Harmful Effects of the Azathioprine Metabolite 6-Mercaptopurine in Vascular Cells: Induction of Mineralization

<div><p>Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells <i>in vitro</i>. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in <i>in vitro</i> and <i>ex vivo</i> models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.</p></div

Effects of a Single Intravitreal Injection of Aflibercept and Ranibizumab on Glomeruli of Monkeys

<div><p>Purpose</p><p>It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.</p><p>Methods</p><p>The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm.</p><p>Results</p><p>Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased.</p><p>Conclusion</p><p>Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.</p></div

Example of a montage of transmission electron micrographs by using multiple image arrangements (MIAs) in order to provide the entire renal glomerulus.

<p>These images were taken using the image analysis software iTEM at the magnification of ×3000. Here one day after aflibercept injection.</p

Quantification and normalisation of the VEGF staining.

<p>Results of the analysis of aoi of glomeruli from kidneys of monkeys one and seven days after ranibizumab and aflibercept treatment and the corresponding controls after anti-VEGF staining; t-test against control: * for p<0.05, *** for p<0.0001; t-test ranibizumab day 1 <i>versus</i> aflibercept day 1 and ranibizumab day 7 <i>versus</i> aflibercept day 7: # for p<0.05, ### for p<0,0001; t-test aflibercept day 1 <i>versus</i> aflibercept day7: +++ for p<0.0001.</p

Involvement of ROS.

<p>(A,B,E,F) VSMCs were stimulated as indicated for 30 min before labeling cells with DHE. Superoxide production was (A) visualized via fluorescence microscopy (representative images from 3 independent experiments) or (B,E,F) quantified in a fluorescence plate reader (n≥6). (C,D) Hydrogen peroxide production is measured in H₂DCFDA-labeled cells via flow cytometry. (C) Representative histograms of flow data (grey: control, white: 6-MP). (D) Quantification of fluorescence intensity by % of labeled control (n≥6). (E) Stimulation with 6-MP (100 µmol/L) or its metabolites (each 10 µmol/L). (F) Stimulation with 6-MP (100 µmol/L) alone or costimulation with inhibitors (tiron [1 mmol/L] and allopurinol [1 µmol/L]). DHE: dihydroethidium, 6-MP: 6-mercaptopurine, 6-MTIMP: 6-methylthioinosine monophosphate, 6-TGDP: 6-thioguanosine diphosphate, 6-TGMP: 6-thioguanosine monophosphate, 6-TGN: 6-thioguanine nucleotide, 6-TGTP: 6-thioguanosine triphosphate, 6-TU: 6-thiouric acid.</p

Semi-quantitative computer assisted method used for the quantification and normalisation of the VEGF staining.

<p>Glomeruli were defined as the area of interest (AOI), and then the AOI were isolated using the image j software. The nuclei in the AOI were then counted and finally the stained area of each AOI was determined.</p

mRNA expression of osteogenic proteins.

<p>(A–C) VSMCs were stimulated with 6-MP as indicated and mRNA expression was detected after 48 h. Data represent means±SEM, n≥6,*p<0.05 vs. control. (D) VSMCs were stimulated with 6-MP for 48 h. Nuclear proteins were extracted. Cbfa1, Cbfa1-phospho and TATA-bp were detected via Western blot. Representative images and relative band intensities of 3 independent blots of Cbfa1-phospho are shown. (E) MEK1 and ERK1/2 activation was detected via Bio-Plex (n≥6). Values are given as % of control and are normalized to total kinase. (F) mRNA expression of cbfa1 after 48 h treatment with 6-MP (100 µmol/L) ± U0126 (1 µmol/L) (n>6). Data represent means±SEM, *p<0.05 vs. control. ALP: alkaline phosphatase, cbfa1: core binding factor alpha-1, 6-MP: 6-mercaptopurine, OCN: osteocalcin.</p

Examples of transmission electron micrographs used for the quantification of the glomerular endothelial cells fenestrations.

<p>The red line drawn a long lamina rara interna, the length of line is in µm, the red crosses point out fenestrations. (<b>A</b>) after injection of the vehicle; (<b>B</b>) in the untreated control; (<b>C</b>) one day after injection of ranibizumab; (<b>D</b>) seven days after injection of ranibizumab; (<b>E</b>) one day after injection of aflibercept; (<b>F</b>) seven days after injection of aflibercept. Magnification ×20000.</p

Examples of representative transmission electron micrographs of (A) a fenestrated glomerular endothelium and of (B) peripheral <i>versus</i> mesangial portions of the glomerular endothelium (both one day after aflibercept injection).

<p>(<b>A</b>) Blood lumen on the upper part, urinary space on the lower part of the image. The healthy glomerular filtration barrier consists of three layers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113701#pone.0113701-Eremina2" target="_blank">[6]</a>: the fenestrated glomerular endothelial cells, the intervening glomerular basement membrane and the podocyte processes and slit diaphragm. GBM =  glomerular basement membrane, CL =  capillary lumen, POD =  podocyte. Arrows mark glomerular endothelial cells fenestrae (note the absence of diaphragm), asterisks mark podocyte foot processes, arrowheads mark podocyte slit diaphragm. (<b>B</b>) At this magnification, podocyte foot processes (asterisks) allow the clear identification of the capillary lumen (CL). In accordance with our definition, the peripheral portion begins where the endothelium and the glomerular endothelial basement membrane (GBM) run approximately parallel (marked by arrows). Arrows mark direction into which peripheral endothelium begins. In between the arrows the mesangium (Mes) and the mesangial portion of the capillary endothelium (MesE) is located. Note that in the mesangial portion there is no GBM adjacent to the fenestrated endothelium so that the described counting method is not applicable and the endothelium does not show the typical single- layered configuration. Magnification ×20000.</p

6-MP-induced calcification <i>in vitro</i> and <i>ex vivo</i>.

<p>(A–D) VSMCs were cultured in control medium or CM±6-MP (100 µmol/L) for 21 days. (A) Mineral deposits were visualized via Alizarin red staining. One representative experiment is shown (n = 5). (B) VSMCs were treated with 6-MP (1 µmol/L–1 mmol/L) for up to 21 days and viability/proliferation was measured. (C) Calcium content (n>6) or (D) ALP enzyme activity (n>6) was quantified and normalized to protein content. (E,F) Rat aortic rings were incubated in control medium or CM ± 6-MP (100 µmol/L) for 14 days. (E) One aortic ring treated with each type of stimulation was used for histochemical analysis. Slices were stained with Alizarin Red to visualize calcium deposition. (F) Calcium content was quantified and normalized to the dry weight of aortic rings (n>6). Data represent means±SEM, *p<0.05 vs. control. ^#p<0.05 vs. CM. ALP: alkaline phosphatase, CM: calcifying medium, 6-MP: 6-mercaptopurine.</p