Vasopeptidase Inhibition Restores Renovascular Endothelial Dysfunction in Salt-Induced Hypertension

J Am Soc Nephrol 12: 2280–2287, 2001 Vasopeptidase Inhibition Restores Renovascular Endothelial Dysfunction in Salt-Induced Hypertension

THOMAS QUASCHNING,* LIVIUS V. D’USCIO,* SIDNEY SHAW,† HERMANN-JOSEF GRO¨ NE,‡ FRANK RUSCHITZKA,§ and THOMAS F. LU¨ SCHER§ *Cardiovascular Research, Institute of Physiology, University of Zu¨rich, Zu¨rich, Switzerland; †Clinical Research, Inselspital, University of Bern, Bern, Switzerland; ‡Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany; and §Cardiovascular Center and Cardiology, University of Zu¨rich, Zu¨rich, Switzerland.

Abstract. Renovascular hemodynamics plays a pivotal role in 6%; P Ͻ 0.05) as well as contractions to endothelin-1 (ET-1) the regulation of BP. The effect of the vasopeptidase inhibitor (98 Ϯ 5% versus 128 Ϯ 5%; P Ͻ 0.05) and big ET-1 (47 Ϯ 6% omapatrilat (O) and the ACE-inhibitor captopril (C) on endo- versus 116 Ϯ 7%; P Ͻ 0.05) were markedly reduced as thelial function in the renal circulation in salt-induced hyper- compared with control animals, whereas standardized aortic tension were investigated. Dahl salt-sensitive rats (n ϭ 6 per weight and heart weight (4.9 Ϯ 0.4 versus 3.2 Ϯ 0.3 g/kg; group) on standard or salt-enriched chow were treated for 8 wk P Ͻ 0.05) increased. Treatment with O restored endotheli- with O (36 Ϯ 4 mg/kg per d), C (94 Ϯ 2 mg/kg per d), or um-dependent relaxations (88 Ϯ 6%; P Ͻ 0.05 versus C) placebo. Renal arteries were suspended in organ chambers for and contractions to ET-1 (120 Ϯ 6%) and big ET-1 (98 Ϯ isometric tension recording. Vascular hypertrophy was as- 9%). O prevented vascular hypertrophy (0.23 Ϯ 0.019 mg/ sessed by determination of standardized heart weight and aortic mm2 versus 0.31 Ϯ 0.018 mg/mm2 in high-salt diet; P Ͻ weight, and morphologic analysis of glomerular injury was 0.05), but, in contrast to C, it only had a modest effect on performed. Systolic BP of salt-fed, placebo-treated animals glomerular injury. In conclusion, O restored renovascular increased to 196 Ϯ 6 mmHg, which was reduced by O (162 Ϯ endothelial function and prevented vascular hypertrophy in 5 mmHg; P Ͻ 0.05) and C (164 Ϯ 7 mmHg; P Ͻ 0.05) to a salt-induced hypertension and therefore may advance as a comparable degree. In salt-induced hypertension, endothelium- beneficial approach in the therapy of various forms of dependent relaxations in renal arteries (56 Ϯ 6 versus 100 Ϯ hypertension.

Inhibition of the angiotensin-converting enzyme (ACE) is a (10). Hence the overall effect of NEP inhibition on vascular well-established treatment option for patients with hyperten- tone will depend on the effects of a compound on the proces- sion and improves morbidity and mortality in large clinical sion of these different vasoactive substances and is—espe- studies (1, 2). The mechanisms involved in the vasculoprotec- cially in molecules, which inhibit other systems as well— tive effects of ACE inhibitors appear—in large part—to be difficult to predict. Nevertheless, omapatrilat (O), a new related to their effects on endothelial function. Indeed, in vasopeptidase inhibitor, effectively lowers BP in salt-depen- human coronary arteries and saphenous veins, endothelium- dent and volume-dependent as well as in renin-dependent dependent relaxations to bradykinin are enhanced after prein- forms of hypertension (11). The combination of ACE and NEP cubation with an ACE inhibitor (3, 4). inhibition may be particularly useful in the treatment of hy- Vasopeptidase inhibition represents a new therapeutic prin- pertension (5, 12–14) and heart failure (15–19). ciple in hypertension (5–7) and heart failure (8), which in- Vasopeptidase inhibitors lower BP in a broader range of cludes inhibition of neutral endopeptidase (NEP) in addition to conditions than inhibition of ACE or NEP alone, and their ACE inhibition. NEP catalyzes the degradation of a number of effectiveness seems to be independent of the activity of the endogenous vasodilator peptides, including atrial natriuretic renin-angiotensin system or the degree of salt retention (20). O peptide, brain natriuretic peptide, C-type natriuretic peptide, is a new vasopeptidase inhibitor that induces long-lasting an- substance P, and bradykinin, as well as vasoconstrictor pep- tihypertensive effects in experimental hypertension (12), tides, including endothelin-1 (ET-1) (9) and angiotensin II greater than those elicited by selective inhibition of either enzyme alone (11). Furthermore, O lowers BP and attenuates cardiac hypertrophy in diabetic hypertensive rats (21). Mean- Received May 9, 2000. Accepted February 27, 2001. while, first clinical data are available, demonstrating hemody- Correspondence to Dr. Thomas F. Lu¨scher, Professor and Head of Cardiology, namic benefits of treatment with O in patients with hyperten- University Hospital, CH-8091 Zu¨rich, Switzerland. Phone: 0041 1 255 21 21; Fax: 0041 1 255 42 51; E-mail: [email protected] sion (14, 22–24) and heart failure (8, 25–27). The first large- 1046-6673/1211-2280 scale clinical study on heart failure indicates reduced morbidity Journal of the American Society of Nephrology and mortality on treatment with O as compared with ACE Copyright © 2001 by the American Society of Nephrology inhibitor treatment (28). J Am Soc Nephrol 12: 2280–2287, 2001 Omapatrilat and Endothelial Function 2281

Despite obvious clinical benefit of vasopeptidase inhibitors mol/L) were obtained in quiescent preparations. To avoid the devel- in heart failure and hypertension, their mechanism of action is opment of tachyphylaxis, only single concentrations of angiotensin I Ϫ7 still poorly understood. A positive influence of O on vessel and angiotensin II were used (10 mol/L). All drugs used in the stiffness (19) and vascular remodeling (29, 30) has been shown organ bath were obtained from Sigma Chemical Co (Buchs, Switzer- before. Also, long-term vasopeptidase inhibition exerts bene- land) apart from ET-1 and big ET-1, which were purchased from Novabiochem/Calbiochem AG (La Jolla, CA). ficial effects in the renal circulation (26). Influences of vasopeptidase inhibitors on endothelial function in the renal artery may substantially improve renal hemodynamics and Vascular and Cardiac Hypertrophy therefore contribute to their beneficial systemic effects. For assessment of vascular hypertrophy, aortic rings were blotted Therefore, this study was designed to investigate the effects dry and weighed, and the arterial surface area of opened rings was measured as described (33). Aortic surface area was calculated using of long-term treatment with the vasopeptidase inhibitor O on formulas for diameter and radius of a cylinder for each individual ring, renovascular endothelial function as well as its effects on and values were averaged. After exsanguination of the animals, hearts vascular hypertrophy in a model of salt-induced hypertension. were removed, isolated, and snap-frozen in liquid nitrogen. Wet weight of hearts was measured, standardized for body weight, and Materials and Methods reported as mg heart weight/kg body weight. Animals Male Dahl salt-sensitive rats 12 wk of age were obtained from Morphologic Analysis of Glomerular Injury Charles River WIGA GmbH (Sulzfeld, Germany) and randomly as- Renal injury was assessed as described previously (34). Briefly, signed to one of four treatment regimens: (1) standard chow (control); paraffin-embedded sections of whole kidneys (5 to 7 ␮m) stained with (2) salt-enriched (4% NaCl) chow (Harlan Teklad, Madison, WI), periodic acid-Schiff reagent were viewed by light microscopy at a ϩ which was given alone (salt diet); (3) together with O (salt O); or magnification of ϫ40 using a Zeiss microscope (Carl Zeiss GmbH, ϩ (4) with captopril (C) (salt C). O and C were provided by Bristol- Jena, Germany). One hundred glomeruli per slide were evaluated. Myers Squibb Pharmaceutical Research Institute (Princeton, NJ). The Morphologic evaluation of glomerular injury was performed using rats were treated for 8 wk, and chow and drug intakes were monitored semi-quantitative scoring methods. Lesions were graded by glomer- during the entire study. Systolic arterial BP and heart rate (HR) were ulosclerosis (grade 1 to 4: 1 to 25%, 26 to 50%, 51 to 76%, and 76 to measured by the tail-cuff method with a pulse transducer (model LE 100% sclerosis, respectively). The glomerular injury score was cal- 5000; Letica, Barcelona, Spain) (31). The study design and the ex- culated by summarizing the products of severity grade times the perimental protocols were approved by the institutional animal care percentage of glomeruli displaying the same degree of severity. committee (Kommission fu¨r Tierversuche des Kantons Zu¨rich, Switzerland). Calculations and Statistical Analyses Tissue Harvesting Relaxations to agonists in isolated arteries are reported as percent precontraction in rings precontracted with norepinephrine to about Animals were anesthetized with pentobarbital (50 mg/kg intraperi- 70% of contraction induced by KCl (100 mmol/L). The contractions toneally) after 8 wk treatment, and blood samples were collected were expressed as a percentage of 100 mmol/L KCl–induced contrac- through puncture of the right ventricle. The renal arteries were re- tions, which were obtained at the beginning of each experiment. moved, dissected free from adherent connective tissue, and placed Results are presented as mean Ϯ SEM. Functional endothelin-con- immediately into cold (4°C) modified Krebs-Ringer bicarbonate so- verting-enzyme (ECE) activity was calculated as the ratio of the lution: 118.6 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl , 1.2 Ϫ 2 contraction to big ET-1 (10 7 mol/L) divided by the contraction to mmol/L MgSO , 1.2 mmol/L KH PO , 25.1 mmol/L NaHCO , 0.026 Ϫ 4 2 4 3 ET-1 (10 7 mol/L). In all experiments, n equals the number of rats per mmol/L ethylenediaminetetraacetic acid, and 10.1 mmol/L glucose. experiment. For statistical analyses, the sensitivity of the vessels to the Under a microscope (Leica Wild M3C, Heerbrugg, Switzerland), drugs was expressed as the negative logarithm of the concentration vessels were cleaned of adherent tissue and cut into 3-mm-long rings. that caused half-maximal relaxation or contraction (pD2). Maximal relaxation (expressed as a percentage of precontraction) or contraction Organ Chamber Experiments was determined for each individual concentration-response curve by Renal artery rings were suspended to fine tungsten stir-ups (diam- nonlinear regression analysis with MatLab software (Math Works eter, 50 ␮m), placed in an organ bath filled with 25 ml Krebs solution, Inc., Natick, MA). For comparison between two values, the unpaired and connected to force transducers (UTC 2, Gould Statham, CA) for t test or the nonparametric Mann-Whitney test was used when appro- isometric tension recording as described (32). After an equilibration priate. For multiple comparisons, results were analyzed by ANOVA period of 60 min, renal artery rings were progressively stretched to followed by Bonferroni’s correction (35). Pearson correlation coeffi- their optimal passive tension (2.0 Ϯ 0.2 g) as assessed by the response cients were calculated by linear regression. Significance was defined to 100 mmol/L KCl in modified Krebs solution (33). Rings were as P Ͻ 0.05. precontracted with norepinephrine (approximately 70% of 100 mmol/L KCl) and relaxations to acetylcholine (ACH; 10Ϫ10 to 10Ϫ5 Ϫ11 Ϫ5 Results mol/L) or sodium nitroprusside (SNP; 10 to 10 mol/L) were Characteristics of Animals obtained. Relaxations to ACH were assessed with and without preincubation of indomethacin (30 min, 10Ϫ7 mol/L) and in the presence or Systolic BP increased after chronic administration of a high- absence of the nitric oxide synthase (NOS) inhibitor NG-nitro-L- salt diet (4% NaCl) in salt-sensitive Dahl rats as compared with Ϫ4 arginine methyl ester (L-NAME) (preincubation for 30 min, 3 ϫ 10 rats on a standard chow at days 14, 28, and 56 after introduc- mol/L). In additional experiments, cumulative concentration-response tion of the diet (Table 1). Treatment with either O or C curves to ET-1 (10Ϫ10 to 10Ϫ7 mol/L) and big ET-1 (10Ϫ9 to 10Ϫ7 prevented the salt-induced BP increase (P Ͻ 0.05 versus rats 2282 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2280–2287, 2001

Table 1. Systolic BP (mmHg) of salt-sensitive Dahl rats during 56 d of treatment with different regimensa

Group Day 0 Day 14 Day 28 Day 56

Control 143 Ϯ 5 146 Ϯ 5 148 Ϯ 6 148 Ϯ 9 Salt diet (4%) 143 Ϯ 4 177 Ϯ 6b 197 Ϯ 6b 196 Ϯ 8b Salt ϩ omapatrilat 140 Ϯ 7 151 Ϯ 6c 156 Ϯ 6c 162 Ϯ 8c Salt ϩ captopril 144 Ϯ 5 149 Ϯ 5c 157 Ϯ 6c 164 Ϯ 7c

a Data are given as mean Ϯ SEM of six rats in each group. Day 0 indicates BP before treatment. b P Ͻ 0.01 versus control rats (ANOVA and Bonferroni’s correction). c P Ͻ 0.05 versus rats on salt diet.

on high-salt diet alone). O, at a mean daily dose of 36.2 Ϯ 4 mg/kg, was equipotent in lowering BP as 94.1 Ϯ 2 mg/kg of C. Changes in heart rate during treatment and differences in heart rate among the treatment groups did not reach statistical significance.

Vascular Relaxations In hypertensive animals, maximal endothelium-dependent relaxations to ACH and sensitivity (pD2 value) in renal arteries were markedly impaired when compared with control rats (Figure 1A; P Ͻ 0.05). Both O and C improved endothelium- dependent relaxations, but the maximal relaxation achieved by O was significantly higher than by C (Figure 1A; P Ͻ 0.05 versus C) and was comparable to the control animals. Prein- cubation with the NOS inhibitor L-NAME blunted relaxations to ACH in all groups completely (Figure 2). In contrast to endothelium-dependent relaxations, maximal endothelium-independent relaxations to the NO donor SNP (Figure 1B) in renal arteries were comparable in all groups. Preincubation with indomethacin (10Ϫ7 mol/L) did not alter maximal relaxations or sensitivity (pD2 value) to either ACH or SNP.

Vascular Contractions Contractions of renal arteries to ET-1 were reduced in Dahl rats on a high-salt diet (Figure 3A; P Ͻ 0.05) and were normalized by long-term administration of O or C, respectively (P Ͻ 0.05 versus placebo-treated, salt-fed Dahl rats for maximal response; Figure 3A). In addition, renal artery contractions to big ET-1 were markedly reduced in salt-sensitive Ͻ Figure 1. Endothelium-dependent (A) and endothelium-independent hypertension (Figure 3B; P 0.05). Treatment with O but not (B) relaxations to acetylcholine (ACH) in renal artery rings of salt- Ͻ with C (P 0.05 for maximal contractions versus O) normal- sensitive Dahl rats after 8 wk of treatment with different regimens. ized contractions to big ET-1 (Figure 3B). Therefore, func- Results are shown as mean Ϯ SEM (n ϭ 6 per group) and are tional ECE activity, expressed as the ratio of the contraction to expressed as percent relaxation of the contraction to norepinephrine (3 Ϫ Ϫ 10 7 mol/L big ET-1 divided by the contraction to 10 7 mol/L ϫ 10Ϫ7 mol/L). ET-1, was significantly lowered in salt-sensitive hypertension (Figure 4; P Ͻ 0.05 versus controls). ECE activity was normalized by O (Figure 3; P Ͻ 0.05 versus placebo treatment) and C (0.28 Ϯ 0.04 versus 0.33 Ϯ 0.06, respectively; NS). but was not significantly affected by C. In addition, ECE Also, ACE activity, as determined by the ratio of the contrac- activity was blunted by incubation with O (10Ϫ7 mol/L) in tion to angiotensin I (10Ϫ7 mol/L) divided by the contraction to vitro (data not shown). angiotensin II (10Ϫ7 mol/L), was significantly reduced by The effectiveness of ACE inhibition, as assessed by deter- either C or O compared with the control group (0.74 Ϯ 0.08; mination of functional ACE activity, did not differ between O P Ͻ 0.01). J Am Soc Nephrol 12: 2280–2287, 2001 Omapatrilat and Endothelial Function 2283

Figure 2. Endothelium-dependent relaxations to ACH in renal artery rings of salt-sensitive Dahl rats after 8 wk of treatment with different Ϫ4 regimens after preincubation with L-NAME (3 ϫ 10 mol/L). Re- sults are shown as mean Ϯ SEM (n ϭ 6 per group) and are expressed as percent relaxation of the contraction to norepinephrine (3 ϫ 10Ϫ7 mol/L).

Vascular and Cardiac Hypertrophy After 8 wk of salt feeding, standardized heart weight of salt-sensitive rats was significantly elevated (4.9 Ϯ 0.4 versus 3.2 Ϯ 0.3 g/kg in control rats; P Ͻ 0.05; Figure 5A), indicating cardiac hypertrophy. Increase in heart weight was prevented by O (3.7 Ϯ 0.4 g/kg; P Ͻ 0.05 versus high-salt diet; Figure 5A) but not by C (4.3 Ϯ 0.3 g/kg, NS). In parallel, aortic weight increased in salt-induced hypertension (0.31 Ϯ 0.018 versus 0.22 Ϯ 0.02 mg/mm2 in control rats; P Ͻ 0.05, Figure 5B). Figure 3. Concentration-dependent contractions to endothelin-1 Vascular hypertrophy was prevented by O (0.23 Ϯ 0.019 (ET-1) (A) and big ET-1 (B) in renal artery rings of salt-sensitive Dahl mg/mm2; P Ͻ 0.05 versus high-salt diet; Figure 5B) but not by rats after 8 wk of treatment with different regimens. Contractions are C (0.28 Ϯ 0.02 mg/mm2, NS). The difference in aortic hyper- expressed as percentage of 100 mmol/L KCl. Results are shown as Ϯ ϭ trophy between C-treated and O-treated animals reached sta- mean SEM (n 6 per group). tistical significance (P Ͻ 0.05).

Morphologic Analysis of Glomerular Injury prevented cardiac and vascular hypertrophy but only tended to High-salt diet induced marked glomerulosclerosis in salt- improve glomerular injury. sensitive Dahl rats (glomerulosclerosis index, 22.0 Ϯ 4.6 ver- Salt-sensitive hypertension is associated with impaired en- sus 9.4 Ϯ 3.2 in control rats on standard chow). Both O and C dothelial function in the aorta (33, 36). Here we extend this reduced glomerular alterations (glomerulosclerosis index, observation to renal arteries in which we documented impaired 16.6 Ϯ 2.6 and 9.6 Ϯ 2.0, respectively), but only the reduction endothelium-dependent relaxations to ACH as well as reduced of glomerular damage by C reached statistical significance (P contractile responses to ET-1 and big ET-1. Long-term treat- Ͻ 0.05 versus salt diet). Both compounds tended to lower the ment with the ACE inhibitor C improved vascular responsive- percentage of sclerotic glomeruli (Figure 6), but a significant ness but did not restore endothelial function to a degree com- reduction of affected glomeruli was only achieved by C in parable to O—even though achieved BP and inhibition of grade 1 and grade 4 sclerotic glomeruli (Figure 6; P Ͻ 0.05 functional and biochemical ACE activity in renal arteries was versus salt diet). comparable in the two treatment groups (21). In vitro inhibi- tory constants of O against ACE and NEP are in the nanomolar Discussion range (11); therefore, sufficient inhibition of both enzymes can In this study, we demonstrated normalization of endothelial be assumed. Thus, with equipotent ACE inhibition, the differ- function in renal arteries of salt-sensitive Dahl rats by the ent effects of the two drugs on renovascular endothelial func- vasopeptidase inhibitor O. O not only improved endothelium- tion must be related to properties of O other than ACE dependent relaxations and renovascular reactivity to ET-1, but inhibition. it also restored renovascular ECE activity. Furthermore, O Vasopeptidase inhibitors simultaneously block ACE and 2284 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2280–2287, 2001

Figure 4. Functional endothelin-converting-enzyme (ECE) activity (given as contraction to 10Ϫ7 mol/L big ET-1 divided by contraction to 10Ϫ7 mol/L ET-1) in renal artery rings of salt-sensitive Dahl rats after 8 wk of treatment with different regimens. Results are shown as mean Ϯ SEM (n ϭ 6 per group). * P Ͻ 0.01 versus control rats; ** P Ͻ 0.01 versus rats on salt diet.

NEP (10), therefore, the metabolism of several vasoactive peptides, such as angiotensin, natriuretic peptides, bradykinin, and ET-1, and their clearance is altered. In accordance with recent findings (33), plasma ET levels were elevated in placebo-treated animals with salt-induced hypertension (37), although functional ECE activity was decreased. Thus, clearance of ET must be reduced in this model. As pure NEP inhibitors cause vasoconstriction due to decreased breakdown of ET-1 (38), selective blockade of this enzyme may not be appropriate under these conditions. In contrast, the combined ACE and NEP inhibitor O normalized both plasma ET-1 levels (37) as well as functional ECE activity. It has to be remembered, Figure 5. Standardized heart weight (A) and standardized aortic however, that the predictive value of ET-1 plasma levels on weight (B) in salt-sensitive Dahl rats after 8 wk of treatment with local, and in particular renal, ET tissue levels is rather limited different regimens. Results are shown as mean Ϯ SEM (n ϭ 6 per (39). However, these findings certainly reflect the complex group). * P Ͻ 0.05 versus rats on salt diet. ** P Ͻ 0.05 versus rats on influence of vasopeptidase inhibition on the endothelin system, omapatrilat (O) treatment. C, captopril. including inhibition of ET-1 degradation (38) as well as inhibition of ET-1 generation from big ET-1 (40). In any case, lowering of plasma ET-1 (37) and elevation of ECE activity vascular bed involved (41,42). As in large renal arteries, how- demonstrate normalization of this altered paracrine system by ever, endothelium-dependent relaxations of renal resistance O in this model of hypertension, and this may be one constit- vessels are impaired in salt-sensitive Dahl rats (43). Corre- uent that contributes to renovascular protection of vasopepti- spondingly, improvement of vasorelaxation in large renal ar- dase inhibitors. teries may contribute to normalization of renal plasma flow and In this study, we only investigated large renal arteries. therefore may be beneficial for both renal function and lower- Whether or not these alterations also occur in renal arterioles is ing of BP. uncertain, particularly because the endothelin system exhibits As in the aorta of salt-sensitive Dahl rats (33), ACH-induced tissue specificity. Distribution of endothelin isoforms and their relaxations in renal arteries were blunted in the presence of the receptors differs in the renal cortex and medulla (39). Also, NOS inhibitor L-NAME and therefore are mediated by NO. In alterations in vascular structure and function in salt-induced renal arteries of hypertensive salt-sensitive Dahl rats, endothe- hypertension are heterogenous, depending on the size and lium-dependent relaxations to ACH, but not the response to J Am Soc Nephrol 12: 2280–2287, 2001 Omapatrilat and Endothelial Function 2285

salt-sensitive hypertension with equipotent dosages of O or C. In contrast to C, O completely restored renovascular NO- mediated relaxation and ECE activity and prevented vascular hypertrophy. Therefore, vasopeptidase inhibition may repre- sent an interesting, new approach in the treatment of hypertension and renovascular disease. In addition to recently published data on vasopeptidase inhibition in hypertension (14, 22–24, 46) and heart failure (8, 26–28), a number of large clinical studies—in part already under way (25, 47)—will be necessary to further evaluate the future clinical role of vasopeptidase inhibitors in the treatment of cardiovascular and renovascular disease and to better understand their beneficial effects.

Acknowledgments This work has been supported by grants from the Swiss National Research Foundation (grant Nr.32–51069.97/1), the Deutscher Aka- Figure 6. Morphologic assessment of glomerular injury in salt-sensi- demischer Austauschdienst (to T.Q.), the ADUMED Foundation and tive Dahl rats. Results are shown as mean Ϯ SEM (n ϭ 5or6per Novartis Foundation (to L.V.d’U.), and Bristol-Myers Squibb Phar- group). * P Ͻ 0.05 versus rats on salt diet. Grade 1 denotes 1 to 25% maceutical Research Institute (Princeton, NJ). sclerosis, grade 2 denotes 26 to 50% sclerosis, grade 3 denotes 51 to 75% sclerosis, and grade 4 denotes 76 to 100% sclerosis. References 1. Hansson L, Lindholm LH, Niskanen L, Lanke J, Hedner T, Niklason A, Luomanmaki K, Dahlof B, de Faire U, Morlin C, SNP, were impaired; therefore, reduced bioavailability of NO Karlberg BE, Wester PO, Bjorck JE: Effect of angiotensin- must be involved, which is in accordance with recent findings converting-enzyme inhibition compared with conventional ther- of reduced endothelial NOS protein expression in this model apy on cardiovascular morbidity and mortality in hypertension: (44). Therefore, restoration of NO bioavailability may contrib- The Captopril Prevention Project (CAPPP) randomised trial. ute to the normalization of endothelium-dependent relaxations Lancet 353: 611–616, 1999 in both treatment groups, but it cannot account for the greater 2. AIRE Study Investigators: Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clin- endothelial protection by O as compared with C. Hence, other ical evidence of heart failure. Lancet 821: 828, 1993 properties of O, such as the reduced breakdown of natriuretic 3. Yang Z, Arnet U, von Segesser L, Siebenmann R, Turina M, peptides, are most likely involved. Lu¨scher TF: Different effects of angiotensin-converting enzyme Besides functional changes, structural vascular and paren- inhibition in human arteries and veins. J Cardiovasc Pharmacol chymal alterations, such as cardiac and vascular hypertrophy 22[Suppl 5]: S17–S22, 1993 (33), occur in salt-induced hypertension. O prevented vascular 4. Auch-Schwelk W, Bossaller C, Claus M, Graf K, Gra¨fe M, Fleck hypertrophy to a greater extent than C did. This is in line with E: ACE inhibitors are endothelium dependent vasodilators of the effect of O in resistance arteries of stroke-prone spontane- coronary arteries during submaximal stimulation with bradyki- ously hypertensive rats (30) and salt-sensitive Dahl rats (29). nin. Cardiovasc Res 27: 312–317, 1993 Surprisingly, assessment of glomerular morphology re- 5. Wallis EJ, Ramsay LE, Hettiarachchi J: Combined inhibition of vealed only moderate improvement of glomerulosclerosis by O neutral endopeptidase and angiotensin-converting enzyme by sampatrilat in essential hypertension. Clin Pharmacol Ther 64: but a significant reduction of the glomerulosclerosis index by 439–449, 1998 C. The lack of renal tissue protection by O contrasts with the 6. Norton GR, Woodiwiss AJ, Hartford C, Trifunovic B, Middle- marked improvement of vascular remodelling. Although the most S, Lee A, Allen MJ: Sustained antihypertensive actions of morphologic assessment may be prone to greater variation than a dual angiotensin-converting-enzyme neutral endopeptidase in- that of larger vessels, this finding requires further investigation hibitor, sampatrilat, in black hypertensive subjects. Am J Hyper- of the mechanisms by which vasopeptidase inhibition influ- tension 12: 563–571, 1999 ences glomerular scarring. Differential, tissue-specific effects 7. Burnett Jr JC: Vasopeptidase inhibition: A new concept in blood of O reflect its complex interaction with local regulatory sys- pressure management [Review]. J Hypertens Suppl 17: S37–S43, tems. Possibly the beneficial effect of O on ET plasma levels 1999 is not paralleled by similar changes of intrarenal ET levels; if 8. Ikram H, McClean DR, Rousseau MF, Fleck E, Juilliere Y, so, elevated renal ET-1 may promote glomerular damage (34). Gronda E, Bryson C, Hanyok J: Omapatrilat, a vasopeptidase inhibitor, produces long-term beneficial haemodynamic and neu- Furthermore, characteristics of the model used in this study rohormonal effects in heart failure. Eur Heart J 20[Suppl 76]: have to be taken into account because O has very recently been 256, 1999 demonstrated to effectively reduce glomerulosclerosis in Gold- 9. Murphy LJ, Corder R, Mallet AI, Turner AJ: Generation by the blatt hypertension (45). phosphoramidon-sensitive peptidases, endopeptidase-24.11 and In conclusion, this study demonstrates marked improvement thermolysin, of endothelin-1 and c-terminal fragment from big of renovascular endothelial function by long-term treatment of endothelin-1. Br J Pharmacol 113: 137–142, 1994 2286 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2280–2287, 2001

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