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

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.

Cardiovascular and renal effects of high salt diet in GDNF+/- mice with low nephron number.

AIMS: To test the suggested association of low nephron number and later development of renal and cardiovascular disease we investigated the effects of high sodium diet in heterozygous GDNF+/- mice. METHODS: Aged wild type and GDNF+/- mice were grouped together according to high sodium (HS, 4%) or low sodium (LS, 0.03%) diet for 4 weeks. The heart, the aorta and the kidneys were processed for morphometric and stereological evaluations and TaqMan PCR. RESULTS: On HS GDNF+/- mice showed significantly higher drinking volume and urine production than wt and mean arterial blood pressure tended to be higher. Heart weight was higher in GDNF+/- than in wt, but the difference was only significant for LS. HS significantly increased cardiac interstitial tissue in GDNF+/-, but not in wt. On LS GDNF+/- mice had significantly larger glomeruli than wt and HS led to an additional two fold increase of glomerular area compared to LS. On electron microscopy glomerular damage after HS was seen in GDNF+/-, but not in wt. Dietary salt intake modulated renal IL-10 gene expression in GDNF+/-. CONCLUSION: In the setting of 30% lower nephron number HS diet favoured maladaptive changes of the kidney as well as of the cardiovascular system.

Early glomerular alterations in genetically determined low nephron number.

An association between low nephron number and subsequent development of hypertension in later life has been demonstrated. The underlying pathomechanisms are unknown, but glomerular and postglomerular changes have been discussed. We investigated whether such changes are already present in prehypertensive "glial cell line-derived neurotrophic growth factor" heterozygous mice (GDNF+/-) with lower nephron number. Twenty-six-week-old mice [22 GDNF+/-, 29 C57B6 wild-type control (wt)] were used for in vivo experiments with intra-arterial and tail cuff blood pressure measurements. After perfusion fixation, kidneys were investigated with morphological, morphometric, stereological, and immunohistochemical techniques and TaqMan PCR analysis. As expected at this age, blood pressure was comparable between GDNF+/- and wt. Nephron number per kidney was significantly lower in GDNF+/- than in wt (-32.8%, P < 0.005), and mean glomerular volume was significantly higher (+49.5%, P < 0.001). Renal damage scores, glomerular and tubular proliferation, analysis of intrarenal arteries and peritubular capillaries, expression of relevant tubular transporter proteins, as well as gene expression of profibrotic, proinflammatory, or prohypertensive markers were not significantly different between GDNF+/- and wt. Compensatory glomerular hypertrophy in GDNF+/- was accompanied by higher numbers of endothelial and mesangial cells as well as PCNA-positive glomerular cells, whereas podocyte density was significantly reduced. Further electron microscopic analysis showed marked thickening of glomerular basement membrane. In conclusion, lower nephron number is associated with marked early glomerular structural changes, in particular lower capillary supply, reduced podocyte density, and thickened glomerular basement membrane, that may predispose to glomerular sclerosis.

Angiopoietin 1 and 2 gene and protein expression is differentially regulated in acute anti-Thy1.1 glomerulonephritis.

Capillary neoformation is important in repair of glomerular injury of various origins. VEGF was shown to be crucial for glomerular capillary repair in glomerulonephritis (GN). We reasoned that other angiogenic factors are likewise involved in glomerular capillary remodeling and found angiopoietin 1 and -2 (ANG1 and ANG2) mRNA to be upregulated in cDNA microarrays of microdissected glomeruli of anti-Thy1.1 GN of the rat. We then studied glomerular in situ gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 in the course of anti-Thy1.1 GN, which was induced by injection of OX-7 antibody. Animals were perfusion fixed at days 6 and 12 after GN induction and compared with nonnephritic controls receiving PBS. Capillary damage and repair were quantitatively analyzed using stereological techniques. Gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 was analyzed using real-time quantitative PCR from microdissected glomeruli, nonradioactive in situ hybridization, double immunofluorescence, and Western blot analysis. Glomerular capillarization assessed as length density was significantly lower at day 6 of anti-Thy1.1 GN than in controls; it was back to normal values at day 12. ANG1 and ANG2 gene expression was markedly upregulated at day 6 of the disease compared with controls. Protein expression of ANG1 and ANG2 was confined to podocytes and that of Tie-2 to endothelial cells. At day 12 of anti-Thy1.1 GN when capillary restoration was nearly completed, ANG1 and ANG2 gene expression returned to basal levels, whereas Tie-2 expression was still high. With the use of a combined molecular and in situ approach, the spatial and temporal gene and protein expression of the angiopoietins and their receptor was analyzed in anti-Thy1.1 GN. The results indicate that glomerular expression of ANG1 and ANG2 and Tie-2 is differentially regulated and may contribute to healing and endothelial cell stabilization in experimental GN.