Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

A major complication of chronic kidney disease is the development of anemia [1,2], resulting in part from decreased renal erythropoietin release and thus impairment of erythropoiesis [3,4]. Moreover, erythropoiesis in uremic patients may be compromized by iron deficiency [5,6]. The anemia in uremic patients is, however, at least in part the result of accelerated clearance of circulating erythrocytes [7], which could be caused by enhanced eryptosis, the suicidal death of erythrocytes [8,9]. Eryptosis is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface [8,9]. The percentage of phosphatidylserine exposing erythrocytes has been reported to be twice as high in patients on dialysis than in healthy individuals [10]. Phosphatidylserine exposing erythrocytes are rapidly cleared from circulating blood in vivo [9] and the increased percentage of phosphatiylserine exposing erythrocytes in circulating blood is expected to be paralleled by the respective decrease of erythrocyte life span.

Eryptosis may be triggered by enhanced cytosolic Ca²⁺ concentration ([Ca²⁺]_i) [8,9] resulting from Ca²⁺ entry through Ca²⁺-permeable cation channels [9]. An increase of [Ca²⁺]_i is followed by cell shrinkage due to activation of Ca²⁺-sensitive K⁺ channels [9], K⁺ exit, hyperpolarization, Cl^- exit and thus cellular KCl and water loss [9]. Increased [Ca²⁺]_i further triggers cell membrane scrambling with phosphatidylserine translocation to the cell surface [9].

Further stimulators of eryptosis include ceramide [9], energy depletion [9], caspase activation [9,11,12] and deranged activity of kinases such as AMP activated kinase AMPK [9], casein kinase 1α [13,14], cGMP-dependent protein kinase [9], Janus-activated kinase JAK3 [15], p38 kinase [16], protein kinase C [9], as well as sorafenib [17] and sunitinib [18] sensitive kinases. Eryptosis is further triggered by a wide variety of xenobiotics and is enhanced in a variety of clinical disorders [9,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43].

Triggers of eryptosis in renal insufficiency are incompletely understood. Eryptosis has previously been shown to be stimulated by the uremic toxins acrolein [44], methylglyoxal [9], indoxyl sulfate [45] and vanadate [9]. A major complication of chronic kidney disease is the increase of plasma phosphate concentration leading to vascular calcification and increased cardiovascular mortality [46,47]. The present study explored, whether eryptosis is modified by alterations of extracellular phosphate concentration. To this end, the sensitivity of [Ca²⁺]_i, cell volume and phosphatidylserine abundance at the erythrocyte surface to alterations of extracellular phosphate concentration was determined.

Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO₄, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl₂; pH 7.4 at 37°C for 48 h. Where indicated, 1 mM CaCl₂ was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), CaCl₂ increased to 1.7 mM, or SB203580 (2 µM, Tocris, Bristol, UK) or pyrophosphate (10µM, Sigma-Aldrich, Steinheim, Germany) added. Erythrocytes were exposed to 0-5 mM phosphate by addition of sodium phosphate buffer (pH 7.4).

For the determination of hemolysis the samples were centrifuged (10 min at 2000 RPM, room temperature) after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.

After incubation under the respective experimental condition, 50 µl cell suspension was washed in Ringer solution containing 5 mM CaCl₂ and then stained with Annexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany).

After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl₂ and 5 µM Fluo-3/AM. The cells were incubated at 37°C for 30 min and washed twice in Ringer solution containing 5 mM CaCl₂. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µl Ringer. Then, Ca²⁺-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.

Data are expressed as arithmetic means ± SEM. Statistical analysis was made using repeated measures ANOVA (Tukey-test). N denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.

In order to explore whether an increase of phosphate concentration in renal insufficiency could participate in the triggering of suicidal erythrocyte death or eryptosis in uremia, erythrocytes were exposed for 48 hours to Ringer solution with phosphate concentrations ranging from 0 to 5 mM. Eryptosis was evidenced from cell membrane scrambling leading to phosphatidlyserine translocation to the cell membrane surface. Phosphatidylserine exposing erythrocytes were identified by annexin V binding. As illustrated in Fig. 1, the percentage of annexin V binding erythrocytes increased with increasing extracellular phosphate concentration. To a smaller extent, increasing phosphate concentrations augmented hemolysis, an effect that reached statistical significance at 2 and 5 mM phosphate concentrations.

Further experiments addressed the effect of phosphate on erythrocyte volume, which was estimated from forward scatter in flow cytometry. As shown in Fig. 2, the forward scatter of erythrocytes increased slightly but significantly following an increase of phosphate concentration from 0 to 0.5 mM. The forward scatter following exposure to any of the other phosphate concentrations was not significantly different from the forward scatter following exposure in the absence of extracellular phosphate.

Fluo3 fluorescence was employed to test, whether phosphate influences cytosolic Ca²⁺ concentration. As illustrated in Fig. 3, the Fluo3 fluoresence did not change significantly up to 2 mM phosphate concentration and slightly but significantly decreased following exposure to 5 mM phosphate concentration.

Further experiments were performed to test, whether the effect of phosphate was sensitive to extracellular Ca²⁺ concentration. To this end, erythrocytes were exposed to 2 mM phosphate for 48 hours either in the presence of extracellular Ca²⁺ (1 mM) or in the nominal absence of Ca²⁺ and presence of the Ca²⁺ chelator EGTA (1 mM). As shown in Fig. 4, the effect of phosphate on annexin-V-binding was virtually abolished in the nominal absence of extracellular Ca²⁺. Further experiments were performed elucidating the effect of phosphate at enhanced extracellular Ca²⁺ concentration. To this end, erythrocytes were exposed to 2 mM phosphate for 48 hours either at 1 mM Ca²⁺ concentration or at 1.7 mM extracellular Ca²⁺ concentration As illustrated in Fig. 5, the effect of enhanced phosphate concentration was augmented by the additional increase of extracellular Ca²⁺ concentration. Those observations pointed to a role of CaHPO₄ precipitations. Since those precipitations could be inhibited by pyrophosphate, additional experiments were made in the presence of pyrophosphate. As illustrated in Fig. 5, addition of pyrophosphate significantly blunted the annexin-V-binding following exposure to 2 mM phosphate.

Further experiments explored the involvement of p38 kinase in the triggering of cell membrane scrambling by phosphate. To this end, erythrocytes were treated with 2 mM phosphate in the presence or absence of p38 kinase inhibitor SB203580. As shown in Fig. 6, the effect of phosphate on annexin-V-binding was significantly blunted by addition of 2 µM SB203580.

The present study reveals a novel effect of phosphate, i.e. an influence on erythrocyte cell membrane scrambling, a hallmark of suicidal erythrocyte death or eryptosis. The effect of phosphate was dependent on the presence of extracellular Ca²⁺ and was enhanced following an increase of extracellular Ca²⁺ concentration.

An increase of phosphate concentration from 0 to 0.5 mM was followed by a slight increase of cell volume which, however, remained virtually constant following further increases of extracellular phosphate concentration. Along those lines intracellular Ca²⁺ concentration ([Ca²⁺]_i) was rather decreased following increases of extracellular phosphate concentration. Other triggers of eryptosis shrink erythrocytes by increase of cytosolic Ca²⁺ concentration with subsequent activation of Ca²⁺ sensitive K⁺ channels [9,48], K⁺ exit, cell membrane hyperpolarisation, Cl^- exit and thus cellular loss of KCl with osmotically obliged water [9].

The effect of increasing phosphate concentration on cell membrane scrambling require the presence of extracellular Ca²⁺. Other Ca²⁺-sensitive triggers of eryptosis are effective by increasing [Ca²⁺]_i[9] with subsequent Ca²⁺ dependent stimulation of cell membrane scrambling. The effect of high phosphate concentration on erythrocyte cell membrane scrambling is, however, paralleled by decreasing [Ca²⁺]_i and obviously not due to enhanced entry of extracellular Ca²⁺. Instead, the effect of phosphate requires extracellular Ca²⁺. Furthermore, the effect was blunted by addition of pyrophosphate. Pyrophosphate inhibits hydroxyapatite formation and tissue calcification [49,50]. Calcium-phosphate crystals induce cell death in vascular smooth muscle cells [51]. In view of the present observations, it is tempting to speculate that the effect of phosphate is at least in part due to calcium phosphate supersaturation and precipitation. In osteoarthritic synovial fibroblasts, basic calcium phosphate crystals stimulate p38 kinase [52]. Along those lines, addition of the p38 inhibitor SB203580 blunted the effects of phosphate treatment on suicidal erythrocyte cell death.

The present paper did not elucidate the p38 kinase dependent mechanisms mediating calcium phosphate induced eryptosis. It is noteworthy, though, that p38 kinase targets include phospholipase 2 [53], which plays a dual role in the stimulation of eryptosis [9]. Phospholipase A2 has been shown to generate platelet activating factor, which activates sphingomyelinase and thus ceramide formation [9]. Ceramide sensitizes erythrocytes to the scrambling effect of Ca²⁺[9]. Whether or not phosphate stimulates ceramide formation, however, remains to be shown. Phospholipase A2 further generates arachidonic acid, which is converted by cycloxygenase to prostaglandin E₂, a stimulator of the Ca²⁺ permeable cation channels [9]. Since cytosolic Ca²⁺ activity did not change, this pathway is apparently not activated by phosphate. At least in theory, phosphate could in addition be effective by modifying further regulators of eryptosis, such as ATP [9], AMP activated kinase AMPK [9], casein kinase 1α [13,14], cGMP-dependent protein kinase [9], Janus-activated kinase JAK3 [15], protein kinase C [9] and caspases [9,11,12].

In healthy individuals, extraosseus hydroxyapatite formation is prevented by calcification inhibitors, most notably pyrophosphate but also Fetuin-A and matrix Gla protein [54]. In chronic kidney disease (CKD), the inhibitory mechanisms are depleted or overridden by marked hyperphosphatemia resulting in hydroxyapatite formation [49,54]. The serum calcification propensity is a predictor of mortality in CKD [55]. The stimulation of eryptosis by hyperphosphatemia or calcium phosphate supersaturation could therefore well contribute to the decreased life span of circulating erythrocytes in uremic patients. Chronic kidney disease is associated with increased levels of phosphatidylserine exposing erythrocytes [10,56]. Phosphatidylserine exposing erythrocytes adhere to phagocytosing cells and are thus rapidly cleared from circulating blood [9]. In renal insufficiency, anemia develops in part due to accelerated loss of erythrocytes, and in part due to impaired formation of new erythrocytes [57]. According ot the present observations the effect of hyperphosphatemia contributes to the eryptotic effects of uremic toxins. Further substances or disorders presumably contribute to the triggering of eryptosis and development of anemia in uremic patients.

As phosphatidylserine exposing erythrocytes adhere to the vascular wall [58] eryptosis could interfere with blood flow [9,58]. Phosphatidylserine exposing erythrocytes are further known to stimulate blood clotting [9,59,60]. Uncritical use of erythropoietin or other erythropoiesis stimulating agents [61,62,63] may thus foster the turnover of erythrocytes thus increasing the concentration of eryptotic erythrocytes with subequent impairment of microcirculation.

Increasing extracellular phosphate concentration fosters erythrocyte cell membrane scrambling and thus eryptosis, the suicidal death of erythrocytes. Phosphate thus shares the ability of some organic uremic toxins to trigger eryptosis.

All authors of this manuscript declare that they have no competing interests.

The study was supported by the Deutsche Forschungsgemeinschaft and the Open Access Publishing Fund of Tuebingen University.