Departments of 1 Nephrology and 2 Pathology and Laboratory Medicine, Groningen University Medical Center, Groningen, The Netherlands
Correspondence and offprint requests to: G. J. Navis, Groningen University Medical Center, Department of Nephrology, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Email: g.j.navis{at}int.azg.nl
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Abstract |
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Methods. Renal ACE activity (Hip-His-Leu cleavage by cortical homogenates) was determined by renal biopsy in 27 adult male Wistar rats. After 1 week of recovery, proteinuria was induced by adriamycin [1.5 mg/kg intravenously (i.v.) n = 18; controls, saline i.v. n = 9]. Proteinuria was measured every 2 weeks. After 12 weeks, rats were sacrificed and their kidneys harvested.
Results. As anticipated, adriamycin elicited nephrotic range proteinuria, renal interstitial damage and mild focal glomerulosclerosis. Baseline renal ACE positively correlated with the relative rise in proteinuria after adriamycin (r = 0.62, P<0.01), renal interstitial -smooth muscle actin (r = 0.49, P<0.05), interstitial macrophage influx (r = 0.56, P<0.05), interstitial collagen III (r = 0.53, P<0.05), glomerular
-smooth muscle actin (r = 0.74, P<0.01) and glomerular desmin (r = 0.48, P<0.05). Baseline renal ACE did not correlate with focal glomerulosclerosis (r = 0.22, NS). In controls, no predictive values for renal parameters were observed.
Conclusion. Individual differences in renal ACE activity predict the severity of adriamycin-induced renal damage in this outbred rat strain. This supports the assumption that differences in renal ACE activity predispose to a less favourable course of renal damage.
Keywords: ACE; adriamycin nephrosis; proteinuria; renal ACE activity; renal damage; Wistar rats
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Introduction |
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In man, the I/D polymorphism of the ACE gene accounts for half of the variance of circulating and tissue ACE levels, with the highest ACE levels in DD homozygotes, the lowest in II homozygotes, and intermediate values in heterozygotes [4,5]. The D allele of the ACE genotype was reported to be associated with a worse renal prognosis in several renal conditions [6], albeit not uniformly so [7,8]. The initiation of renal disease, however, does not appear to be associated with the D allele. Based on these data, it has been hypothesized that genetically determined individual differences in renal ACE activity are relevant to the extent of renal damage that develops in response to injury [9] and, more specifically, that a higher renal ACE activity predisposes to a more progressive course of renal damage. In line with this assumption, established renal damage was associated with elevated renal ACE activity in different rat models of renal disease [10,11]. These data, however, invariably were obtained after development of renal damage. Therefore, they do not allow the identification of renal ACE as a factor predisposing to renal damage, as the elevated ACE activity might just as well have been the consequence of renal damage.
Therefore, to test whether higher renal ACE activity predisposes to a more progressive course of renal damage, we prospectively determined renal (and plasma) ACE activity in healthy Wistar rats prior to induction of nephrosis by a single injection of adriamycin, and investigated its predictive value for the subsequent development of renal damage. Adriamycin nephrosis provides a well-characterized model of progressive renal damage, induced by a uniform challenge at a single point in time. This results in proteinuria and subsequent structural renal damage with a relatively large variability between individual animals. In healthy outbred Wistar rats, the plasma and tissue ACE levels display a relatively large inter-individual variability, which allows to test for the predictive value of naturally occurring differences in ACE activity.
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Methods |
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Renal biopsy
After baseline values had been obtained, renal biopsy was performed under isoflurane/O2/N2 anaesthesia. Tissue was resected from the lower pole of the right kidney through a dorsolateral incision. Local haemostasis was obtained by application of gelfoam (Spongostan®, Ferrosan, Copenhagen, Denmark). The median amount of renal tissue removed was 0.33 g (0.290.39). Renal tissue was processed further for determination of ACE activity as described below. During the same procedure, blood samples were obtained by orbital puncture for determination of plasma ACE activity and creatinine.
Nephrosis induction
After 1 week of recovery from biopsy, nephrosis was induced by a single intravenous (i.v.) injection of adriamycin under light anaesthesia. Nineteen rats received 1.5 mg/kg adriamycin by tail vein. Nine rats serving as controls received saline.
Termination protocol
Twelve weeks after the induction of nephrosis, animals were anaesthetized and blood pressure was measured. Subsequently, the abdomen was opened through a midline incision. A 2 ml blood sample was obtained via aortic puncture for determination of plasma ACE activity and creatinine. Kidneys were saline perfused and harvested and animals were sacrificed. Renal cortical tissue from the upper pole was processed for ACE determination. Mid-coronal renal tissue slices were processed for histological and immunohistochemical examination.
Measurements
Urinary protein excretion was measured by means of a third generation nephelometer (Dade Behring, Mannheim, Germany) by using a 20% trichloroacetic acid solution. Systolic blood pressure (SBP) was measured by tail cuff. Plasma creatinine level was determined colorimetrically (Sigma Chemical Co., St Louis, MO).
ACE activity was determined as described previously [12]. Renal cortex tissue was homogenized in a 50 mM K2PO4 buffer at pH 7.5. Subsequently, 100 µl of the diluted sample was pipetted into a 0.5 M K2PO4 buffer. Then, the substrate [100 µl of 12.5 mM Hip-His-Leu (Sigma)], which is cleaved by ACE, was added. This was incubated at 37°C for exactly 15 min. At this amount the substrate is present in excess, and thus is not rate limiting for the reaction. The conversion of the substrate was stopped by adding 1.45 ml of 280 mM sodium hydroxide. Then, 100 µl of 1% phtaldialdehyde, which adheres to the formed bipeptide His-Leu, was added. The amount of tagged His-Leu was determined fluorimetrically at 364 nm excitation wavelength and 486 nm emission wavelength. This yields a measure of the amount of His-Leu generated in the sample. In blank samples, sodium hydroxide was added to prevent conversion. The substrate was added after the incubation period. The coefficient of variation was 6% for these measurements of ACE activity using this method.
Histological procedures
Renal tissue was fixed in 4% paraformaldehyde and processed for paraffin embedding. For morphological evaluation, 4 µm sections were cut. One series was stained with periodic acidSchiff (PAS). Another series was stained with polyclonal rabbit anti-rat collagen III antibody (Biogenenesis Ltd, Poole, UK). An automated staining system was used on series for macrophages (ED-1; Serotec Ltd, Oxford, UK), the pre-fibrotic markers -smooth muscle actin (
-SMA; clone 1A4; Sigma Aldrich, St Louis, MO) and desmin (clone D33; DAKO Cytomation, Glostrup, Denmark). Sections were first dewaxed and subjected to heat-induced antigen retrieval by overnight incubation in 0.1 M TrisHCl buffer at 80°C. Endogenous peroxidase was blocked with 0.075% H2O2 in phosphate-buffered saline (PBS) for 30 min. Antibody dilutions were made in PBS supplemented with 1% bovine serum albumin. Antibody binding was detected using sequential incubations with peroxidase-labelled rabbit anti-mouse and peroxidase-labelled goat anti-rabbit antibodies (RAMPO/GARPO Dakopatts, DAKO). Normal rat serum (1%) was added to the secondary antibodies. Peroxidase activity developed by using 3,3-diaminobenzidine tetrachloride for 10 min.
PAS-stained sections were used for determination of focal glomerular sclerosis (FGS) and mesangial matrix expansion (MME). The degree of FGS and MME was assessed by scoring 50 glomeruli per kidney semi-quantitatively on a scale of 04. FGS was scored positive when mesangial matrix expansion and adhesion of the glomerular visceral epithelium to Bowman's capsule were present in the same segment. If 25% of the glomerulus was affected, a score of 1 was adjudged, 50% was scored as 2, 75% as 3, and 100% as 4. The ultimate score is obtained by multiplying the degree of change by the percentage of glomeruli with the same degree of injury and adding these scores, thus rendering a theoretical range of 0200.
The number of glomerular ED-1-positive cells was determined by manual counting of 50 glomeruli per kidney. Interstitial macrophage number was determined by manual counting of 30 cortical fields per kidney.
Interstitial -SMA and collagen III were measured using computer-assisted morphometry on 30 cortical fields per kidney, excluding vessels and glomeruli. The surface area found positive was divided by the total area of the field measured, providing a percentage of
-SMA-positive tissue. Glomerular
-SMA was determined on 50 glomeruli per kidney. The glomerular visceral epithelial desmin staining was determined by manual counting of 50 glomeruli per kidney, on a scale of 0100%, with intervals of 10%.
Statistical analysis
Statistical analyses were performed on a total of 27 animals: 18 adriamycin-treated animals and nine controls. The analysis on predictive value of endogenous renal ACE activity was performed on data obtained from the right kidney at biopsy and at termination. Left kidneys obtained at termination were compared with the right kidney termination data to test for possible harmful consequences of the biopsy.
Data are expressed as median and 95% confidence interval of the median, calculated according to the ranks method by Altman. Differences between experimental and control groups were analysed with MannWhitney U test. Wilcoxon's non-parametric test for paired samples was used to compare parameters from baseline with termination. Pearson's coefficients were calculated to account for bivariate correlation. Statistical significance was assumed at the 5% level. Analyses were performed by SPSS version 10.0.
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Results |
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Finally, the between-individual differences in renal and plasma ACE activity were consistent between the start and the end of the study. This is illustrated in Figure 3, providing the correlation between renal ACE at baseline and at termination (upper panel) and the correlation between plasma ACE at baseline and at termination (lower panel, n = 16 for adriamycin-treated and n = 8 for control animals).
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Discussion |
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The differences in baseline renal ACE activity can be considered to reflect naturally occurring differences between these outbred animals. Interestingly, in these adriamycin-treated rats, the between-individual differences found at baseline were consistent throughout the study from the pre-induction values in normal kidney to the termination values in diseased kidney. This consistency strongly suggests that the values reflect a true difference between the animals rather than a random fluctuation at the time of biopsy.
It could be argued that the renal biopsy affected the renal outcome in our study. However, no morphological or immunohistochemical differences between the biopsied and the non-biopsied kidney were found at termination. Renal ACE activity at termination was similar in biopsied kidney and non-biopsied kidney. Thus, whereas effects of the renal biopsy cannot be excluded, they do not modify the conclusions of our study.
In the time controls, median proteinuria was low and stable throughout the study, and values at termination were not predicted by baseline ACE. Considering the absence of substantial abnormalities at termination in these animals, the likelihood of detecting a predictive effect within the time frame of the study was low. It would be of interest, therefore, to study long-term age-related changes in this strain in relation to baseline renal ACE activity.
Baseline renal ACE predicted the severity of interstitial and glomerular pre-fibrotic changes, but not focal glomerular sclerosis and MME. Within the time frame of our study, the glomerular sclerotic changes, however, remained mild. Accordingly, the quantitative resolution to detect a relationship with the glomerular sclerotic changes may have been too limited. The predictive value of renal ACE for expression of the pre-fibrotic glomerular markers suggests that a consistent dissociation between interstitial and glomerular changes is unlikely. In this model, interstitial morphological changes have been noted to prevail over glomerular changes [13]. Nevertheless, during advanced stages of renal damage in this model, usually interstitial and glomerular changes go hand in hand [14]. Therefore, it would be worthwhile to investigate whether during long-term follow-up, baseline ACE also predicts the progression of glomerular sclerosis.
Our data show the predictive value of baseline ACE for renal damage, but do not provide proof for a causal role of renal ACE activity in the differences in renal damage, as we cannot exclude that it is an epiphenomenon to (unknown) associated factors. Study designs with intervention in renal ACE activity, either pharmacological or genetic, would be needed for conclusive proof. The renoprotective effect of ACE inhibition in this model, as well as other proteinuric models [2,11,15,16], is well established, and in line with a role for renal ACE in renal damage, albeit not conclusively, as the relationship between baseline renal ACE and pharmacodynamics of ACE inhibition is complicated, and has not been well characterized.
Recent data in mice with extra copies of the ACE gene provide specific data on this issue. These genetically engineered mice have modestly elevated plasma ACE and kidney ACE mRNA, but do not develop spontaneous renal damage. After induction of diabetes, they develop more severe hypertension and renal damage than animals with one or two copies [17]. Thus, a genetically elevated ACE activity does not elicit renal damage but, when damage is inflicted, the course is more severe, which is in line with our data in outbred rats.
By what mechanism could renal ACE activity modify the course of renal damage? Egido's group [10] postulated that elevated ACE activity in tubular and interstitial cells leads to generation of angiotensin II, which contributes to tubulo-interstitial damage by its pro-inflammatory and pre-fibrotic effects. In experimental renal disease (protein overload) and human diabetic nephropathy [18], these authors found higher renal ACE activity when renal damage was present. This is in line with a prior study from our group in adriamycin nephrosis [11]. These data were obtained after development of renal damage, however. The higher renal ACE in association with more severe damage might be the result of renal damage, rather than a predisposing factor. Our present data show that individual renal ACE levels in the healthy condition precede, and thus predict, the course of renal damage. Moreover, the predictive value was observed despite stable renal ACE levels throughout the study, showing that the predictive effect of baseline renal ACE does not depend on a disease-associated rise in renal ACE level. The absence of a rise in renal ACE activity in our study is somewhat at variance with increased renal ACE activity in some other studies [10]. This may be due to differences between the models, or to the relatively mild extent of damage in our study.
In conclusion, our data show that naturally occurring individual differences in renal ACE levels predict the susceptibility to adriamycin-induced renal injury. This suggests that differences in renal ACE activity predispose to a more aggressive course of renal damage. Future studies are needed to investigate the underlying mechanisms, and to explore whether this predictive effect also applies to other models of renal damage and to human renal disease.
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Acknowledgments |
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Conflict of interest statement. None declared.
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Notes |
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References |
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