Eryptosis - the Neglected Cause of Anemia in End Stage Renal Disease.

End stage renal disease (ESRD) invariably leads to anemia which has been mainly attributed to compromised release of erythropoietin from the defective kidneys with subsequent impairment of erythropoiesis. However, erythropoietin replacement only partially reverses anemia pointing to the involvement of additional mechanisms. As shown more recently, anemia of ESRD is indeed in large part a result of accelerated erythrocyte loss due to suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface. Phosphatidylserine exposing erythrocytes are bound to and engulfed by macrophages and are thus rapidly cleared from circulating blood. If the loss of erythrocytes cannot be fully compensated by enhanced erythropoiesis, stimulation of eryptosis leads to anemia. Eryptotic erythrocytes may further adhere to the vascular wall and thus impair microcirculation. Stimulators of eryptosis include complement, hyperosmotic shock, energy depletion, oxidative stress, and a wide variety of xenobiotics. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, and cyclin-dependent kinase 4. Eryptosis is inhibited by AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and some illdefined tyrosine kinases. In ESRD eryptosis is stimulated at least in part by a plasma component, as it is triggered by exposure of erythrocytes from healthy individuals to plasma from ESRD patients. Several eryptosis-stimulating uremic toxins have been identified, such as vanadate, acrolein, methylglyoxal, indoxyl sulfate, indole-3-acetic acid and phosphate. Attempts to fully reverse anemia in ESRD with excessive stimulation of erythropoiesis enhances the number of circulating suicidal erythrocytes and bears the risk of interference with micocirculation, At least in theory, anemia in ESRD could preferably be treated with replacement of erythropoietin and additional inhibition of eryptosis thus avoiding eryptosis-induced impairment of microcirculation. A variety of eryptosis inhibitors have been identified, their efficacy in ESRD remains, however, to be shown.

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein.

BACKGROUND: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2+]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. METHODS: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. RESULTS: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca2+]i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. CONCLUSION: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation.

Stimulation of Phospholipid Scrambling of the Erythrocyte Membrane by 9-Cis-Retinoic Acid.

BACKGROUND/AIMS: The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca2+ and/or ceramide. METHODS: Flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Exposure to 9-cis-retinoic acid (1 µg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis. CONCLUSIONS: 9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and ceramide.

Enhanced eryptosis following auranofin exposure.

BACKGROUND/AIMS: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. METHODS: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. RESULTS: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). CONCLUSIONS: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

Effect of saponin on erythrocytes.

Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.

Stimulation of erythrocyte cell membrane scrambling by mitotane.

UNLABELLED: background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+) concentration ([Ca(2+)]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. METHODS: [Ca(2+)]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. RESULTS: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca(2+)]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca(2+). Within 30 min Ca(2+) ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). CONCLUSIONS: Mitotane increases [Ca(2+)]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca(2+) induced cell shrinkage.

Suicidal erythrocyte death in end-stage renal disease.

UNLABELLED: Anemia in end-stage renal disease (ESRD) results mainly from erythropoietin and iron deficiency. Anemia could be confounded, however, by accelerated clearance of circulating erythrocytes because of premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+) concentration ([Ca(2+)]i), oxidative stress, and ceramide. The present study explored whether and how ESRD influences eryptosis. Blood was drawn from healthy volunteers (n = 20) as well as ESRD patients (n = 20) prior to and after hemodialysis. Phosphatidylserine exposure was estimated from annexin V binding, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, and ceramide from fluorescein-isothiocyanate-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes from healthy volunteers exposed in vitro for 24 h to plasma from healthy volunteers or ESRD patients prior to and following dialysis. The patients suffered from anemia (hemoglobin 10.1 ± 0.5 g/100 ml) despite 1.96 ± 0.34 % reticulocytes. The percentage of phosphatidylserine-exposing erythrocytes was significantly higher in ESRD patients (0.84 ± 0.09 %) than in healthy volunteers (0.43 ± 0.04 %) and was significantly increased immediately after dialysis (1.35 ± 0.13 %). The increase in phosphatidylserine exposure was paralleled by increase in [Ca(2+)]i, oxidative stress, and ceramide abundance. As compared to addition of plasma from healthy individuals, addition of predialytic but not of postdialytic plasma from ESRD patients increased phosphatidylserine exposure, [Ca(2+)]i, ROS, and ceramide abundance. In conclusion, both, dialyzable components of uremic plasma and dialysis procedure, trigger eryptosis at least in part by increasing erythrocyte [Ca(2+)]i, ROS, and ceramide formation. KEY MESSAGES: Anemia in uremia results in part from eryptosis, the suicidal erythrocyte death. Eryptosis in uremia is triggered in part by a dialyzable plasma component. Eryptosis in uremia is further triggered by dialysis procedure. Eryptosis in uremia is in part due to increased cytosolic Ca(2+) concentration. Eryptosis in uremia is further due to oxidative stress and ceramide formation.

Stimulation of erythrocyte cell membrane scrambling by mushroom tyrosinase.

BACKGROUND: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter. CONCLUSIONS: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation.

Breakdown of phosphatidylserine asymmetry following treatment of erythrocytes with lumefantrine.

BACKGROUND: Lumefantrine, a commonly used antimalarial drug, inhibits hemozoin formation in parasites. Several other antimalarial substances counteract parasitemia by triggering suicidal death or eryptosis of infected erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), formation of ceramide, oxidative stress and/or activation of p38 kinase, protein kinase C (PKC), or caspases. The present study explored, whether lumefantrine stimulates eryptosis. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, content of reduced glutathione (GSH) from mercury orange fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 48 h exposure to lumefantrine (3 µg/mL) was followed by a significant increase of annexin-V-binding without significantly altering forward scatter, [Ca2+]i, ROS formation, reduced GSH, or ceramide abundance. The annexin-V-binding following lumefantrine treatment was not significantly modified by p38 kinase inhibitors SB203580 (2 µM) and p38 Inh III (1 µM), PKC inhibitor staurosporine (1 µM) or pancaspase inhibitor zVAD (1 or 10 µM). CONCLUSIONS: Lumefantrine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+, ceramide formation, ROS formation, glutathione content, p38 kinase, PKC or caspases.

Stimulation of suicidal erythrocyte death by ribavirin.

Ribavirin is widely used in the treatment for viral disease such as chronic viral hepatitis. Side effects limiting the use of the drug include haemolytic anaemia. If challenged by stimulators of haemolysis, erythrocytes may enter suicidal death or eryptosis, thus preventing the release of haemoglobin into circulating blood. Eryptosis is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase in cytosolic Ca2+ activity ([Ca2+]i). This study explored whether ribavirin modifies [Ca2+]i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, haemolysis from haemoglobin release and [Ca2+]i from Fluo-3 fluorescence. A 48-hr exposure to ribavirin (≥8 µg/ml) was followed by a significant increase in [Ca2+]i, a significant decrease in forward scatter and a significant increase in annexin V binding. The annexin V binding after ribavirin treatment was significantly blunted but not abolished in the nominal absence of extracellular Ca2+. In conclusion, ribaverin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+.

Effect of nitazoxanide on erythrocytes.

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis-like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca(2+) -activity ([Ca(2+) ]i ). The Ca(2+) -sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca(2+) ]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48-hr exposure to nitazoxanide (1-50 µg/ml) did not significantly modify [Ca(2+) ]i but significantly increased ceramide formation, decreased forward scatter (≥10 µg/ml), increased the percentage of annexin-V-binding erythrocytes (≥10 µg/ml) and, at higher concentrations (≥20 µg/ml), stimulated haemolysis. The stimulation of annexin-V-binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.

Stimulation of suicidal erythrocyte death by artesunate.

BACKGROUND: The artemisinin derivative artesunate is effective in the treatment of severe malaria and is considered for the treatment of malignancy. Artesunate triggers tumor cell apoptosis, an effect at least in part mediated by mitochondria. Even though lacking mitochondria, erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and breakdown of the phospholipid asymmetry of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), ceramide formation, and oxidative stress. The present study explored whether artesunate stimulates eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. RESULTS: A 48 h exposure of human erythrocytes to artesunate significantly increased the percentage of annexin-V-binding cells (≥ 9 µg/ml) without significantly influencing forward scatter. Artesunate significantly increased [Ca(2+)]i. The stimulation of annexin-V-binding by artesunate (15 µg/ml) was significantly blunted but not abolished by removal of extracellular Ca(2+). Artesunate increased the ceramide abundance at the cell surface and the 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. CONCLUSIONS: Artesunate stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to increase of [Ca(2+)]i, stimulation of ceramide formation and generation of oxidative stress.

Oxidative stress and suicidal erythrocyte death.

SIGNIFICANCE: Eryptosis, the suicidal erythrocyte death, is characterized by cell shrinkage, membrane blebbing, and phosphatidylserine translocation to the outer membrane leaflet. Phosphatidylserine at the erythrocyte surface binds endothelial CXCL16/SR-PSOX (CXC-Motiv-Chemokin-16/Scavenger-receptor-for-phosphatidylserine-and-oxidized-low-density-lipoprotein) and fosters engulfment of affected erythrocytes by phagocytosing cells. Eryptosis serves to eliminate infected or defective erythrocytes, but excessive eryptosis may lead to anemia and may interfere with microcirculation. Clinical conditions with excessive eryptosis include diabetes, chronic renal failure, hemolytic uremic syndrome, sepsis, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, glutamate cysteine ligase modulator deficiency, and Wilson's disease. RECENT ADVANCES: Eryptosis is triggered by a wide variety of xenobiotics and other injuries such as oxidative stress. Signaling of eryptosis includes prostaglandin E2 formation with subsequent activation of Ca(2+)-permeable cation channels, Ca(2+) entry, activation of Ca(2+)-sensitive K(+) channels, and cell membrane scrambling, as well as phospholipase A2 stimulation with release of platelet-activating factor, sphingomyelinase activation, and ceramide formation. Eryptosis may involve stimulation of caspases and calpain with subsequent degradation of the cytoskeleton. It is regulated by AMP-activated kinase, cGMP-dependent protein kinase, Janus-activated kinase 3, casein kinase 1α, p38 kinase, and p21-activated kinase 2. It is inhibited by erythropoietin, antioxidants, and further small molecules. CRITICAL ISSUES: It remains uncertain for most disorders whether eryptosis is rather beneficial because it precedes and thus prevents hemolysis or whether it is harmful because of induction of anemia and impairment of microcirculation. FUTURE DIRECTIONS: This will address the significance of eryptosis, further mechanisms underlying eryptosis, and additional pharmacological tools fostering or inhibiting eryptosis.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 µM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Triggering of suicidal erythrocyte death by penta-O-galloyl-ß-D-glucose.

The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-ß-D-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-ß-D-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-ß-D-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-ß-D-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-ß-D-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-ß-D-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.

Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.

BACKGROUND/AIMS: Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. METHODS: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca(2+)-concentration utilizing photometry. Erythrocytes were exposed to Ca(2+)-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. RESULTS: Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. CONCLUSIONS: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca(2+)-entry, energy depletion, hyperosmotic shock and oxidative stress.

Triggering of suicidal erythrocyte death by uremic toxin indoxyl sulfate.

BACKGROUND: Anemia in end stage renal disease is attributed to impaired erythrocyte formation due to erythropoietin and iron deficiency. On the other hand, end stage renal disease enhances eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i) and by ceramide, which sensitizes erythrocytes to [Ca2+]i. Mechanisms triggering eryptosis in endstage renal disease remained enigmatic. The present study explored the effect of indoxyl sulfate, an uremic toxin accumulated in blood of patients with chronic kidney disease. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, ceramide abundance by specific antibodies, hemolysis from hemoglobin release, and [Ca(2+)]i from Fluo3-fluorescence. RESULTS: A 48 hours exposure to indoxyl sulfate significantly increased [Ca(2+)]i (≥ 300 µM), significantly decreased forward scatter (≥ 300 µM) and significantly increased annexin-V-binding (≥ 50 µM). Indoxyl sulfate (150 µM) induced annexin-V-binding was virtually abolished in the nominal absence of extracellular Ca(2+). Indoxyl sulfate (150 µM) further enhanced ceramide abundance. CONCLUSION: Indoxyl sulfate stimulates suicidal erythrocyte death or eryptosis, an effect in large part due to stimulation of extracellular Ca(2+)entry with subsequent stimulation of cell shrinkage and cell membrane scrambling.

Tannic acid induced suicidal erythrocyte death.

BACKGROUND: The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca(2+) by ceramide. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fuorescence and ceramide utilizing fluorescent antibodies. RESULTS: A 48 h treatment with tannic acid was followed by significant decrease of forward scatter (≥ 1 µg/ml) and significant increase of annexin-V-binding (≥ 10 µg/ml). Tannic acid did not significantly modify [Ca(2+)]i (up to 50 µM) but significantly increased ceramide formation (50 µM). The annexin-V-binding following tannic acid treatment (50 µM) was significantly blunted in the nominal absence of extracellular Ca(2+). CONCLUSIONS: Tannic acid stimulates eryptosis, an effect at least partially due to ceramide formation with subsequent sensitization of erythrocytes to cytosolic Ca(2+).

Effect of thioridazine on erythrocytes.

BACKGROUND: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²âº-concentration ([Ca²âº](i)) and activation of p38 kinase. The present study explored, whether thioridazine elicits eryptosis. METHODS: [Ca²âº](i) has been estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, and hemolysis from hemoglobin release. RESULTS: A 48 hours exposure to thioridazine was followed by a significant increase of [Ca²âº](i) (30 µM), decrease of forward scatter (30 µM), and increase of annexin-V-binding (≥12 µM). Nominal absence of extracellular Ca²âº and p38 kinase inhibitor SB203580 (2 µM) significantly blunted but did not abolish annexin-V-binding following thioridazine exposure. CONCLUSIONS: Thioridazine stimulates eryptosis, an effect in part due to entry of extracellular Ca²âº and activation of p38 kinase.

Effect of miltefosine on erythrocytes.

BACKGROUND: Miltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca(2+)-level ([Ca(2+)]i). The present study explored, whether miltefosine elicits eryptosis. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence. RESULTS: A 48 h exposure to miltefosine (≥ 4.9 µM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca(2+)]i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca(2+). CONCLUSION: Miltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca(2+) entry.