Darbepoetin alpha reduces oxidative stress and chronic inflammation in atherosclerotic lesions of apo E deficient mice in experimental renal failure

Abstract

Background: Cardiovascular morbidity and mortality is very important in patients with chronic renal failure. This occurs even in mild impairment of renal function and may be related to oxidative stress and chronic inflammation. The nephrectomized apo E knockout mouse is an accepted model for evaluating atherosclerosis in renal dysfunction. Erythropoietin derivates showed anti-oxidative and anti-inflammatory effects. Therefore, this study evaluates the effects of Darbepoetin on markers of oxidative stress and chronic inflammation in atherosclerotic lesions in apo E knockout mice with renal dysfunction.

Methods: Apo E knockout mice underwent unilateral (Unx, n = 20) or subtotal (Snx, n = 26) nephrectomy or sham operation (Sham, n = 16). Mice of each group were either treated with Darbepoetin or saline solution, a part of Snx mice received a tenfold higher dose of Darbepoetin. The aortic plaques were measured and morphologically characterized. Additional immunhistochemical analyses were performed on tissue samples taken from the heart and the aorta.

Results: Both Unx and Snx mice showed increased expression of markers of oxidative stress and chronic inflammation. While aortic plaque size was not different, Snx mice showed advanced plaque stages when compared to Unx mice. Darbepoetin treatment elevated hematocrit and lowered Nitrotyrosin as one marker of oxidative stress, inflammation in heart and aorta, plaque stage and in the high dose even plaque cholesterol content. In contrast, there was no influence of Darbepoetin on aortic plaque size; high dose Darbepoetin treatment resulted in elevated renal serum parameters.

Conclusion: Darbepoetin showed some protective cardiovascular effects irrespective of renal function, i.e. it improved plaque structure and reduced some signs of oxidative stress and chronic inflammation without affecting plaque size. Nevertheless, the dose dependent adverse effects must be considered as high Darbepoetin treatment elevated serum urea. Elevation of hematocrit might be a favorable effect in anemic Snx animals but a thrombogenic risk in Sham animals.

Figure 1. Plaque stages and cholesterol content.

Left row shows scatter plots of scores of plaque stage and plaque cholesterol content of the seven treatment groups. Significant differences are marked with the certain significance level. Right row shows histological examples. Plaque stage of Darbepoetin alpha treated Unx animal equals score 2 (arrows show cholesterol crystals) while plaque stages of Snx animal equals score 3 (arrows show chondrocyte-like cells and necrosis). Cholesterol content of control Snx animal equals score 2 and that of Snx animal treated with high dose Darbepoetin alpha equals score 1. Arrows show cholesterol crystals.

Figure 2. Nitrotyrosine plaque and myocardial artery score.

Upper row shows nitrotyrosine staining score of plaque and intramyocardial arteries. Significant differences are marked with the certain significance level. Lower row shows histological examples. Plaque staining increases stepwise from Sham to Snx. Darbepoetin alpha treated examples show lower scores in each group.

Figure 3. Nitrotyrosine aortic endothelium score.

Table shows nitrotyrosine staining score of aortic endothelia (0 or 1) and percentage of positively stained endothelia per group. Significant differences are marked with the certain significance level. Right row shows histological examples. The score of the Unx control animal equals 1 (arrows show stained areas of the endothelium) while the score of the Darbepoetin alpha treated Unx animal equals 0.

Figure 4. ICAM and VCAM plaque and myocardial artery score.

Scatter plots of ICAM and VCAM staining score of plaque and intramyocardial arteries of the seven treatment groups. Significant differences are marked with the certain significance level.

Figure 5. CRP plaque and myocardial artery score.

Upper row shows CRP staining score of plaque and intramyocardial arteries. Significant differences are marked with the certain significance level. Lower row shows histological examples. Myocardial artery staining increases from Sham to Snx. Darbepoetin alpha treated examples show lower scores in each group.

Figure 6. CD 40 and CD 154 plaque and myocardial artery score.

Upper rows show CD40 and CD154 staining scores of plaque and intramyocardial arteries. Significant differences are marked with the certain significance level. Lower row shows histological examples of CD40. The Darbepoetin alpha treated Snx animal shows a dose dependently lower CD40 myocardial artery staining score than the control Snx animal.