Increased inorganic phosphate induces human endothelial cell apoptosis in vitro

G. S. Di Marco, M. Hausberg, U. Hillebrand, P. Rustemeyer, W. Wittkowski, D. Lang, H. Pavenstädt
2008 AJP - Renal Physiology  
Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (Ͼ2.5 mM) induced apoptosis in two
more » ... l cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at ϳ2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease. atherosclerosis; ROS; mitochondrial dysfunction; endothelial dysfunction VASCULAR CHANGES IN CHRONIC kidney disease and end-stage renal failure are associated with increased atherosclerosis, ischemic heart disease, and vascular stiffening. Prospective studies and registry data suggest a strong role of hyperphosphatemia and an increased calcium-phosphate product as significant predictors of cardiovascular mortality, potentially acting as progression factors of unwanted atherosclerosis and calcifications in uremia (26). In addition, it has been very recently shown that an increased phosphate concentration is an independent risk factor for higher mortality during the predialysis phase, suggesting that phosphate levels within the normal range are likely of vital importance in predialysis patients (32). Moreover, it is now widely accepted that oxidative stress and the ensuing endothelial dysfunction play a key role in the pathogenesis of atherosclerosis and cardiovascular disease. Specifically, endothelial dysfunction caused by an excess of reactive oxygen species (ROS) precedes and promotes atherosclerosis (18, 25). The high turnover of endothelial cells in atherosclerosis also suggests that apoptosis may contribute to the pathology (4). In this context, common risk factors for atherosclerosis are associated with increased generation of ROS and mitochondrial dysfunction, which can lead to activation of the mitochondrial apoptotic pathway (4, 18, 25). Thus mitochondria have been proposed as an important link among risk factors, oxidative damage, endothelial dysfunction, and apoptosis, and the initiation and development of atherosclerotic lesions. If a number of observations confirm the importance of endothelial cell apoptosis in the pathogenesis of atherosclerosis (8, 14, 25, 29) , there are only sparse data on the effects of phosphate, in concentrations as observed in uremia-related hyperphosphatemia, on endothelial cell function and death. Thus, in the present study, we aimed to ascertain whether increased phosphate concentrations alone or in combination with increased calcium concentrations are able to modulate endothelial cell apoptosis and to explore the mechanisms involved in this process. MATERIALS AND METHODS All experiments were performed under institutional ethical approval of the protocol. Endothelial cell culture. Human endothelial cells [EAhy926; a permanent human cell line derived from human umbilical vein endothelial cells that express highly differentiated functions characteristic of human vascular endothelium] (1, 5, 6, 24) and bovine aortic endothelial GM-7373 cells (DSMZ, Braunschweig, Germany) (24) were used. EAhy926 cells were grown in DMEM (Biochrom, Berlin, Germany) containing 5% FCS (PAA Laboratories, Pasching, Austria), 2 mM L-glutamine, and 50 U/ml each of penicillin/streptomycin at 37°C in an atmosphere of 5% CO 2 in air. GM-7373 cells were cultured in MEM supplemented with 20% FCS, nonessential amino acids, MEM vitamins, and penicillin/streptomycin as recommended by the manufacturer. Cell cultures between passages 10 and 20 and 8 and 13 were used, respectively. For most of the experiments, cells were seeded in 24-well plates. Twenty-four hours before apoptosis induction, the growth medium was replaced; and just before each specific treatment, cells were rinsed with fresh medium and treated for the indicated times. Treatment protocols. Medium supplemented with 5% FCS was set as the control (phosphate and calcium concentrations in the culture medium are 1.0 and 1.8 mM, respectively). In treated groups, control medium was supplemented with 133 l of a stock solution of sodium phosphate (1.0 M Na ϩ ; 0.6 M PO 4 2Ϫ ) and 217 l of a stock solution of calcium gluconate/calcium saccharate (0.23 M Ca 2ϩ ) to raise the phosphate concentration to 2.5 mM and calcium concentration to 2.8
doi:10.1152/ajprenal.00003.2008 pmid:18385273 fatcat:k6ifspvowjfdresehwob2tmeva