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ACE Inhibition in Anti-Thy1 Glomerulonephritis Limits Proteinuria but Does Not Improve Renal Function and Structural Remodeling.
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PMID:  22479264     Owner:  NLM     Status:  PubMed-not-MEDLINE    
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BACKGROUND/AIMS: ACE inhibitor (ACE-I) treatment effectively inhibits proteinuria and ameliorates the course of various renal diseases. In experimental glomerulonephritis, however, angiotensin II (AngII) infusion has also been shown to be renoprotective. We evaluated the long-term (28 days) course of anti-Thy1 glomerulonephritis in animals with suppressed AngII formation by ACE-I treatment.
METHODS: Brown Norway rats received perindopril (2.8 mg/kg/day, n = 12), dihydropyridine calcium-antagonist amlodipine (Ca-A; 13 mg/kg/day, n = 6) or were left untreated (n = 14). All animals were monitored for blood pressure, proteinuria, and creatinine clearance after anti-Thy1 injection. Renal histology was assessed at day 7 and 28.
RESULTS: Systolic blood pressure was equally reduced by ACE-I and Ca-A treatment. AngII suppression prevented development of proteinuria, but did not protect against glomerular microaneurysm formation or reduction in creatinine clearance. After resolution of the microaneurysms, animals with suppressed AngII production showed a modest increase in glomerulosclerosis and vasculopathic thickening of intrarenal vessels.
CONCLUSIONS: In anti-Thy1 glomerulonephritis, suppression of AngII formation does not protect against the induction of glomerular damage and is associated with mild aggravation of adverse renal fibrotic remodeling. Proteinuria, however, is effectively prevented by ACE-I treatment. Ca-A treatment did not affect the course of glomerulonephritis, indicating that ACE-I effects are blood pressure independent.
Authors:
Peter E Westerweel; Jaap A Joles; Krista den Ouden; Roel Goldschmeding; Maarten B Rookmaaker; Marianne C Verhaar
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Publication Detail:
Type:  Journal Article     Date:  2012-01-26
Journal Detail:
Title:  Nephron extra     Volume:  2     ISSN:  1664-5529     ISO Abbreviation:  Nephron Extra     Publication Date:  2012 Jan 
Date Detail:
Created Date:  2012-04-05     Completed Date:  2012-08-23     Revised Date:  2013-05-29    
Medline Journal Info:
Nlm Unique ID:  101571879     Medline TA:  Nephron Extra     Country:  Switzerland    
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Languages:  eng     Pagination:  9-16     Citation Subset:  -    
Affiliation:
Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
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Journal ID (nlm-ta): Nephron Extra
Journal ID (iso-abbrev): Nephron Extra
Journal ID (publisher-id): NNE
ISSN: 1664-5529
Publisher: S. Karger AG, Allschwilerstrasse 10, P.O. Box · Postfach · Case postale, CH–4009, Basel, Switzerland · Schweiz · Suisse, Phone: +41 61 306 11 11, Fax: +41 61 306 12 34, karger@karger.ch
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Copyright © 2012 by S. Karger AG, Basel
open-access:
collection publication date: Season: Jan-Dec Year: 2012
Electronic publication date: Day: 26 Month: 1 Year: 2012
pmc-release publication date: Day: 26 Month: 1 Year: 2012
Volume: 2 Issue: 1
First Page: 9 Last Page: 16
ID: 3318936
PubMed Id: 22479264
DOI: 10.1159/000335750
Publisher Id: nne-0002-0009

ACE Inhibition in Anti-Thy1 Glomerulonephritis Limits Proteinuria but Does Not Improve Renal Function and Structural Remodeling
Peter E. Westerweela
Jaap A. Jolesa
Krista den Oudena
Roel Goldschmedingb
Maarten B. Rookmaakera
Marianne C. Verhaara*
aDepartment of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands
bDepartment of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands
Correspondence: *Prof. Marianne C. Verhaar, MD, PhD, University Medical Center Utrecht, Department of Nephrology and Hypertension, F03.227, Heidelberglaan 100, NL–3584 CX Utrecht (The Netherlands), Tel. +31 88 755 7380, E-Mail m.c.verhaar@umcutrecht.nl

Introduction

The anti-Thy1 glomerulonephritis model is characterized by marked transient proteinuria and involves both renal injury and repair. It therefore offers the potential to study various components of renal disease. Thy1 is expressed on glomerular mesangial cells. Binding of the anti-Thy1 antibody delivered by single injection results in complement-mediated mesangiolysis with secondary endothelial damage [1, 2]. The model is self-limiting and fully reversible in healthy animals, while uninephrectomized rats injected with anti-Thy1.1 antibody develop progressive glomerulosclerosis [3].

The potent antiproteinuric effects of ACE inhibitors (ACE-I) are well established. Angiotensin II (AngII) is a pathogenic factor in various renal diseases. However, AngII may also have renoprotective effects in early stages of kidney disease development. Stimulation of renal angiogenesis by AngII occurs during physiological postnatal kidney development [4] and drives the accelerated glomerular recovery seen after infusion of AngII during early-phase anti-Thy1 glomerulonephritis [5, 6]. Consistently, glomerular endothelial cell proliferation after anti-Thy1 glomerulonephritis was inhibited with AngII receptor blockade [7]. The attenuated glomerular injury with AngII infusion is in apparent contrast with previous studies in the anti-Thy1 model suggesting attenuation of renal damage by treatment with ACE-I or AngII receptor blocker [7, 8, 9, 10, 11, 12, 13]. The latter studies reported improvement mainly in terms of reduced proteinuria and early reduction of matrix expansion. We hypothesized that the suppression of AngII formation would not improve late-stage renal function in anti-Thy1 glomerulonephritis despite possible antiproteinuric effects. Therefore, we evaluated the effect of ACE-I treatment using perindopril on proteinuria, creatinine clearance, induction of glomerular damage and renal histological recovery in nephritic rats. To dissociate direct effects of ACE-I treatment from those on blood pressure reduction, we also included nephritic rats treated with the dihydropyridine calcium-antagonist amlodipine (Ca-A) in our study.


Animals and Methods
Animals

Male 11-week-old Brown Norway/RijHsd (BN) rats weighing 280–300 g (Harlan, Horst, The Netherlands) housed in a 12/12 h light/dark cycle and receiving food and acidified water ad libitum were used for all experiments. The Animal Ethics Committee of our institution approved all protocols.

Experimental Design

Anti-rat Thy1.1 monoclonal antibody (ER4, 1 mg/kg body weight) was injected intravenously on day 0 in all rats. Rats treated with ACE-I perindopril (gift from Servier; 33 mg/l in the drinking water, resulting in an average dose of 2.8 mg/kg body weight/day; n = 12) since day 3 before anti-Thy1 injection were compared to controls (n = 14) and rats treated with Ca-A (gift from Servier; 150 mg/l in the drinking water, resulting in an average dose of 13 mg/kg body weight/day; n = 6). Rats were placed in metabolic cages with free access to food and water at several time points to collect 24-hour urine samples. Systolic blood pressure was measured in conscious rats by tail-cuff sphygmomanometry (IITC, San Diego, Calif., USA). Rats were sacrificed at day 7 (n = 4 control vs. n = 6 ACE-I-treated animals) and day 28 (n = 10 control vs. n = 6 ACE-I-treated and n = 6 Ca-A-treated animals), and kidneys were excised after perfusion at 120 mm Hg with ice-cold 0.9% saline. Kidney specimens were transversely cut in 1-mm slices, fixed in 4% buffered formaldehyde and embedded in paraffin.

Measurements of Renal Function Parameters

Urinary protein concentration was determined by Bio-Rad Protein assay (Bio-Rad Laboratories GmbH, München, Germany) in 24-hour urine samples. Plasma and urinary creatinine levels were determined colorimetrically (Sigma Diagnostics Inc., St. Louis, Mo., USA). The creatinine clearance, calculated by the standard formula, was used as an estimate of glomerular filtration rate (GFR).

Renal Histology

Renal morphology was evaluated using Periodic Acid Schiff (PAS)-stained 5-μm paraffin sections, based on at least 50 glomeruli per kidney section. The percentage of glomeruli containing microaneurysms was counted in sections from rats terminated at day 7. The Glomerular Sclerosis Index (GSI) was calculated as previously described [14] in sections from rats terminated at day 28. For the GSI, glomeruli were scored based on the percentage of the glomerular area that was sclerotic (normal = 0, <25% = 1, 25–50% = 2, 50–75% = 3, 75– 100% = 4). Renal artery wall thickness of the cortical intrarenal arteries in PAS-stained kidney sections was measured using AnalySIS 3.0 software (Soft Imaging Systems, Münster, Germany), based on lamina externa and interna diameters and using the average of at least five vessels per kidney section.

Statistical Analysis

All values are expressed as mean ± SEM. Data were analyzed using SPSS version 11.0 software. Differences between groups were analyzed using the Student t test, and one- or two-way ANOVA with Kruskal-Wallis post-hoc test if appropriate. A p value <0.05 was considered significant.


Results
ACE-I and Ca-A Lowered Systolic Blood Pressure

Baseline systolic blood pressure was 112 ± 3 mm Hg. At the time of anti-Thy1 injection, systolic blood pressure was reduced by 19 ± 2 mm Hg in ACE-I-treated rats (p < 0.001) and by 22 ± 3 mm Hg in Ca-A-treated rats (p = 0.001), and this level of blood pressure reduction was sustained. The difference in blood pressure reduction for Ca-A and ACE-I treatment was not significant.

ACE-I, but Not Ca-A, Inhibited the Development of Proteinuria after Anti-Thy1 Injection

In control rats, induction of glomerulonephritis caused an increase in urinary protein excretion peaking at day 7 (fig. 1). ACE-I treatment significantly reduced the development of proteinuria and resulted in normalization of proteinuria at day 14 when proteinuria was still high in controls (p < 0.01 for treatment interaction in two-way ANOVA for time and treatment interaction). Ca-A treatment did not affect the development of proteinuria (fig. 1).

ACE-I Did Not Attenuate the Reduction of Creatinine Clearance after Anti-Thy1 Glomerulonephritis

Creatinine clearance was reduced at day 28 in all nephritic rats (table 1). In perindopril-treated animals, creatinine clearance was not beneficially affected.

ACE-I Does Not Prevent Microaneurysm Formation in Early-Phase Anti-Thy1 Glomerulonephritis

Consistent with previous reports [2, 15, 16], injection of anti-Thy1 caused acute mesangiolysis, capillary ballooning and microaneurysm formation at day 7 (fig. 2). ACE-I treatment did not affect the percentage of microaneurysmatic glomeruli (40 ± 4 vs. 41 ± 5% in controls; p = not significant) at that time point. At day 28, microaneurysms were only sporadically observed in sections from both treated and untreated nephritic animals.

ACE-I Treatment Aggravated Adverse Fibrotic Remodelling during Late-Stage Anti-Thy1 Glomerulonephritis

At day 28, nearly all microaneurysms had resolved, but around one third of glomeruli showed segmental glomerular sclerosis (fig. 2). At day 28, ACE-I-treated animals had a slightly higher average glomerulosclerosis score than controls, which was statistically significant (GSI 0.49 ± 0.07 vs. 0.36 ± 0.03, p < 0.05; fig. 3). To further evaluate possible adverse fibrotic remodeling, we assessed intrarenal intima-media thickness as this was pathologically increased with ACE inhibition in experimental kidney transplantation [17]. Indeed also in our model, ACE-I treatment was associated with signs of renal artery vasculopathy, with a significantly increased wall thickness of the cortical arteriesat day 28 (intima-media surface area 4,046 ± 419 vs. 2,689 ± 195 μm2, p < 0.05; fig. 4). GSI in Ca-A-treated animals did not differ from controls (0.39 ± 0.06 vs. 0.36 ± 0.03, p = not significant; fig. 3), and cortical artery wall thickness was unchanged in untreated controls and Ca-A-treated animals at day 28 (fig. 4).


Discussion

A single intravenous injection of anti-Thy1 antibody is known to cause reversible glomerulonephritis characterized by marked transient proteinuria and multiple stages of structural glomerular pathology. Here, we show that ACE-I treatment during anti-Thy1 glomerulonephritis effectively reduces the development of proteinuria, but does not protect against the induction of glomerular damage or decline in renal function. ACE-I treatment could not prevent glomerular microaneurysm formation and was even associated with a modest but statistically significant accentuation of glomerulosclerosis and vasculopathic thickening of intrarenal vessels. Our study indicates that the multiple pathophysiological components during the course of anti-Thy1 glomerulonephritis are differentially affected by the suppression of AngII formation.

We show a potent protective effect of ACE inhibition on the proteinuria occurring during the development of glomerular damage. Several previous studies have similarly shown a reduction in proteinuria during anti-Thy1 glomerulonephritis by treatment with ACE-I or AngII receptor blockers [7, 8, 9, 10, 11, 12, 13]. Consistently, proteinuria tended to stay elevated for a longer period of time when rats with anti-Thy1 glomerulonephritis received AngII infusion [6]. Proteinuria is a practical and powerful indicator of the course of renal disease [18]. Preventing or reducing proteinuria by ACE-I is well-established and likely to make a major contribution to the beneficial effects of ACE-I on the course of renal disease. Proteinuria is thought to not only reflect renal damage, but also play a pathophysiological role in the progression of renal damage itself [19]. Indeed, in the aforementioned studies of ACE inhibition in anti-Thy1 glomerulonephritis, early matrix remodelling was inhibited. In our study, glomerular microaneurysm formation was not reduced by ACE inhibition. As glomerular microaneurysm formation reflects the degree of endothelial damage, the antiproteinuric effects do not appear to be attributable to endothelial protection but may be mediated through another mechanism. Such a specific antiproteinuric effect of ACE-I treatment may be explained by direct effects on the selective properties of the glomerular membrane size [20] independent of the effects on the glomerulonephritic disease process.

Our study shows that in anti-Thy1 glomerulonephritis, inhibition of AngII formation promoted late-stage adverse sclerotic remodelling in both the glomerulus and intrarenal vasculature. This is in line with previous reports indicating that AngII infusion accelerated glomerular endothelial recovery [6], reduced influx of inflammatory cells, reduced mesangial proliferation, and limited matrix production [5]. Similar deterioration of renal function and aggravated graft vasculopathy was observed in association with ACE-I treatment in the Fisher-to-Lewis experimental rat model of chronic renal transplant failure, even though the development of proteinuria and glomerulosclerosis was prevented [17]. Furthermore, in both healthy and adriamycin-induced proteinuric rats on a low-sodium diet, ACE-I treatment caused interstitial renal damage and vasculopathic hypertrophy of intrarenal arteries [21].

We did not observe any effect of dihydropyridine Ca-A treatment on the course of anti-Thy1 nephritis. Unlike ACE-I, Ca-A treatment did not adversely affect the disease course, despite giving comparable blood pressure reduction. Thus, the adverse effects observed with ACE-I treatment appear to be independent of the systemic blood pressure-reducing action. Nevertheless, we cannot exclude a role for altered renal blood flow and filtration pressure, which are affected differently by dihydropyridine Ca-A and ACE-I [22].

In conclusion, our study suggests that ACE inhibition is effective in reducing proteinuria, but does not beneficially affect other pathological components involved in anti-Thy1 glomerulonephritis. This is in line with the concept that AngII may have specific renoprotective effects [23, 24]. Also, these results suggest that in specific conditions, proteinuria may thus not be a reliable indicator of the course of renal disease treated with ACE inhibitors.


Acknowledgements

This work was supported by the Dutch Kidney Foundation (grant PC127) and the Netherlands Organization for Scientific Research (NWO VENI grant 016.036.041 and VIDI grant 917.96.359).

The technical assistance of Paula Martens, Ria de Winter, and Nel Willekes is gratefully acknowledged.


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Article Categories:
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Keywords: Key Words Anti-Thy1 glomerulonephritis, Angiotensin II, ACE inhibitor, Glomerulosclerosis.

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