtained from the remnant kidneys of losartan-treated and captopril-treated uremic rats only tended to be higher compared to untreated uremic animals (8467 6 678 and 7133 6 1348 glomeruli per kidney, respectively). Plasma ir-ET-1 concentration was similar in the different groups (Figure 3A). In contrast, ir-ET-1 concentration in the mesenteric arterial bed, the thoracic

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captopril, significantly reduced ir-ET-1 concentration in glomeruli (P , .05). In addition, losartan totally prevented the increase in ir-ET-1 excretion in uremic rats, whereas the decrease in ir-ET-1 excretion by captopril was not statistically significant (Figure 4B). DISCUSSION

FIGURE 2. Changes in urinary volume (Uvol) (A), urinary protein (Upro) (B), and urinary sodium (UNa) (C) excretion in control (n 5 10), uremic-untreated (n 5 8), losartan-treated (n 5 9), and captopril-treated (n 5 7) rats measured in 24-h urine samples collected before the initiation of treatments and at the end of the experimental protocol. **P , .01 v other groups of rats.

aorta, and renal preglomerular arteries was significantly greater in uremic rats compared to the controls (Figure 3). Treatment of uremic rats with losartan or captopril decreased ir-ET-1 concentration in the thoracic aorta and preglomerular arteries, but not in the mesenteric arterial bed. The ir-ET-1 reduction in the thoracic aorta of uremic rats was, however, less with losartan compared to captopril (P , .05 and P , .01, respectively, v untreated uremic rats; Figure 3C). Changes in ir-ET-1 concentration in the thoracic aorta were correlated with the decrease in systolic blood pressure (r 5 0.672, P , .01). Ir-ET-1 concentration was also increased in the glomeruli of uremic rats compared to the controls (Figure 4A). Interestingly, treatment of uremic rats with losartan, but not with

Similar to our observations in a previous study,8 ET-1 concentration was significantly increased in blood vessels and glomeruli as well as in the urine of rats with reduced renal mass. In contrast, plasma ET-1 concentration was similar in the different groups of rats supporting that locally produced ET-1, rather than circulating peptide levels, plays a role in the aggravation of hypertension and the progression of renal insufficiency. Indeed, we and others have reported that increased ET-1 production in vascular and renal tissues is associated with hypertension and proteinuria as well as with glomerulosclerosis.6 – 8 This is supported by the study of Benigni and coworkers20 where chronic treatment of uremic rats with a selective ETA receptor antagonist significantly blunted the increase of blood pressure and attenuated the development of glomerulosclerosis. Recently, we reported that increased ET-1 production in blood vessels of hypertensive rats occurs exclusively in the endothelium.24,25 In the vessel wall, ET-1 is released from the endothelial cells predominantly on the abluminal side toward the smooth muscle cells where it acts in an autocrine and paracrine fashion to modulate vascular tone.26 In conditions with enhanced ET-1 production such as in this rat remnant kidney model, small increases in ET-1 released into the intimal space may cause important changes in its concentration in the immediate vicinity of its receptors on smooth muscle cells. Similar phenomena may also occur in the glomerulus where the glomerular capillary endothelial cells as well as the mesangial cells are able to produce ET-1.27,28 Among humoral factors that have been implicated in renal disease progression, Ang II is an excellent candidate for the regulation of ET-1 production. In fact, Ang II is a potent stimulator of ET-1 production and release in endothelial cells and in mesangial cells in vitro12,13 as well as in vascular and renal tissues in vivo.11,14 –17 In this study, we show for the first time that AT1 receptor blockade with losartan or ACE inhibition with captopril significantly attenuated the rise of ir-ET-1 concentration in blood vessels of rats with reduced renal mass. Losartan also reduced irET-1 concentration in glomeruli and normalized urinary ir-ET-1 excretion, thus indicating that Ang II modulates ET-1 production through the AT1 receptor subtype. Furthermore, this study shows that the beneficial effects of losartan could be attributable, at least in part, to the attenuation of ET-1 production in this rat remnant kidney model of chronic renal failure.

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TABLE 2. BLOOD VESSEL WET WEIGHT, AND HEART WET WEIGHT TO BODY WEIGHT RATIO IN CONTROL, UREMIC-UNTREATED, LOSARTAN-TREATED, AND CAPTOPRIL-TREATED RATS Tissue Wet Weight (mg) Group

Mesenteric Arterial Bed

Control Uremic Uremic 1 losartan Uremic 1 captopril

35 6 1† 52 6 3 55 6 2 48 6 5

Thoracic Aorta

Renal Preglomerular Arteries

Heart Wet Weight/ Body Weight Ratio (1023)

38 6 2 56 6 3† 45 6 2 42 6 3

49 6 5† 114 6 6 96 6 12 86 6 3§

2.73 6 0.08‡* 3.54 6 0.15 3.35 6 0.18 2.99 6 0.10§

Values are means 6 SEM obtained from 7 to 10 mesenteric arterial bed, thoracic aorta, and heart preparations, and from three to five renal preglomerular arterial preparations (values are in mg/kidney). * P , .01 v uremic rats; † P , .01 v other groups of rats; ‡ P , .05 v losartan-treated rats; § P , .05 v uremic rats.

The effects of losartan and captopril on ir-ET-1 concentration in blood vessels and glomeruli of uremic rats, in the presence of significantly reduced plasma renin activity, further support a role for a tissue reninangiotensin system in chronic renal failure.29 Increased renin-induced Ang II formation and reduced degradation have been reported in the hindlimb of uremic rats,30 where vascular Ang II formation was completely inhibited by captopril. In the remnant kidney, renin immunostaining was increased in the vasculature and glomeruli of tissues adjacent to the renal scar.31 Moreover, enhanced expression of the renin gene has been reported in glomeruli of uremic rats.32 Hence, tissue Ang II formation may be higher in rats with reduced renal mass, which may contribute to local ET-1 production. Consistent with this hypothesis, our study shows that ir-ET-1 concentration in

FIGURE 3. Immunoreactive endothelin-1 (ir-ET-1) concentration in plasma (A), the mesenteric arterial bed (B), the thoracic aorta (C), and preglomerular arteries (D) in control (n 5 10), uremic-untreated (n 5 8), losartan-treated (n 5 9) and captopril-treated (n 5 7) rats. *P , .01 v uremic-untreated rats; **P , .01 v other groups of rats; 1P , .05 v uremic-untreated rats.

blood vessels and glomeruli was significantly reduced by treatment of uremic rats with the AT1 receptor antagonist losartan. Furthermore, the inhibition of Ang II formation by the ACE-I captopril also attenuated ET-1 production in blood vessels. However, captopril had no effect on ir-ET-1 concentration in glomeruli of uremic rats. It may be speculated that these differences are related to differential tissue distribution of captopril and losartan. Because both treatments normalized systolic blood pressure and totally prevented the increase in proteinuria, and have also been shown to be equipotent in attenuating the vascular remodeling and the glomerulosclerosis,18,19,21 the effects of losartan and captopril on ET-1 production were most likely tissue specific. Hence, the renal protective actions of AT1 receptor antagonists and ACE-I in chronic renal failure appear to be mediated by

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FIGURE 4. Immunoreactive endothelin-1 (ir-ET-1) in glomeruli (A) and 24-h urinary ir-ET-1 (Uir-ET-1) (B) at the end of the experimental protocol in control, uremic-untreated, losartantreated, and captopril-treated rats. *P , .05 v uremic-untreated rats; **P , .01 v uremic-untreated and captopril-treated rats; 1P , .05 v control and uremic-untreated rats. Ir-ET-1 concentration in glomeruli was determined in three to five separate preparations and urinary ir-ET-1 was measured in 7 to 10 samples per group.

different mechanisms. The effects of losartan may be attributable, at least in part, to the specific blockade of Ang II-induced ET-1 production, whereas those of ACE-I may be related to enhanced bradykinin-induced nitric oxide (NO) release.33 This is supported by a recent study showing that administration of a NO synthesis inhibitor blunted the effects of captopril on blood pressure and the progression of renal insufficiency and damage in uremic rats.33 NO release could attenuate the hemodynamic effects of Ang II and ET133–36 as well as their effects on mesangial cell proliferation.37 The decrease in blood pressure may also play a role in the attenuation of ir-ET-1 concentration in blood vessels of uremic rats. At the end of the study, the reduction in systolic blood pressure with losartan was slightly less than with captopril but was not significantly different. Similarly, the diminution of ir-ET-1 concentration in the thoracic aorta was less marked in losartan-treated uremic rats compared to captopriltreated uremic animals. These differences may be related to the decrease of hemodynamic shear stress on the vascular endothelium, which could also modulate ET-1 production.38 In addition, ACE-I may potentiate the bradykinin-induced release of NO, which also in-

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hibits endothelial ET-1 production.39 In preglomerular arteries, however, both drugs were equipotent in reducing ir-ET-1 concentration. This is possibly attributable to the fact that preglomerular arteries were prepared from the remnant kidneys of two animals per group of uremic rats. Therefore, the results obtained from this pool of preglomerular artery preparations may attenuate the differences between losartantreated and captopril-treated uremic rats. In contrast, neither losartan nor captopril affected ir-ET-1 concentration in the mesenteric arterial bed of uremic rats. Under these experimental conditions, ET-1 production in the mesenteric arteries may depend on other factors that remain to be elucidated. This study also supports a role for ET-1 in the pressor response and the vascular and renal adaptive changes previously attributed to Ang II in chronic renal failure.1,2,29 Recent investigations have shown that ET-1 partially or totally mediates the vasoconstrictor response to Ang II in some vessel preparations in vitro,15 and the hemodynamic effects of Ang II in normal and hypertensive rats in vivo.16 The reduction of Ang II-induced ET-1 production in blood vessels of uremic rats may significantly attenuate its systemic and renal pressor action.3,9 On the other hand, we have reported recently that elevated ET-1 levels in blood vessels are associated with vascular media hypertrophy.24,40,41 In these studies, the degree of vessel wall thickening correlated with the increased in tissue wet weight. Similarly, we found increased tissue wet weight of vessels of uremic rats that exhibited higher ir-ET-1 concentration. In addition, the wet weight of the thoracic aorta and preglomerular arteries was significantly diminished in losartan-treated and captopril-treated uremic rats in which ir-ET-1 concentration was reduced, suggesting that Ang II-induced ET-1 production was also involved in vascular hypertrophy in these animals. This notion is supported by previous investigations showing that an AT1 receptor antagonist and ACE-I prevent vessel wall thickening in rats with reduced renal mass.21 The decrease in wall thickening was significantly less in mesenteric arteries than in other vessels. This is also consistent with the finding that wet weight of the mesenteric arterial bed and ir-ET-1 concentration were similar in untreated and treated uremic rats. In the glomerulus, the beneficial effects of the AT1 antagonist losartan on renal hyperfiltration and glomerular injury such as glomerulosclerosis could be attributable to the reduction in local ET-1 production.19,21 Indeed, increased ET-1 production in glomeruli of uremic rats may induce mesangial cell growth and extracellular matrix protein accumulation.10,11,17 This hypothesis is in keeping with recent studies showing that increased glomerular ET-1 production in glomerulonephritis is associated with mesangial ex-

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pansion.42 Moreover, the effects of Ang II on mesangial matrix protein expression and cell growth are attenuated by a monoclonal antibody against ET-1 or by an ETA receptor antagonist.10,11 Interestingly, these effects of Ang II are totally blocked by a transforming growth factor-b (TGF-b) antiserum, suggesting that Ang II-induced ET-1 production in mesangial cells is mediated by TGF-b. TGF-b is a potent stimulator of ET-1 production in vascular endothelial and glomerular mesangial cells.43 Exagerated TGF-b expression has also been reported in rats with reduced renal mass and is significantly reduced by losartan.44 Finally, it has been suggested that urinary ET-1 excretion reflects its renal production.6,7,45 Recently, we have proposed that increased urinary ET-1 excretion in uremic rats is related to its overproduction in preglomerular arteries and glomeruli but not in the renal papilla where ET-1 production was significantly reduced.8 However, we could not exclude that ET-1 excretion derives from the inner medullary collecting duct epithelial cells and medullary interstitial cells which are normally major sites of ET-1 production in the kidney.28 In the present study, we found that treatment of uremic rats with losartan totally prevented the increase of urinary ir-ET-1 excretion due to the reduction of ir-ET-1 concentration in preglomerular arteries and glomeruli. Consequently, much smaller amounts of ET-1 were filtered by the remaining nephrons and were possibly degraded by neutral endopeptidase localized in the brush border of proximal tubules,46 thus preventing the increase of urinary ET-1 levels. However, treatment of uremic rats with captopril slightly reduced urinary ir-ET-1 excretion due to the decrease of ir-ET-1 concentration in preglomerular arteries only. Therefore, these findings support our hypothesis that urinary ET-1 excretion reflects its production in part in preglomerular arteries and predominantly in glomeruli of rats with chronic renal failure. In summary, this study shows that Ang II is an important modulator of ET-1 production in blood vessels and glomeruli of rats with reduced renal mass. The vascular and renal protective effects of the AT1 receptor antagonist losartan and the ACE-I captopril in this model of chronic renal failure may be attributable, at least in part, to a reduction of ET-1 production.

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2.

Brown MA, Whitworth JA: Hypertension in human renal disease. J Hypertens 1992;10:701–712.

3.

Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411– 415.

4.

Koyama H, Nishzawa Y, Morii H, et al: Plasma endothelin levels in patients with uremia. Lancet 1989;991– 992.

5.

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We thank Danielle Lizotte, Claude Villeneuve, and Luc Caron for their excellent technical assistance and Elisabeth Lemay for her secretarial help.

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