Altered antioxidant defence in a mouse adriamycin model of glomerulosclerosis

An Deman1, Bart Ceyssens1, Marina Pauwels1, Jigang Zhang1, Katherina Vanden Houte2, Dierik Verbeelen3 and Christiane Van den Branden,1

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Antioxidant enzyme status changes in experimental models of chronic renal disease with glomerulosclerosis. Most of the studies are performed in rats. We now investigate whether a mouse model with more rapid development of glomerulosclerosis is suitable for the study of radical-associated renal disease.

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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Progressive sclerosis of glomeruli and tubulointerstitial tissue are characteristics of evolutive renal diseases. These processes are accompanied by an increase of reactive oxygen intermediates (ROI) and of changes in the antioxidant capacity of the cell [1]. The precise mechanism of these disturbances and the question of whether they play a major role in the pathogenesis of renal failure remain largely unknown.

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.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Experimental design
Female BALB/c mice (Iffa Credo, Brussels, Belgium) with body weights of 17–19 g were injected intravenously with a single dose of adriamycin (doxorubicin (Pharmacia and Upjohn, Puurs, Belgium) diluted to 50% with 0.9% saline; 10 mg/kg; n=8). Control mice were injected with the same volume of saline (n=7). Intake of food was made easier by offering the animals a wet porridge of A04 meal (UAR, Epinay, France) instead of pelleted chow. Mice were sacrificed on day 20. Blood samples were taken by transthoracic cardial puncture and the kidneys were rapidly removed and weighed. Transversal slices of renal tissue were fixed in 4% buffered formaldehyde at room temperature for 24 h and embedded in paraffin for evaluation of sclerosis and ferric iron deposits. The remaining cortex of the same kidney was homogenized in appropriate buffers for determination of glutathione concentration and AOE activities. The second kidney cortex was snap frozen in liquid nitrogen and used for RNA extraction.

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% agarose–3% 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 [{alpha}-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)–haematoxylin–saffron. 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 Mann–Whitney test. A P-value <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
General observations and clinical parameters
All animals survived and remained in good shape during the experiment. A daily urine evaluation with the Multistix strip showed massive proteinuria in the adriamycin-treated mice starting from day 5; none of them had haematuria. After 20 days, the 24-h urine collection confirmed the significant difference of proteinuria between adriamycin and control animals. Serum cholesterol concentration was increased in the adriamycin-treated mice. Serum urea, creatinine and protein concentrations were unchanged (Table 1Go).


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Table 1. Clinical parameters in control and adriamycin-treated mice, 20 days after adriamycin treatment

 

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 10–60% 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 1AGo and BGo).



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Fig. 1. (A and B) PAS–haematoxylin–saffron staining. Glomerulosclerosis and tubulointerstitial lesions are present in the cortex of adriamycin-treated mice (B). This is not the case in control cortex (A). (C and D) Berlin-blue staining. Ferric iron deposition is present in the tubulointerstitial compartment of the renal cortex of adriamycin-treated mice (D). In control cortex (C), no ferric iron deposits are observed. Magnification: x200.

 
Ferric iron was absent in control animals. It was found in the tubulointerstitial compartment (often in close relation with the proteinaceous casts) of all adriamycin mice, but never in the glomeruli (Figure 1CGo and DGopar;.

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 2Go).


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Table 2. Antioxidant enzyme activities and glutathione concentration in renal cortex from control and adriamycin-treated mice, 20 days after treatment

 

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).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Nephrotoxic action of adriamycin is considered to be (at least partly) due to a drug-induced ROI generation. Adriamycin generates semiquinone radicals, which in turn react with molecular oxygen and provide other ROI at an early stage after administration. Once the initial ROI boost of adriamycin has disappeared, locally infiltrated neutrophils and activated glomerular mesangial cells continue ROI production [12]. Increased concentration of reduced glutathione in the renal cortex supports the idea of ROI involvement in our experimental conditions. GSH synthesis was shown to be induced in cells exposed to oxidative stress as an adaptive process [13]. Although one would expect increased GSH levels to protect against ROI induced injury, in cultured mouse mesangial cells increased GSH concentration enhances the generation of collagens and the expression of collagen and TGF-ß genes [14]. In our experiment also, increased GSH level does not prevent development of progression of renal injury. Adriamycin administration can have different effects on GSH levels, depending on the animal species, the organ studied, the dosage of the drug and the length of the treatment. Chronic administration causes increase in cardiac GSH levels, whereas both increased and decreased GSH levels are observed in acute studies [15]. Ishiyama et al. [16] consider the presence of ferric iron in the tubulointerstitial compartment of rats on a high cholesterol diet as proof for the metabolization of O2-. to OH., resulting in tissue damage. Adriamycin-treated mice also show an important hyperlipidemia. Increased O2-. production resulting from hyperlipidemia and inflammatory cell infiltration causes increased metabolization to OH., a reaction catalysed by iron. The presence of ferric iron in the tubulointerstitial compartment of adriamycin-treated mice is a second argument for ROI involvement in our experiment.

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.



   Acknowledgments
 
We thank the Voorzorgskas van Geneesheren, the Research Council of the Vrije Universiteit Brussel and the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen for grants supporting this work. We are indebted to Prof. J. L. Tilly, (Baltimore, MD, USA) and to Dr R. V. Guntaka (Columbia, MO, USA) for allowing us the use of the antioxidant enzymes and 18S ribosomal cDNA probes, respectively. We are grateful to Dr E. Wyffels for skilful help in handling experimental animals.



   Notes
 
Correspondence and offprint requests to: Dr Christiane Van den Branden, Human Anatomy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. Back



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 Results
 Discussion
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Received for publication: 1.12.99
Revision received 28. 8.00.