Volume 83, Issue 2, Pages 213-222 (February 2013) High phosphate directly affects endothelial function by downregulating annexin II  Giovana Seno Di Marco, Maximilian König, Christian Stock, Anne Wiesinger, Uta Hillebrand, Stefanie Reiermann, Stefan Reuter, Susanne Amler, Gabriele Köhler, Friedrich Buck, Manfred Fobker, Philipp Kümpers, Hans Oberleithner, Martin Hausberg, Detlef Lang, Hermann Pavenstädt, Marcus Brand  Kidney International  Volume 83, Issue 2, Pages 213-222 (February 2013) DOI: 10.1038/ki.2012.300 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 High phosphate levels downregulate annexin II (Annx II) expression and inactivate Akt in endothelial cells. EAhy926 cells were incubated in the presence of 1.0 (control), 2.5, and 5.0mmol/l phosphate. Annexin protein expression was analyzed by (a) western blotting and (b) flow cytometry (extracellular protein), whereas (c) Akt phosphorylation was assessed by western blotting. Results are mean±s.e.m. *P<0.05 compared with control; #P<0.05 compared with inorganic phosphate (Pi) 2.5mmol/l. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Downregulation of annexin II in endothelial cells by predialysis sera as a function of phosphate concentration. EAhy926 cells were incubated with patient sera and protein expression was analyzed by flow cytometry. Extracellular annexin expression was plotted against serum phosphate, evidencing a negative correlation between them. Results were normalized by values obtained from cells incubated with fetal calf serum (internal control values). Note that 7.7mg/dl corresponds to 2.5mmol/l phosphate. r=Spearman correlation coefficient. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Shedding of microparticles from endothelial cells exposed to high phosphate. Human coronary artery endothelial cells were cultured in medium containing 1.0 (control) or 2.5mmol/l phosphate. Microparticles were obtained from the supernatants by serial centrifugation, stained with specific antibodies, and analyzed by flow cytometry. Besides increased release of microparticles bearing (a) phosphatidylserine (which binds annexin V) and (b) CD105, a greater amount of (c) annexin II–positive microparticles is also found under high phosphate concentration. Results are given in scatter plots; n=8. *P<0.05 compared with control. (d) Microparticles were further characterized by immunofluorescence microscopy. They appeared as vesicles with a diameter <1 μm and stained for annexin II (upper panel). Omission of the first antibody was used as negative control (lower panel). Bar=1 μm. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Effect of high phosphate levels on tube formation. EAhy926 cells were incubated in the presence of 1.0 (control) or 2.5mmol/l phosphate. Endothelial tube formation was analyzed after seeding cells on a polymerized thin layer of matrigel. High phosphate levels and application of an antibody against annexin II (anti-Annx II) led to a similar decrease in tube formation. Quantification was performed by using the NIH Image J Software. Results are mean±s.e.m. *P<0.05 compared with control. Pi, inorganic phosphate. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Antiangiogenic effect of high phosphate levels on the chicken chorioallantoic membrane (CAM). (a, b) Locally, 10μl of a 2.5-mmol/l phosphate solution (high phosphate) or phosphate-buffered saline (control) was applied on the CAM. (c, d) Systemic hyperphosphatemia was induced by intravitellus injection of 1ml of a 125-mmol/l phosphate solution in in ovo–cultured embryos (high phosphate; inorganic phosphate (Pi) concentration 9.5±0.9mg/dl). Water-injected embryos served as control (Pi concentration 5.8mg/dl). (a, c) Six hours after exposure to high phosphate levels, vascular density was analyzed macroscopically (antiangiogenic score: 1 for clearly decreased number of blood vessels, 0.5 for equivocal, and 0 for no differences compared with nontreated area/embryos; left panels) or microscopically (Sambucus nigra lectin staining; right panels). (b, d) A great density of capillary vessels is observed in control CAM, whereas phosphate-treated CAM presented a significant reduction in the number of vessels. The microscopic field corresponds to 35,000μm2. Results are mean±s.e.m. Arrows indicate blood vessel (endothelial cell) staining. *P<0.05 compared with control. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 High phosphate levels stiffen endothelial cells in vitro and in vivo. For in vitro experiments, EAhy926 cells in culture (a, b) or a small patch of control rat aorta with the endothelial surface facing upward (c) were incubated in the presence of 1.0 (control) and 2.5mmol/l phosphate. Aortas were also isolated from rats exposed to 2h of continuous in vivo treatment with high phosphate (inorganic phosphate (Pi); blood phosphate level 9.3±0.85mg/dl) or vehicle (NaCl; blood phosphate level 6.4±1.0mg/dl) (d). Endothelial stiffness was measured as the force necessary to compress a cell for a certain distance (nN/nm) by atomic force microscopy (AFM), and expressed as control-fold increase. In a, AFM images of EAhy926 cells under control condition and after 24h of incubation with high phosphate levels; in b, each mean value represents 20 individual experiments. In c and d, each mean value was calculated from 30–36 single experiments in three different aortae. Results are given in scatter plots. *P<0.05 compared with control. Bar=20μm. Kidney International 2013 83, 213-222DOI: (10.1038/ki.2012.300) Copyright © 2013 International Society of Nephrology Terms and Conditions