Angiotensin Type 1 Receptor Antagonism and ACE Inhibition Produce Similar Renoprotection in Nω-Nitro-l>-Arginine Methyl Ester/Spontaneously Hypertensive Rats
Abstract—This study was conducted to determine potentially differential effects between an angiotensin II type 1 (AT1) receptor antagonist and an ACE inhibitor on systemic, renal, and glomerular hemodynamics and pathological changes in spontaneously hypertensive rats (SHR) with Nω-nitro-l>-arginine methyl ester (L-NAME)–exacerbated nephrosclerosis. The hemodynamic, renal micropuncture, and pathological studies were performed in 9 groups of 17-week-old male SHR treated as follows: group 1, controls (n=16); group 2, candesartan (10 mg/kg per day for 3 weeks) (n=7); group 3, enalapril (30 mg/kg per day for 3 weeks) (n=8); group 4, candesartan (5 mg/kg per day) plus enalapril (15 mg/kg per day for 3 weeks) (n=9); group 5, L-NAME (50 mg/L in drinking water for 3 weeks) (n=17); group 6, L-NAME (50 mg/L) plus candesartan (10 mg/kg per day for 3 weeks) (n=7); group 7, L-NAME (50 mg/L) for 3 weeks followed by candesartan (10 mg/kg per day) for another 3 weeks (n=8); group 8, L-NAME (50 mg/L) plus enalapril (30 mg/kg per day for 3 weeks) (n=7); and group 9, L-NAME (50 mg/L) plus enalapril (30 mg/kg per day) and the bradykinin antagonist icatibant (500 μg/kg SC per day via osmotic minipump for 3 weeks) (n=7). Both candesartan and enalapril similarly reduced mean arterial pressure and total peripheral resistance index. These changes were associated with significant decreases in afferent and efferent glomerular arteriolar resistances as well as glomerular capillary pressure. Histopathologically, the glomerular and arterial injury scores were decreased significantly, and left ventricular and aortic masses also were diminished significantly in all treated groups. L-NAME–induced urinary protein excretion was prevented by both candesartan and enalapril. Thus, both AT1 receptor and ACE inhibition prevented and reversed the pathophysiological alterations of L-NAME–exacerbated nephrosclerosis in SHR. Itatibant only blunted the antihypertensive effects of enalapril but did not attenuate the beneficial effects of ACE inhibition on the L-NAME–induced nephrosclerosis. Thus, the AT1 receptor antagonism and ACE inhibition have similar renal preventive effects, which most likely were achieved through reduction in the effects of angiotensin II, and ACE inhibition of bradykinin degradation demonstrated little evidence of renoprotection.
- angiotensin antagonist
- angiotensin-converting enzyme inhibitors
Angiotensin-converting enzyme (ACE) inhibition promotes renoprotection chiefly through inhibition of the renal effects of the renin-angiotensin system.1 2 3 The angiotensin II type 1 (AT1) receptor antagonists may also protect the kidney by antagonizing angiotensin II directly at the receptor site.4 5 However, ACE inhibitors may promote additional renoprotective benefits through their effect of inhibiting bradykinin degradation,6 7 although some reports have suggested that the AT1 receptor blockers may be more protective than the ACE inhibitors.8 9 Unfortunately, only a few studies have used renal micropuncture technology in chronically treated animals to evaluate and compare the agents of 2 classes of renin-angiotensin system inhibitors.4 5
The present study was therefore designed to compare the systemic, renal, and intrarenal effects of an AT1 antagonist and an ACE inhibitor in spontaneously hypertensive rats (SHR) alone, in the presence of NO synthetase inhibition, and when a bradykinin antagonist (HOE140) was used with an ACE inhibitor to determine whether bradykinin contributes to the renal preservation afforded by the ACE inhibitor.
Male SHR (Charles River Laboratories, Wilmington, Mass) aged 17 weeks, housed in plastic cages, and maintained at 20°C in a light-controlled room were permitted free access to standard rat chow feed (PMI Feeds Inc) and tap water. All experimental studies had been approved in advance by our institutional animal care and use committee. The rats were divided into 9 groups (Table 1⇓). All rats (except for those of the control group) were given a once-daily dose of either candesartan or enalapril administered by gastric gavage. Nω-Nitro-l>-arginine methyl ester (L-NAME, Sigma Chemical Co) was administered in the drinking water (50 mg/L),3 10 11 12 13 and the icatibant (HOE140) was administered subcutaneously by means of a minipump (model 2ML4, Alza Co). Two SHR groups, groups 1 and 5, were included in an earlier study11 and are included in the present study so that they may serve as a frame of reference for the evaluation of the other 7 groups. In our preliminary studies, combination therapy with one-half dose each of candesartan and enalapril was demonstrated to reduce mean arterial pressure (MAP) to the comparable extent as full doses of either candesartan or enalapril alone. When we used full doses in the combination therapy (both candesartan and enalapril), the MAP was markedly reduced, causing severe hypotension. Therefore, we chose to use one half the dose of each agent in group 4 (candesartan plus enalapril) to evaluate the effects of combination therapy on renal and glomerular hemodynamics. The urinary protein (UprotV, Lowry method)14 and sodium (UNaV, Beckman Astra 8 frame photometer) excretion rates were obtained before all renal micropuncture studies.3 10 11 12 13
Rats were anesthetized with thiobutabarbital (Inactin, 100 mg/kg IP, Byk-Gulden) and then placed on a temperature-regulated table to maintain rectal temperature at 37°C. After a tracheostomy, an indwelling polyethylene catheter (PE-50) was inserted into the right femoral artery to permit blood sampling and hemodynamic measurements. Arterial pressure was recorded through a transducer (model P23 Dd, Statham Instruments) connected to a multichannel polygraph (Sensor Medics R612, Beckman Instruments). The right carotid artery was cannulated with a thermistor microprobe (Type IT-18, Physitemp Instruments Inc) and connected to a thermodilution device (Cardiotherm 500, Columbus Instruments) for determination of cardiac output. The right jugular vein was also cannulated with a polyethylene catheter (PE-50) for infusion of solutions. The calculated cardiac output was normalized for body weight and expressed as cardiac index (CI, in milliliters per minute per kilogram); the total peripheral resistance index (TPRI) was calculated as the quotient of MAP and CI. The bladder was cannulated with a polyethylene catheter (PE-100) for urine collection. The left kidney was then exposed through a flank incision and suspended in a Lucite cup (packed with cotton) while warm agar was dripped around it to form a saline (0.9% NaCl) well at room temperature. The left ureter was cannulated with a PE-10 catheter for timed urine collection. The right jugular vein was used for [3H]methoxyinulin (850 μCi/mL) infusion (0.1 mL/100 g body wt per hour). The right femoral vein was cannulated for 12% albumin infusion during the first 45 minutes of the surgical procedure (0.4 mL/100 g body wt per hour) and thereafter with saline containing 1% albumin and 1.5% p-aminohippurate (0.4 mL/100 g body wt per hour, Merck Sharp and Dohme).3 11 After an appropriate equilibration period, urine was collected over 4 sequential 30-minute periods; blood samples were withdrawn at their midpoints.
Two or 3 “star vessels” were punctured directly for sampling efferent glomerular arteriolar blood. To determine single nephron glomerular filtration rate (SNGFR), precisely timed (90-second) samples of fluid were collected from 4 to 6 superficial proximal tubules. Efferent (PE), tubular (PT), and stopped-flow (SFP) pressures were measured directly by using a servo-null system (Instrumentation for Physiology and Medicine).3 10 11 12 13 The PT and PE were obtained from the proximal tubule and the star vessel, respectively. The glomerular capillary pressure (PG) was calculated from the sum of SFP and afferent colloid osmotic pressure (πA). Arterial plasma protein concentration was determined refractometrically; πA and efferent colloid osmotic pressure (πE) were calculated from the Landis-Pappenheimer equation (see Falchuk and Berliner15 ). The pressure gradient (ΔP) across the glomerular capillary wall was calculated as ΔP=PG−PT, and the transmembrane colloid osmotic pressure difference (Δπ) was calculated according to the equation of Deen et al,16 as modified by Arendshorst and Gottschalk.17 The PT, PE, and SFP measurements were made in triplicate, and their averages were determined.
[3H]Inulin radioactivity of all tubular fluid, urine, and plasma samples was counted to determine SNGFR and glomerular filtration rate (GFR). These measurements were used to calculate πA and πE, afferent (RA) and efferent (RE) glomerular arteriolar resistances, and the glomerular capillary ultrafiltration coefficient (Kf). At the termination of each study, blood was withdrawn for measurement of serum creatinine and uric acid concentrations by using a 747-100 Analyzer (Boehringer-Mannheim/Hitachi).
After fixation in 10% buffered formalin and embedding in paraffin for light microscopy, the kidneys were cut at a thickness of 2 to 3 μm and stained with hematoxylin and eosin, periodic acid–Schiff, and periodic acid–methenamine–silver.3 10 11 12 13 Histological examination was conducted in a blinded fashion, and glomerular (GIS) and arteriolar (AIS) injury scores were calculated.3 10 11 12 13
All data are expressed as mean±1 SEM. A 1-way ANOVA followed by the Duncan multiple range test was performed for between-group significance.18 The 5% confidence level was considered to be of statistical significance.
Effects of Treatments in the Absence of L-NAME
There were no differences in body weight among the groups (Table 2⇓). Left ventricular and aortic masses were reduced significantly by candesartan, enalapril, and the combination of both agents (at least, P<0.05). In contrast, neither right ventricular nor renal masses were changed in groups 2 and 3, although the combination therapy (group 4) reduced right ventricular mass (P<0.01). Compared with enalapril, candesartan reduced left ventricular mass to a greater extent (P<0.01), whereas aortic mass reduction was greater with enalapril (P<0.01).
Systemic and Renal and Glomerular Hemodynamics
Treatment with either candesartan or enalapril reduced MAP, TPRI, and renal vascular resistance (RVR) (at least, P<0.05; Table 3⇓). The combination therapy had a greater effect on reducing arterial pressure. Candesartan, enalapril, and the combination of both increased stroke index (SI), effective renal plasma flow (ERPF), and GFR (at least, P<0.05). ERPF was also significantly increased by candesartan and the combination treatment.
Of particular note, the hematocrit decreased with candesartan and the treatment combination but not with enalapril. To attempt to determine the basis of this finding, plasma and renal tissue erythropoietin concentrations were analyzed in a separate group of SHR treated chronically with candesartan, but no significant changes were found. (The analysis was performed in the laboratory of James W. Fisher, PhD, Department of Pharmacology, Tulane University, New Orleans, La.) Hence, at this time we are at a loss to explain the hematocrit reduction in the present and other19 studies.
SFP, ΔP, PG, RA, and RE were significantly reduced by all 3 treatments. Enalapril and combination therapy reduced the serum creatinine concentration significantly, whereas candesartan also reduced the serum creatinine concentration, but not significantly.
Effects of Treatments in the Presence of L-NAME
Left ventricular and aortic masses were increased by L-NAME and were reduced significantly when either candesartan or enalapril was administered with L-NAME and when candesartan was given after L-NAME was discontinued (P<0.01, Table 2⇑). In contrast, left kidney masses did not change with treatment (Table 2⇑). Right ventricular mass decreased in the group treated with L-NAME together with candesartan and with enalapril and icatibant (HOE140) (P<0.01). Body weight increased in group 7 rats because, perforce, they were 3 weeks older.
Systemic, Cardiac, and Whole-Kidney Hemodynamics
Cotreatment of either candesartan or enalapril with L-NAME prevented the L-NAME–induced alterations in MAP, TPRI, GFR, and RVR (at least, P<0.05; Table 3⇑). Moreover, when candesartan followed the 3-week administration of L-NAME, the increases in MAP, TPRI, and RVR and decreases in ERPF and GFR were reversed compared with the administration of L-NAME alone (at least, P<0.05; Table 3⇑). Hematocrit was decreased significantly by candesartan with and after L-NAME treatment. Candesartan (either administered with or after L-NAME) increased CI slightly, but enalapril did not.
L-NAME decreased SNGFR, SNPF, and Kf, whereas it increased SFP, ΔP, PG, RA, and RE (Table 4⇓). However, when either candesartan or enalapril was added to L-NAME, it prevented these L-NAME–induced changes (Table 4⇓). Furthermore, when candesartan was administered after the 3-week course of L-NAME treatment, SNGFR, single nephron plasma flow (SNPF), ΔP, PG, RA, RE, and Kf were all reversed, beneficially and significantly (at least, P<0.05). Finally, the UprotV and plasma creatinine concentration were increased by L-NAME, and these were also prevented by both candesartan or enalapril (Table 5⇓).
Glomerular and Arteriolar Injury Scores
Histological analysis demonstrated that L-NAME exacerbated both GIS and AIS compared with the control condition. The GIS of both subcapsular and juxtamedullary cortical glomeruli was more severe in the L-NAME–treated SHR than in their controls, and candesartan or enalapril significantly improved the subcapsular and juxtamedullary cortical GIS (P<0.01). Moreover, the AIS was also improved significantly by both drugs (Table 6⇓).
To determine whether the L-NAME–induced changes could be reversed naturally during a 3-week period after the administration of L-NAME (given only tap water), we studied 8 additional SHR. These SHR demonstrated a reduction only in body weight during that period (304±20 g). Their MAP values (183±6 mm Hg) were slightly lower, and systemic hemodynamics (CI 188±8 mL · min−1 · kg−1, SI 0.485±0.02 mL · beat−1 · kg−1, GFR 0.5±0.1 mL · min−1 · g−1, ERPF 1.3±0.2 mL · min−1 · g−1, and RVR 85.4±27.1 U), UprotV (43.4±8 mg/24 h), and LV mass (LV index 3.61±0.1 mg/g) did not return to pre–L-NAME treatment levels. Furthermore, their histopathologic changes were similar to those produced by L-NAME (total GIS 113±28 versus 91±16). These results support those findings in our previously published report.10 Therefore, the L-NAME–induced nephrosclerosis in SHR did not reverse naturally and spontaneously during a 3-week period after L-NAME withdrawal. It has been reported that NO is upregulated in SHR. This upregulation disappears when L-NAME is given.20 Our present data demonstrate that this possible contribution to L-NAME injury in the SHR nephrosclerosis was not reversible 3 weeks after its discontinuation.
The results of the present study demonstrate that the AT1 receptor antagonist candesartan and the ACE inhibitor enalapril, each alone or in combination (the latter in half dose of each), normalized systemic, renal, and glomerular hemodynamics when used without L-NAME in SHR. Moreover, as we have reported previously with quinapril12 and enalapril,3 candesartan and enalapril, as used in the present study, both prevented and reversed the severe L-NAME–induced nephrosclerosis in SHR. Thus, the adverse systemic and renal hemodynamics were significantly improved, and the severely compromised glomerular dynamics and pathophysiological alterations induced by L-NAME were prevented and reversed. Of particular note, these findings were confirmed in enalapril-treated SHR when the bradykinin receptor antagonist icatibant (HOE140) was administered, simultaneously providing strong evidence that the beneficial effects of the ACE inhibitor on the renal pathophysiological changes induced by L-NAME were not mediated by bradykinin.
The effects of AT1 receptor antagonists and ACE inhibitors have been extensively investigated and compared in experimental and clinical studies. Most studies found similar beneficial pathophysiological changes, including amelioration of proteinuria and glomerulosclerosis.21 22 23 24 A few reports have suggested that AT1 receptor antagonism may be more protective than the ACE inhibitors in five-sixths nephrectomized rats.8 9 However, the present report provides further evidence that the ACE inhibitors and AT1 receptor antagonists provide similar renoprotection (by using micropuncture and histological analysis) in SHR with or without L-NAME.
The principal mechanisms underlying the beneficial effects of the ACE inhibitors and AT1 receptor antagonists provide renoprotection from the adverse pathophysiological effects of angiotensin II.1 2 3 4 5 It is well known that angiotensin II plays an important role in the genesis of proteinuria by adversely altering intrarenal hemodynamics, glomerular capillary permeability, and filtration surface area.1 Moreover, angiotensin II stimulates extracellular matrix protein synthesis in glomerular mesangial cells25 and hypertrophy in glomerular cells.1 Therefore, either by inhibiting angiotensin II generation by ACE inhibition or by antagonizing its effect on the AT1 receptor sites, these agents produce similar renoprotection in SHR. When the differential pharmacological mechanisms of the 2 classes of agents are compared, the issues concerning the effects of bradykinin and angiotensin type 2 receptor have been raised. It is well known that ACE inhibition results in reduced degradation of bradykinin6 7 and that bradykinin may cause selective efferent arteriolar dilatation26 and stimulate endothelial NO formation. This action could provide reverse glomerular capillary hypertension and additional amelioration of renal injury.27 28 To assess this possibility of bradykinin-induced renal protection, we simultaneously administered the bradykinin antagonist icatibant (HOE140) with enalapril. However, the icatibant (HOE140) only blunted the systemic antihypertensive effects of enalapril and failed to attenuate the enalapril-induced effects of renoprotection and the progression of glomerulosclerosis. Thus, even though ACE inhibition increased the available bradykinin, its effects were not unlike those of the AT1 receptor antagonists. On the other hand, the angiotensin II type 2 (AT2) receptor has been shown to exert an antiproliferative effect,29 30 31 which could contribute to the beneficial effect of AT1 receptor antagonism.8 9 Thus, AT1 receptor antagonist feedback could increase plasma renin and angiotensin generation, which, in turn, could stimulate AT2 receptors.7 Although we did not use an AT2 receptor agonist to further evaluate that possibility, our results did demonstrate that the AT1 receptor antagonist and the ACE inhibitor, alone or in combination, exerted similar renoprotection. Hence, these findings provide strong evidence that the beneficial effects of those 2 classes of agents on the kidney were most likely achieved by preventing the adverse effects of angiotensin II rather than an additional effect of bradykinin produced by ACE inhibition or by increased AT2 receptor stimulation produced by further production and generation of angiotensin. However, these findings do indicate that angiotensin II plays a crucial role in L-NAME–induced nephrosclerosis in the SHR by promoting the glomerular pathophysiological changes through the AT1 receptor.
The authors acknowledge Astra Pharmaceuticals for its supplies of candesartan and enalapril and for its support, in part, in the performance of this study. We also acknowledge Aventis Pharma Deutschland GmbH.
Reprint requests to Edward D. Frohlich, MD, Alton Ochsner Distinguished Scientist, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, LA 70121.
- Received July 10, 2000.
- Revision received August 16, 2000.
- Accepted November 7, 2000.
Francischetti A, Ono H, Frohlich ED. Renoprotective effects of felodipine and/or enalapril in spontaneously hypertensive rats with and without L-NAME [published erratum appears in Hypertension. 1998;31:1046]. Hypertension. 1998;31:795–801.
Mackenzie HS, Troy JL, Rennke HG, Brenner BM. TCV 116 prevents progressive renal injury in rats with extensive renal mass ablation. J Hypertens. 1994;12(suppl):S11–S16.
Inada Y, Wada T, Shibouta Y, Ojima M, Daanada T, Ohtsuki K, Itoh K, Kubo K, Kohara Y, Naka T, et al. Antihypertensive effects of a highly potent and long-acting angiotensin II subtype-1 receptor antagonist, (+-)-1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate (TCV-116), in various hypertensive rats. J Pharmacol Exp Ther. 1994;268:1540–1547.
Noda M, Fukuda R, Matsuo T, Ohta M, Nagano H, Imura Y, Nishikawa K, Shibouta Y. Effects of candesartan cilexetil (TCV-116) and enalapril in 5/6 nephrectomized rats. Kidney Int. 1997;63:S136–S139.
Ono H, Ono Y, Frohlich ED. Nitric oxide synthase inhibition in spontaneously hypertensive rats: systemic, renal and glomerular hemodynamics. Hypertension. 1995;26:249–255.
Nakamura Y, Ono H, Frohlich ED. Differential effects of T- and L-type calcium antagonists on glomerular dynamics in spontaneously hypertensive rats. Hypertension. 1999;34:273–278.
Ono H, Ono Y, Frohlich ED. ACE inhibition prevents and reverses L-NAME–exacerbated nephrosclerosis in spontaneously hypertensive rats. Hypertension. 1996;27:176–183.
Lowry OH, Rosenbrough NJ, Farr Al, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.
Falchuk KH, Berliner RW. Hydrostatic pressures in peritubular capillaries and tubules in the rat kidney. Am J Physiol. 1971;220:1422–1426.
Deen WM, Troy JL, Robertson CR, Brenner BM. Dynamics of glomerular ultrafiltration in the rat, IV: determination of the ultrafiltration coefficient. J Clin Invest. 1973;52:1500–1508.
Arendshorst WJ, Gottschalk CW. Glomerular ultrafiltration dynamics: euvolemic and plasma volume-expanded rats. Am J Physiol. 1980;239:F171–F186.
Duncan WG. Multiple range and multiple tests. Biometrics. 1955;11:1–42.
Hayakawa H, Raij L. The link among nitric oxide synthase activity, endothelial function, and aortic and ventricular hypertrophy in hypertension. Hypertension. 1997;29(pt 2):235–241.
Zoja C, Donadelli R, Corna D, Testa D, Facchinetti D, Maffi R, Luzzana E, Colosio V, Bertani T, Remuzzi G. The renoprotective properties of angiotensin-converting enzyme inhibitors in a chronic model of membranous nephropathy are solely due to the inhibition of angiotensin II: evidence based on comparative studies with a receptor antagonist. Am J Kidney Dis. 1997;29:254–264.
Lafayette RA, Mayer G, Park SK, Meyer TW. Angiotensin II receptor blockade limits glomerular injury in rats with reduced renal mass. J Clin Invest. 1992;90:766–771.
Ots M, Mackenzie HS, Troy JL, Rennke HG, Brenner BM. Effects of combination therapy with enalapril and losartan on the rate of progression of renal injury in rats with 5/6 renal mass ablation. J Am Soc Nephrol. 1998;9:224–230.
Kagami S, Border WA, Miller D-E, Noble NA. Ang II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells. J Clin Invest. 1994;93:2431–2437.
Dworkin LD, Hostetter TH, Rennke HG, Brenner BM. Hemodynamic basis for glomerular injury in rats with desoxycorticosterone-salt hypertension. J Clin Invest. 1984;73:1448–1461.
Dworkin LD, Feiner HD. Glomerular injury in uninephrectomized spontaneously hypertension rats: a consequence of glomerular capillary hypertension. J Clin Invest. 1986;77:797–809.
Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci U S A. 1995;92:10663–10667.
Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2 receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651–657.
Tsuzuki S, Matoba T, Eguchi S, Inagami T. Angiotensin II type 2 receptor inhibits cell proliferation and activates tyrosine phosphatase. Hypertension. 1996;28:916–918.