1 Department of Human Anatomy, 2 Division of Pathology and 3 Department of Nephrology, Vrije Universiteit Brussel and Academic Hospital of the Vrije Universiteit Brussel, Brussels, Belgium
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Abstract |
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Methods. Female BALB/c mice are injected intravenously with a single dose of adriamycin (10 mg/kg). The development of glomerular and interstitial injury is evaluated by means of renal function parameters and histology. Renal cortex activities of catalase, Cu/Zn and Mn superoxide dismutase and glutathione peroxidase are measured by enzymatic techniques, and their mRNA levels by Northern blot analysis.
Results. The mice develop proteinuria and hypercholesterolaemia; glomerulosclerosis is present 20 days after adriamycin injection. Involvement of reactive oxygen intermediates in the disease process is supported by an increased cortex level of glutathione (1.77±0.13 vs 1.31±0.12 µmol/g kidney; P=0.021) and ferric iron deposition in the tubulointerstitial compartment. Glomerulosclerosis and tubulointerstitial lesions are accompanied by decreased cortex activities of catalase (0.19±0.01 vs 0.23±0.01 U/mg protein; P=0.024), glutathione peroxidase (0.28±0.01 vs 0.32±0.01 U/mg protein; P=0.049) and Mn superoxide dismutase (6.61±0.91 vs 9.25±0.99 U/mg protein, P=0.020). We find decreased cortex mRNA levels only for glutathione peroxidase.
Conclusion. The fast development of glomerulosclerosis combined with an altered antioxidant status makes this mouse adriamycin model a suitable alternative for the slower rat models.
Keywords: adriamycin; antioxidant enzymes; chronic renal failure; glomerulosclerosis; oxidative stress
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Introduction |
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We previously reported changes of antioxidant enzyme (AOE) activities in chronic renal failure with glomerulosclerosis, using rat models. In both the remnant kidney as well as in adriamycin-induced renal disease, we documented a decrease of antioxidant enzymes and a beneficial effect of different treatments that influence AOE status [25]. A major disadvantage of rat models is that both the remnant kidney and the adriamycin model require several months before severe glomerulosclerosis develops. Recently, Chen et al. [6], showed that 18 days after the administration of adriamycin at a dose of 10 mg/kg body weight, glomerulosclerosis was present in BALB/c mice. In this study we investigate whether in their mouse adriamycin model with glomerulosclerosis, similar antioxidant defence system changes can be observed as in rat models.
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Subjects and methods |
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Clinical parameters
Serum and urine urea, creatinine and total protein concentrations were analysed by the Kodak Ektachem method (Kodak Eastman, Rochester, NY, USA) and serum cholesterol concentration was determined by enzymatic methods [7]; a semi-quantitative measurement of proteinuria and a qualitative measurement of haematuria were performed daily using a Multistix strip (Bayer, Brussels, Belgium). Proteinuria was expressed as the protein:creatinine ratio.
Determination of renal cortex glutathione concentration
The reduced form of glutathione (GSH) was measured in renal cortex homogenates using the BIOTECH GSH-400 kit (OXIS International, Portland, OR, USA). Concentration was expressed as µmol GSH/g kidney cortex.
Determination of renal cortex AOE activities
Cortex homogenates were prepared in 50 mM potassium phosphate buffer containing 0.1 mM EDTA and 1% Triton X-100, pH 7.8. Catalase (CAT) activity was assayed by the method of Aebi [8]. Glutathione peroxidase (GPx) activity (selenium and non-selenium dependent) was determined by the method of Carmagnol et al. [9]. Total superoxide dismutase (SOD) and Mn SOD activities were measured by the method of Marklund and Marklund [10] and Cu/Zn SOD activity was calculated. The results are expressed in units/mg protein. Protein measurement was performed by the bicinchoninic acid method (PIERCE, Rockford, IL, USA).
RNA extraction and Northern blot analysis of AOE
Renal cortex was sonicated and total RNA was extracted using an RNeasy minikit (Qiagen, Leusden, The Netherlands). For Northern hybridization analysis, 20 µg RNA was electrophoresed on a 1% agarose3% paraformaldehyde gel. RNA was transferred to a Hybond N Filter (Amersham, Little Chalfort, UK) by capillary blotting. Filters were pre-hybridized overnight at 42°C. After hybridization with [-32P]uridine triphosphate labelled cRNA probes at 60°C, filters were washed at 65°C to a final stringency of 0.1xsalt sodium citrate/0.1% sodium dodecyl sulphate. Antisense RNA probes complementary to mice Cu/Zn, and Mn SOD, GPx and CAT mRNA sequences were synthesized by in vitro transcription using a RNA labelling kit (Amersham, Little Chalfort, UK). Hybridization signals were detected by a molecular imager system (GS-525; Biorad, Hercules, CA, USA). The mRNA signals were related to the 18S ribosomal RNA.
cDNA-probes
The cDNAs containing CAT, GPx, Cu/Zn SOD and Mn SOD sequences were kindly provided by Prof. J. L. Tilly (Baltimore, MD, USA) [11]. The 5.6 kb 18S ribosomal cDNA probe was a gift from Dr R. V. Guntaka (Columbia, MO, USA).
Evaluation of glomerulosclerosis and tubulointerstitial sclerosis, presence of ferric iron in renal cortex
Paraffin sections of 4 µm were cut and stained with periodic acid Schiff (PAS)haematoxylinsaffron. They were examined by light microscopy and scored in a blinded fashion by a pathologist. Glomerulosclerosis was evaluated semiquantitatively in ±100 glomeruli per animal by counting the number of quadrants showing sclerosis [3]. Tubulointerstitial lesions were studied in a descriptive way (presence of large tubular casts, tubular dilatation and atrophy, lymphocyte infiltration). For the demonstration of ferric iron, 4 µm paraffin sections were stained using the Berlin-blue method.
Statistics
Values are presented as mean±SEM and were compared by a MannWhitney test. A P-value <0.05 was considered statistically significant.
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Results |
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Evaluation of glomerulosclerosis and tubulointerstitial sclerosis, and the presence of ferric iron in renal cortex
None of the control animals had tubulointerstitial changes. The adriamycin group showed large tubular casts (consistent with the important proteinuria) and 1060% of the tubuli showed important dilatation. There was no tubular atrophy, nor lymphocyte infiltration. Glomerulosclerosis was seen in 8.1±1.6% of the glomeruli (6.3±1.1% of glomerular quadrants) in adriamycin-treated mice versus 0.8±0.4% in control animals (P=0.0012). The lesions were most pronounced in the juxtamedullar glomeruli. Glomerular collapse was rare and hyaline deposits were only seen in treated mice. No thrombosis was observed (Figure 1A and B
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Renal cortex glutathione concentration and AOE activities
CAT, GPx and Mn SOD activities were significantly decreased in the renal cortex of adriamycin-treated mice. The activity of Cu/Zn SOD was not significantly decreased. Glutathione concentration, an indicator of oxidative stress, was significantly increased in the cortex of adriamycin-treated mice (Table 2).
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mRNA levels of AOE
For Cu/Zn SOD, MnSOD and CAT the mRNA signals were of comparable intensity in control and adriamycin-treated mice. For GPx however, the mRNA signal was significantly lower in adriamycin-treated animals (GPx -60%; P=0.01).
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Discussion |
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Twenty days after the start of our experiment, the activities of GPx, CAT and Mn SOD were decreased in the renal cortex. The activity of Cu/Zn was not changed. Further analysis of the mechanism leading to decreased CAT, GPx and Mn SOD activities showed that only the GPx mRNA level was lower than in control mice, whereas CAT and Mn SOD mRNA levels were unchanged. Our results are very similar to the results obtained by Gwinner et al. [17] in a puromycin aminonucleoside model of glomerulopathy in the rat. They find decreased CAT and GPx activities and unchanged SOD activities, with, in their case, unchanged mRNA levels for CAT and GPx. In our model, decreased activity of GPx probably originates at the transcription level, whereas decreased activity of CAT and Mn SOD happens at the post-transcriptional level. Several authors have reported upregulation of the different AOE in chronic exposure to oxidative stress or inflammation in vitro [12,18] and in vivo [19], and protection against adriamycin toxicity in the heart by the overexpression of AOE in transgenic mice [18], fitting the concept of an adaptive oxidant/antioxidant balance. Our results, however, show that in a condition of increased ROI production, AOEs are not necessarily induced. In this respect, the adriamycin model is similar to the puromycin aminonucleoside model of glomerulopathy [17].
The mouse adriamycin model used in our experiment combines development of glomerulosclerosis with a decreased antioxidant status and increased concentration of reduced glutathione and ferric iron deposition, 20 days after the onset of the experiment. This development occurs much faster than in rat models and constitutes the major advantage of the mouse model. A few disadvantages of the mouse model are the absence of a good method to isolate mouse glomeruli for separate evaluation of AOE in glomeruli and tubuli, the smaller amounts of tissue available for different evaluations, and a more difficult collection of urine and blood.
In conclusion, the adriamycin model in female BALB/c mice permits the study of enhanced oxidative stress and the initiation of chronic renal disease in a short time span of 20 days. Furthermore, this model seems promising for the study of ROI toxicity, the mechanisms of defence of the kidney and the effects of (antioxidant) drugs. The combined presence of significant glomerulosclerosis and altered antioxidant status makes the model suitable as an alternative for the more widely used rat models in which development of glomerulosclerosis occurs much more slowly.
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Acknowledgments |
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Notes |
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References |
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