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J Am Soc Nephrol 12:2572-2584, 2001
© 2001 American Society of Nephrology

Blood Pressure-Independent Additive Effects of Pharmacologic Blockade of the Renin-Angiotensin and Endothelin Systems on Progression in a Low-Renin Model of Renal Damage

Kerstin Amann*, Aurelia Simonaviciene{dagger}{ddagger}, Tatiana Medwedewa{dagger}{ddagger}, Andreas Koch{ddagger}, Stephan Orth§, Marie-Luise Gross{ddagger}, Christian Haas{dagger}, Alexander Kuhlmann*, Wolfgang Linz, Bernward Schölkens|| and Eberhard Ritz§

Departments of *Pathology and {dagger}Internal Medicine, University of Erlangen, Erlangen, Germany; Departments of {ddagger}Pathology and §Internal Medicine, University of Heidelberg, Heidelberg, Germany; ||Department of Nephrology and Hypertension, Inselspital, Bern, Switzerland; and Aventis Pharma, Frankfurt, Germany.

Correspondence to Dr. Kerstin Amann, Department of Pathology, University Erlangen-Nürnberg, Krankenhausstrasse 8-10, D-91054 Erlangen, Germany. Phone: 49-9131-85-22291; Fax: 49-9131-85-22601; E-mail: kerstin.amann{at}patho.imed.uni-erlangen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Pharmacologic blockade of the renin and endothelin (ET) systems is an established strategy to interfere with progression of renal failure. In the Heyman nephritis model, additive benefits of decreases in BP with the combination of angiotensin-converting enzyme inhibitors (ACE-i) and ETA receptor antagonists (ET-RA) were demonstrated. To further investigate these findings and to exclude confounding effects of BP decreases, this issue was reassessed in a low-renin model of subtotal kidney resection. Subtotally nephrectomized (SNX) and sham-operated rats were left untreated or received an ACE-i, an angiotensin II subtype 1 receptor antagonist (AT1-RA), an ET-RA, or combinations thereof (ACE-i plus ET-RA or AT1-RA plus ET-RA). The parameters studied were the glomerulosclerosis index (GSI), tubulointerstitial index, vascular damage index, glomerular geometry, and albumin excretion. After 12 wk, BP values were comparable. Urinary albumin excretion rates were significantly higher for untreated SNX rats (24.3 ± 31.3 mg/24 h), compared with untreated sham-operated rats (0.71 ± 0.40 mg/24 h). Rates were significantly lower for all treated, compared with untreated, SNX groups. GSI values were significantly higher for untreated SNX rats than for untreated sham-operated rats. ACE-i caused significantly lower GSI in SNX rats (0.46 ± 0.06), compared with AT1-RA (0.60 ± 0.10) or ET-RA (0.65 ± 0.10). GSI values were significantly decreased further with ACE-i plus ET-RA (0.29 ± 0.09) or AT1-RA plus ET-RA (23 ± 0.05) treatment. Changes in the tubulointerstitial index and vascular damage index proceeded in parallel. The results document BP-independent effects of the ACE-i and AT1-RA on the GSI and urinary albumin excretion and an effect of the ET-RA on the GSI. The contrasting results suggest different pathogenetic pathways for glomerulosclerosis and albuminuria. The combination of treatments provided superior effects on the GSI and tubulointerstitial index but not on urinary albumin excretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has been established that both the renin-angiotensin system (RAS) and the endothelin (ET) system play important roles in the progression of renal disease. Both systems are activated in damaged kidneys, and therapeutic interventions with angiotensin-converting enzyme (ACE) inhibitors (ACE-i) and angiotensin II (AngII) subtype 1 receptor antagonists (AT1-RA) retard progression in experimental models (1–4) and in clinical studies (5–7).

Pharmacologic blockade of the RAS has certainly been one of the great therapeutic breakthroughs in nephrology. Nevertheless, in experimental (8) and clinical (9) observations, progression cannot consistently be completely abrogated by blockade of the RAS, particularly when blockade is initiated late. Pharmacologic blockade of the RAS ameliorates the abnormality in glomerular permselectivity (10,11). In contrast, several studies (12–14) suggested that ET receptor antagonists (ET-RA) have little or no effect on proteinuria. The concept that protein loading of proximal tubular cells provokes tubular expression of ET-1 and (preferentially abluminal) secretion of ET-1 has been proposed (15,16). This hypothesis is in good agreement with the findings of recent studies on the expression of ET-1 and ET receptors in human renal biopsy specimens, which demonstrated more pronounced expression in individuals with marked proteinuria (17).

In view of the incomplete efficacy of monotherapy with ACE-i or AT1-RA in blockade of the RAS, an attractive approach is to combine ACE-i or AT1-RA with ET-RA (18,19). Indeed, using the Heyman nephritis model, Benigni et al. (20) demonstrated that the combination of high doses of the ACE-i trandolapril and the ET-RA LU 135252 reduced proteinuria and glomerular damage more than did the respective monotherapies.

To confirm that important study and to obtain information on the generalizability of the results, we used a model with less-pronounced proteinuria and low activity of the systemic RAS, i.e., rats after surgical ablation of a standardized amount of parenchyma. To exclude confounding effects of BP decreases, nonhypotensive doses of ACE-i and ET-RA were used. Because of concerns that very high doses of LU 135252 (100 mg/kg per d) are not absolutely specific for ETA receptors, substantially lower doses (20 mg/kg per d) were chosen. Consequently, in this study we compared the effects of the ACE-i ramipril, the AT1-RA HR 720, and the ET-RA LU 135252, alone and in combination. In many studies, the effects of ET-RA were demonstrated to be greater in animals after salt loading (21,22). In this study, a low-salt diet (0.2%) was adopted to exclude confounding by this factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
Nine-week-old, male, Sprague Dawley rats (200 g; Möllegaard, Ry, Denmark) were housed in single cages under conditions of constant temperature (22°C) and humidity (50%), with a 12-h dark/light cycle. The animals had free access to water and food. After 3 d of adaptation, the animals were randomly allocated to subtotal nephrectomy (SNX) or sham operation. In a first session, the right kidney was removed under ketamine (100 mg/kg)/diazepam (2.5 µg/kg) anesthesia. Seven days later, the left kidney was subtotally resected by removal of cortical tissue corresponding to two-thirds of the weight of the contralateral kidney, having undergone compensatory hypertrophy. The sham operation comprised decapsulation of the kidney, with special care not to damage the adrenal glands. Twenty-four hours after the second operation, the animals were randomly allocated to the different treatment groups.

The animals received either the ACE-i ramipril (Aventis Pharma, Frankfurt, Germany), the AT1-RA HR 720 (fonsartan; Aventis Pharma), or the specific, orally active, ET-RA LU 135252 (Knoll AG, Ludwigshafen, Germany). The drugs were administered in the drinking fluid. Daily food and water consumptions were measured, and the doses of drugs were determined on the basis of the actual amount of water consumed. The diet (Altromin 1320; Altromin Co., Lage/Lippe, Germany) contained 0.2% sodium, 0.9% calcium, 1.0% potassium, 0.7% phosphorus, and 0.2% magnesium.

The protocol of the study included the following groups (n = 7 to 10 animals/group): group 1, sham-operated control animals; group 2, sham-operated control animals treated with ramipril (1 mg/kg per d) (sham operation plus ACE-i); group 3, sham-operated animals treated with HR 720 (10 mg/kg per d) (sham operation plus AT1-RA); group 4, sham-operated animals treated with LU 135252 (20 mg/kg per d) (sham operation plus ET-RA); group 5, untreated SNX animals; group 6, SNX animals treated with ramipril (1 mg/kg per d) (SNX plus ACE-i); group 7, SNX animals treated with HR 720 (10 mg/kg per d) (SNX plus AT1-RA); group 8, SNX animals treated with LU 135252 (20 mg/kg per d) (SNX plus ET-RA); group 9, SNX animals treated with ramipril plus LU 135252 (doses as noted above) (SNX plus ACE-i plus ET-RA); group 10, SNX animals treated with HR 720 plus LU 135252 (doses as noted above) (SNX plus AT1-RA plus ET-RA).

After 5 and 11 wk, animals were placed in metabolic cages and 24-h urine samples were collected for measurement of urine volume and urinary electrolyte and albumin excretion. Urinary albumin excretion was determined as a marker for glomerular proteinuria, by using a sandwich enzyme-linked immunosorbent assay (ELISA), as described in detail elsewhere (23).

BP Measurements
Systolic BP and heart rate were measured twice during the experiment (weeks 4 and 12), by tail plethysmography with conscious rats. For each animal, six consecutive measurements were performed.

Measurements of Pro-Atrial Natriuretic Peptide, Big ET, AngII, and Aldosterone Levels and Plasma Renin Activity
Pro-Atrial Natriuretic Peptide(1-98) Levels.
In clinical studies, measurements of plasma levels of pro-atrial natriuretic peptide(1–98) [proANP(1–98)] proved to be superior to {alpha}-ANP measurements for the early diagnosis of cardiac dysfunction (24) and renal failure (25). The proANP(1–98) test kit is a sandwich ELISA designed for determination of proANP(1–98) levels directly in biologic fluids (BI-20892; Biomedica, Vienna, Austria) (intra-assay mean, 427 ± 27 fmol/ml; coefficient of variation, 6%; interassay mean, 436 ± 29 fmol/ml; coefficient of variation, 7%; detection limit, 50 fmol/ml).

To provide maximal specificity, the kit incorporates a pair of immunoaffinity-purified polyclonal antibodies raised in sheep. The capture antibody, which is specific for proANP(10–19), is coated onto the microtiter plate. The detection antibody, which is specific for proANP(85–90), is labeled with biotin. In the first step, the sample and the detection antibody are simultaneously added to the wells. ProANP(1–98), if present in the sample, binds to the precoated capture antibody and forms a sandwich with the detection antibody. After a washing step, which removes all nonspecifically bound material, a streptavidin-peroxidase conjugate detects the presence of bound detection antibodies. After removal of unbound conjugate through washing, tetramethylbenzidine is added to the wells as a substrate. ProANP(1–98) is quantitated on the basis of an enzyme-catalyzed color change, which is detectable with a standard ELISA reader. The amount of color development is directly proportional to the amount of proANP(1–98) present in the samples or standards.

Big ET(1–38) Levels.
Elevated levels of big ET have been detected in individuals exposed to cardiovascular stress, such as with acute myocardial infarctions (26) or during and after graft rejection (27). The big ET(1–38) test kit is an ELISA designed for determination of big ET levels directly in ethylenediaminetetraacetate-treated plasma (BI-20072; Biomedica) (intra-assay mean, 6.7 ± 0.26 fmol/ml; coefficient of variation, 3.9%; interassay mean, 6.5 ± 0.39 fmol/ml; coefficient of variation, 6.1%; detection limit, 0.05 fmol/ml).

No extraction or concentration steps are necessary. To provide maximal sensitivity, the kit incorporates an immunoaffinity-purified polyclonal capture antibody and a monoclonal detection antibody (rabbit anti-big ET antibody), which are both highly specific for big ET(1–38). In the first step, the sample and the monoclonal detection antibody are simultaneously added to the wells. Big ET, if present in the sample, binds to the precoated capture antibody and forms a sandwich with the detection antibody. After a washing step, which removes all nonspecifically bound material, a peroxidase-conjugated antibody detects the presence of bound detection antibodies. After removal of unbound conjugate through washing, tetramethylbenzidine is added to the wells as a substrate. Big ET is quantitated on the basis of an enzyme-catalyzed color change, which is detectable with a standard ELISA reader. The amount of color development is directly proportional to the amount of big ET present in the sample.

Plasma Renin Activity, AngII Levels, and Aldosterone Levels.
Determination of plasma renin activity (Renin-Maja; Serono Diagnostika, Freiburg, Germany), plasma AngII levels (RIA kit; Nichols Institute Diagnostika, Bad Nauheum, Germany), and plasma aldosterone levels (Coat-a-Count aldosterone RIA kit; Diagnostic Products, Los Angeles, CA) was performed as described elsewhere (28).

Tissue Preparation
After 12 wk, the experiment was terminated by retrograde perfusion fixation, using 3% glutaraldehyde as fixative (23). After perfusion, the kidneys of each animal were removed for determination of weight and volume; tissue sampling was performed by using area-weighted sampling. Paraffin sections were cut and stained with hematoxylin/eosin and periodic acid-Schiff stain. Semithin sections (1 µm) and ultrathin sections (0.08 µm) from several randomly selected animals were prepared and stained with methylene blue and basic fuchsin or lead acetate, respectively.

Semiquantitative, Morphometric, and Stereologic Measurements
Indices of Renal Damage (Glomerulosclerosis and Tubulointerstitial and Vascular Damage).
All semiquantitative, morphometric, and stereologic investigations were performed in a blinded manner by an observer who was unaware of the study protocol. Glomerulosclerosis (in 100 systematically subsampled glomeruli/animal, as an indicator of the progression of renal failure), tubulointerstitial changes (tubular atrophy, dilation, casts, interstitial inflammation, and fibrosis, as indicators of interstitial damage), and vascular damage (mild, moderate, or severe wall thickening, lumen obliteration, and fibrinoid necrosis) were assessed by using a semiquantitative scoring system (score, 0 to 4) (23). The resulting index for each animal was expressed as a mean of all scores obtained.

Glomerular Geometry.
Paraffin sections were used (stained with hematoxylin/eosin and periodic acid-Schiff stain for assessment by light microscopy, using various magnifications). After determination of kidney weight and volume area, the kidneys were dissected in 1-mm-thick slices perpendicular to the longitudinal axis. By using area-weighted sampling, 10 small pieces were selected for embedding in Epon araldite. All remaining kidney slices were embedded in paraffin, yielding one representative section of each slice for morphometric and stereologic investigations (23). By using systematic subsampling, a Zeiss eyepiece with a 100-point grid (magnification, x100 and x400; Integrationsplatte II; Zeiss, Oberkochen, Germany), and the point-counting method (PP = AA = VV), the volume densities of the cortex, medulla, and glomeruli were determined (29). The glomerular number per area (NA) was counted, and the total glomerular volume (Vglom) was calculated by multiplying the volume density of glomeruli by the cortex volume (VVglom x VC). The glomerular number per volume (NV) was determined using the following equation: NV = k/ß x NA1.5/VV0.5, with k = 1.03 and ß = 1.382 (30).

The total glomerular number (Nglom) was calculated by multiplying the glomerular number per volume (NV) by the cortex volume (VC). The mean glomerular volume (v) was then derived from the total glomerular volume and the total number of glomeruli per kidney (Vglom/Nglom) (30).

In a previous study, this method provided comparable results for mean glomerular volume and a slightly greater total number of glomeruli per kidney, compared with the unbiased fractionator technique (31).

Mean Cell Number per Glomerulus.
The numbers of cells per glomerulus and per tuft area were counted in 100 randomly selected glomeruli per animal.

Statistical Analyses
Data are provided as mean ± SD. After testing for normality, ANOVA or the Kruskal-Wallis test was chosen for ANOVA. For assessment of differences between groups, the Duncan multiple-range test was used. The results were considered significant when the probability of error (P) was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animal Data
After 12 wk, body weights were comparable in all groups (Table 1). The absolute (in grams) and relative (in grams per kilogram body weight) weights of the perfused left kidney were comparable for all sham-operated control groups. The weight of the left kidney remnant was significantly higher in untreated, AT1-RA-treated, and ET-RA-treated SNX animals, compared with sham-operated control animals. Systolic BP and heart rate at the end of the experiment were not significantly different among the groups. Serum creatinine and urea concentrations were comparable for all sham-operated control groups; values were significantly higher for all SNX groups, compared with sham-operated control animals (Table 2). Aldosterone concentrations were significantly higher in untreated, ACE-i-treated, and AT1-RA-treated SNX animals than in sham-operated animals. ProANP levels were not statistically different among the experimental groups. Renin activity was lower in untreated SNX animals, compared with sham-operated control animals and all treated SNX groups. Big ET levels were 3.8 ± 3.7 fmol/ml in untreated sham-operated rats and 3.3 ± 1.9 fmol/ml in untreated SNX rats. The concentrations were not significantly different in the treated subgroups. The urinary sodium excretion rate was significantly lower in untreated SNX animals (38.8 ± 13.7 mmol/24 h), compared with untreated sham-operated animals (68.4 ± 23 mmol/24 h). Excretion was significantly greater in SNX animals treated with LU 135252 (55.5 ± 12.1 mmol/24 h) and SNX animals treated with the combination therapies (62.7 ± 17.9 and 71.2 ± 18.5 mmol/24 h, respectively).


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  		Table 1. Animal dataa
 

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  		Table 2. Serum parametersa
 
Urinary Sodium and Albumin Excretion
Urinary sodium excretion rates were significantly lower in untreated SNX animals than in the treated sham-operated control groups (Table 1). Values were significantly higher, however, for ET-RA-treated SNX animals and both groups of SNX animals given combination treatments, compared with untreated SNX rats.

In SNX rats, urinary albumin excretion rates increased progressively between the fifth and 11th weeks of the experiment. Excretion was significantly lower in all treated SNX groups, compared with untreated SNX rats.

Structural Parameters of Renal Damage
Glomerulosclerosis Index.
Glomerulosclerosis index (GSI) values were comparable for all sham-operated groups (Figures 1A and 2). GSI values were significantly higher in untreated SNX rats than in sham-operated and treated SNX groups. ACE-i treatment led to significantly lower values, compared with AT1-RA or ET-RA treatment. The GSI values were lowest, however, in the groups that received the combination therapy.



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  		Figure 1. Effects of treatment with an angiotensin-converting enzyme inhibitor (ACE-i), angiotensin II subtype 1 receptor antagonist (AT1-RA), and endothelin subtype A receptor antagonist (ET-RA), alone or in combination, on the semiquantitative indices of renal damage in subtotally nephrectomized (SNX) rats. Data are given as mean ± SD. *P < 0.05 versus sham-operated groups. +P < 0.05 versus untreated SNX animals. §P < 0.05 versus SNX animals treated with ACE-i. °P < 0.05 versus SNX animals treated with AT1-RA. #P < 0.05 versus SNX animals treated with ET-RA. $P < 0.05 versus SNX animals treated with ACE-i plus ET-RA. (A) Glomerulosclerosis index (GSI). The GSI was significantly higher in untreated and ACE-i-, AT1-RA-, and ET-RA-treated SNX animals, compared with sham-operated control animals. The GSI was significantly lower in all treated SNX groups, compared with untreated SNX animals. (B) Index of tubulointerstitial damage. The index of tubulointerstitial damage was significantly higher in untreated and ACE-i-, AT1-RA-, and ET-RA-treated SNX animals, compared with sham-operated control animals. The index was significantly lower, however, when SNX animals were treated with ACE-i, AT1-RA, ET-RA, or either combination therapy. (C) Index of vascular damage. The index of vascular damage was significantly higher in all SNX groups, compared with sham-operated control animals. The index was significantly lower in all treated SNX animals, compared with untreated SNX animals.

 


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  		Figure 2. Effects of ACE-i, AT1-RA, and ET-RA, in monotherapy or combination therapy, on the development of glomerulosclerosis in SNX rats. Paraffin sections. Magnification, x400. Glomerular enlargement, with a slight increase in cell number and mesangial matrix deposition (*), was evident in untreated SNX rats (B), compared with untreated sham-operated control animals (A). These changes were nearly completely prevented by ACE-i (C) and both combination treatments (F and G). In contrast, mesangial matrix deposits (*) were still present in AT1-RA-treated (D) and ET-RA-treated (E) rats.

 
Tubulointerstitial Index.
Tubulointerstitial indices were comparable for all sham-operated rats (Figure 1B). The indices were significantly higher in untreated SNX animals than in sham-operated or treated SNX animals. Tubulointerstitial indices for the combination-treated groups were significantly lower than those for groups treated with monotherapies and were not different from sham-operated group values. Treatment of SNX animals with ACE-i resulted in significantly less tubulointerstitial damage, compared with ET-RA treatment.

Vascular Index.
The indices of vascular damage were comparable for all sham-operated groups (Figure 1C). Indices were significantly higher in untreated SNX animals than in sham-operated or treated SNX animals. Vascular index values were significantly lower for AT1-RA-treated SNX animals than for ACE-i-treated animals, ET-RA-treated animals, and both combination-treated groups.

Glomerular Geometry
The number of glomeruli per left kidney was significantly lower in all SNX animals, compared with sham-operated rats (data not shown). The mean glomerular volume was significantly higher in SNX animals, either untreated or treated with ACE-i or AT1-RA, compared with sham-operated animals (Table 3). Glomerular enlargement was somewhat decreased in ET-RA-treated rats but was significantly less only in SNX animals treated with ACE-i plus ET-RA or AT1-RA plus ET-RA.


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  		Table 3. Mean glomerular volume and cell number per glomerulus or per glomerular area
 
Number of Glomerular Cells per Glomerulus
The mean cell number per glomerulus tended to be higher in untreated SNX animals (71.5 ± 14.0 cells/glomerulus) than in untreated sham-operated animals (60.0 ± 7.3 cells/glomerulus), but this difference was not statistically significant (Table 3). In contrast, cell density (i.e., the number of glomerular cells per glomerular tuft area) was significantly lower in untreated SNX animals. The mean cell number per glomerulus was slightly but not significantly lower in SNX animals treated with ACE-i, AT1-RA, ET-RA, or ACE-i plus ET-RA. This parameter was significantly lower only in AT1-RA- plus ET-RA-treated SNX animals. The same was true for the mean number of cells per tuft area.

Investigation of Glomerular Structural Changes Using Semithin and Ultrathin Sections
Structural analysis of glomerular changes in untreated SNX rats (Figures 3B and 4B) demonstrated podocyte hypertrophy and degeneration, as well as a slight increase in mesangial cell number, with focal deposition of mesangial matrix, compared with untreated sham-operated control animals (Figures 3A and 4A). These changes were almost completely prevented by ACE-i (Figures 3C and 4C) and both combination therapies (Figures 3, F and G, and 4, F and G) but not by AT1-RA (Figures 3D and 4D) or ET-RA (Figures 3E and 4E). In sham-operated control animals, glomerular morphologic features were not affected by treatment (data not shown).



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  		Figure 3. Effects of ACE-i, AT1-RA, and ET-RA, in monotherapy or combination therapy, on glomerular cells and capillaries in SNX rats. Semithin sections. Magnification, x400. Hypertrophy and degeneration of podocytes (arrows), as well as a slight focal increase in mesangial cell number and focal mesangial matrix deposition (*), were present in untreated SNX rats (B), compared with the normal morphologic features in untreated sham-operated control rats (A). These changes were no longer visible when animals were treated with ACE-i (C) or either combination treatment (F and G). They were present, however, in AT1-RA-treated (D) and ET-RA-treated (E) rats.

 


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  		Figure 4. Effects of ACE-i, AT1-RA, and ET-RA, in monotherapy or combination therapy, on the glomerular ultrastructure in SNX rats. Ultrathin sections. Magnification, x2500. Glomerular enlargement, degenerative podocyte changes (arrows), and mesangial cell activation (*) in untreated SNX rats (B) compared to sham (A) were partly prevented by ACE-i treatment (C) and both combination therapies (F and G) but not by AT1-RA (D) or ET-RA (E) treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study in a low-renin model of controlled surgical renal ablation without salt loading, the main finding was the observation that low doses of the ETA receptor-specific antagonist LU 135252 in monotherapy, which did not decrease BP, caused moderate reduction of albuminuria, glomerular volume, and glomerular damage. This compound caused further marked reduction of urinary albumin excretion, early and late in the experiment, when combined with either ACE-i or AT1-RA. Combination therapy also resulted in further significant reduction of histologic evidence of glomerular, tubulointerstitial, and vascular damage, despite having no effects on BP.

When designing this study, we addressed several problems, as follows. Counting of glomeruli in the kidney remnant documented that consistently approximately 75% of nephrons were resected. As a result, glomerular volume increased only moderately, by a factor of 2. Although glomerular cell proliferation could not be measured by using the proliferating cell nuclear antigen detection technique after tissue fixation, the number of cells per glomerulus remained unchanged. As a result of glomerular enlargement via increased deposition of matrix, the cell density was lower despite unchanged absolute cell numbers (Table 3). Although BP levels were high (possibly because of stress during tail plethysmography), there was no significant increase in BP among SNX animals. This finding was confirmed in repeated BP measurements. The low systemic activity of the RAS in this model was confirmed by measurement of plasma renin activity. The model is characterized by a relatively moderate but progressively increasing urinary albumin excretion rate (i.e., 24.3 mg/d after 12 wk), compared with the marked proteinuria (i.e., 800 mg/d) in the Heymann glomerulonephritis model (20). Because of the known potentiating effects of salt loading on ET receptor blockade (21,22,32), a diet with relatively low sodium content was chosen. Urinary sodium excretion was monitored, and the absence of major salt loading was also documented by measurement of plasma ANP concentrations.

At the time when the experiment was being planned, it was uncertain whether the effects of ACE-i on the kidney could be duplicated by AT1-RA (33). Therefore, we examined low doses of both an ACE-i and an AT1-RA. Meanwhile, the careful study by Ots et al. (34) clearly demonstrated that enalapril and losartan exerted similar effects on renal injury in the hypertensive high-renin model of rat kidney ablation. It is of interest that, despite the marked effects of the ACE-i on indices of glomerular and tubular damage, the compound was less effective with respect to the index of vascular damage, in contrast to the AT1-RA. This finding may be related to the paradoxical increase in vascular abnormalities in knockout models of AngII type 1A and 1B receptors, angiotensinogen, and ACE (35–38). Relatively little effect of ACE-i on vascular changes was also observed by Kakinuma et al. (39). These unanticipated differences certainly warrant further investigations.

The dose of ramipril selected corresponds to 14 times the maximal therapeutic dose for human patients, on a per-kilogram-per-day basis. In a previous study by our group, this dose was demonstrated to produce a sixfold increase in plasma renin activity (40,41). The relatively modest effect on BP is most likely attributable to stress-induced catecholamine release during tail-cuff measurements (40).

In this study, urinary ET-1 excretion was not measured, because these measurements had already been performed in two previous studies using the same animal model, 8 and 12 wk after SNX (14,32). At both time points, urinary ET-1 excretion was significantly higher in untreated SNX animals than in sham-operated control animals (14,32). In addition, in the study by Nabokov et al. (14), renal ET-1 mRNA contents were measured by using the reverse transcription-PCR technique and were observed to be significantly higher in SNX animals than in sham-operated control animals at the end of the 8-wk study.

In our study, the ET-RA had no effect on urinary albumin excretion at 5 wk. At 11 wk, however, the ET-RA caused moderate but significant decreases (50%), compared with the marked decreases (>90%) produced by the ACE-i. However, even in the early phase of the experiment (at 5 wk), the combination of the ET-RA with either the ACE-i or the AT1-RA caused further significant decreases in albuminuria. The modest antialbuminuric effect of ET-RA contrasts with a striking effect on the GSI, which was comparable to that of the AT1-RA and only slightly less than that of the ACE-i. This finding contrasts with the fivefold reduction of GSI in the groups given the combination therapies, i.e., ET-RA plus ACE-i or ET-RA plus AT1-RA. These effects were at least additive, which suggests that the two interventions operate through different pathogenetic pathways.

In a model of extensive renal mass reduction by arterial ligation, Benigni et al. (42) documented that renal ET protein production was higher than that in control animals and was further increased after stimulation with thrombin. In the same model, Orisio et al. (43) observed that ET gene expression was increased in the remnant kidney and was correlated with disease progression. It has been proposed that activation of the renal ET system is primarily stimulated by tubular protein overload (15,16), and this hypothesis was confirmed by our studies of patients with glomerulonephritis (17). However, it must be acknowledged that, in addition to albumin, other proteins excreted under proteinuric conditions could contribute to tubular activation and consequent inflammation. This may be one possible explanation for the finding that the indices of glomerular and tubulointerstitial damage were decreased out of proportion to the reduction of albuminuria by ET-RA and particularly by ET-RA plus ACE-i or AT1-RA. One hint in this direction involves the effects on the mean glomerular volume, which tended to be smaller with ET-RA treatment and was significantly smaller with the combination therapies, compared with the respective monotherapies. This is not completely surprising, in view of the effects of ET on mesangial cells (for review, see reference (44). We acknowledge that the effects on glomerular volume were not paralleled by commensurate effects on glomerular cell numbers per glomerular profile and per area, but we cannot exclude the possibility that this difference is explained by greater precision of the three-dimensional volume measurements.

There has been much recent interest in potential differential roles of ETA and ETB receptor subtypes in ET-mediated target organ injury. Because of concern that the highly bioavailable ET-RA LU 135252 might lose its selectivity for the ETA receptor subtype at very high doses (45), doses were chosen for this experiment that were lower by a factor of 5, compared with the earlier study by Benigni et al. (20). We acknowledge, however, that a previous study in our laboratory demonstrated similar effects of specific and nonspecific ET-RA (14).

In summary, this study confirms the important findings by Benigni et al. (20) that the combination of ET-RA and ACE-i (or AT1-RA) is more effective in reducing indices of renal injury, compared the respective monotherapies. This study extends the previous findings by indicating that this effect is demonstrable even in a model without major proteinuria, is BP independent, and can be observed in the absence of salt loading and at doses at which the ET-RA is specific for ETA receptors. We are certainly aware of important species differences in the ET system. Nevertheless, in view of these consistent observations in rats, it is intriguing to propose that this strategy should be studied in human patients as well.46


   Acknowledgments
 
This work was performed with the support of the Deutsche Forschungsgemeinschaft (Am 93/2-3 and SFB 423 Project B8). Dr. Koch was a member of the Graduiertenkolleg Nieren- und Kreislaufregulation Heidelberg of the Deutsche Forschungsgemeinschaft. Dr. Medwedewa was the recipient of a Jelzin research grant from the University of Moscow. The skillful technical assistance of Z. Antoni, A. Bönisch, H. Derks, K. Herbig, P. Rieger, S. Söllner, M. Weckbach, and H. Ziebart is gratefully acknowledged.


   Footnotes
 
Thomas Coffman served as guest editor and supervised the review and final disposition of this manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 2, 2000. Accepted for publication July 8, 2001.




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