1 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas and 2 Division of Nephrology and Hypertension, University of California at Irvine, Irvine, California, USA
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Abstract |
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Methods. Rats were randomized into four groups after 5/6 nephrectomies to produce CRF. The Epo-treated CRF group received Epo, 150 U/kg, twice weekly for 6 weeks to prevent anaemia; the felodipine and Epo-treated CRF group received Epo but was kept normotensive by felodipine treatment; the placebo-treated CRF group received placebo injections and became anaemic; and the iron-deficient CRF group received Epo but was kept anaemic by dietary iron-deficiency. A group of sham-operated rats was included as normal control. Basal and thrombin-stimulated platelet cytosolic calcium ([Ca2+]i) were determined using a Ca2+-sensitive dye (fura-2).
Results. Platelets from placebo-treated CRF group exhibited a profound attenuation of thrombin-stimulated surge in [Ca2+]i, which is the final pathway of platelet activation. Long-term Epo administration led to a normalization of the thrombin-induced rise in platelet [Ca2+]i in the CRF animals, independent of either haematocrit or blood pressure values. Further studies revealed that improved Ca2+ signalling with Epo is associated with increased Ca2+ uptake and expanded Ca2+ stores in the platelets.
Conclusions. The defective Ca2+ signalling in uraemic animals and its improvement with chronic Epo therapy provides the biochemical basis of the previously reported platelet dysfunction and prolonged bleeding time in uraemic patients and animals, and their amelioration with chronic Epo therapy.
Keywords: anaemia; bleeding time; cytosolic calcium; erythropoietin; hypertension; platelets; uraemia
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Introduction |
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The introduction of recombinant human erythropoietin (Epo) has revolutionized the treatment of anaemia associated with CRF. Besides its effect on the stimulation of erythropoiesis, Epo improves platelet adhesion/aggregation in uraemic patients [6,7]. However, the mechanisms of improved platelet function after Epo therapy has not been fully elucidated [8]. Krawczyk et al. [2] reported that Epo therapy partially normalized uraemic platelet phospholipid contents. Malyszko et al. [9] found that Epo therapy significantly increased both whole blood and platelet serotonin concentrations. Finally, Turi et al. [10] showed that Epo decreased platelet cAMP content but improved ATP release during platelet aggregation. The former was negatively, while the latter was positively, correlated with platelet aggregation.
It is well known that cytosolic calcium plays an essential role in the regulation of platelet function [11,12]. Several studies [13,14] have shown that CRF is associated with increased resting cytosolic calcium. In addition, in a study addressing the mechanisms of Epo-induced hypertension in rats [15], we used platelets as a surrogate of smooth muscle cells with regard to [Ca2+]i regulation. We found that short-term incubation of Epo with platelets lead to Ca2+ influx and expanded intracellular calcium stores [15]. These observations prompted us to examine the hypothesis that the improved platelet function after long-term Epo therapy in CRF is, in part, due to correction of platelet calcium signalling.
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Subjects and methods |
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Animal models
Male SpragueDawley rats (Charles River, Wilmington, MA, USA) with an average body weight of 300 g were used. The animals were fed a standard laboratory diet (Purina Rat Chow; Purina Mills, Brentwood, MO, USA) and water ad libitum. Under general anaesthesia, the animals were subjected to a right nephrectomy followed by a two-thirds left nephrectomy 4 days later to produce CRF. The nephrectomies were carried out extraperitoneally through a dorsal incision. Strict haemostasis and aseptic measures were observed during the surgical procedures. The animals were then randomized into three groups.
Epo-treated CRF group received intraperitoneal injections of recombinant human Epo, 150 U/kg, two times weekly for 6 weeks. The Epo dose employed was based on the preliminary experiments carried out to determine the amount required to maintain normal haematocrit in CRF animals. The placebo-treated CRF group received placebo injections at the same frequency and were allowed to become anaemic. The iron-deficient CRF group received Epo injections as in Epo-treated CRF groups, but were necessarily Epo-resistant due to iron deficiency. Iron deficiency was induced by feeding the animals an iron-free diet (prepared by Purina Test Diets, Richmond, IN, USA) for 6 weeks, beginning at 12 weeks of age (average weight 200 g), which was continued after nephrectomy. A group of sham-operated rats was included as normal controls.
To discern the effect of blood pressure, a subgroup of CRF animals was simultaneously treated with Epo and the calcium channel blocker felodipine (Astra Zeneca, Inc., Wilmington, DE, USA). Felodipine was administered by implanted osmotic pumps (Alza, Inc., Palo Alto, CA, USA) at 7 mg/kg/day. Body weight, blood pressure and haematocrit (microhaematocrit method) were measured weekly. At the end of the 6-week observation period, the animals were placed in metabolic cages for a 24-h urine collection. The following measurements were performed.
Measurement of blood pressure
Arterial blood pressure was measured by tail plethysmography (Harvard Apparatus Inc., Natick, MA, USA) as described previously [15]. Briefly, conscious rats were placed in a restrainer on a heated pad and allowed to rest inside the cage for 15 min prior to blood pressure measurements. The procedure was carried out in a climate-controlled room with an ambient temperature of 70°F. Rat tails were placed inside a tail cuff, and the cuff was inflated and released several times to allow conditioning of the animals to the procedure. A minimum of four consecutive measurements were taken and recorded by a student oscillograph (Harvard Apparatus, Natick, MA, USA). The data were then averaged for presentation.
Platelet cytosolic calcium measurements
At the end of the 6-week observation period, rats were killed, and platelets were isolated using a modification of the procedure described previously [15]. In brief, freshly drawn blood was mixed with a solution containing 2.5 g sodium citrate, 1.5 g citric acid and 2 g dextrose in 100 ml H2O at a 1:6 ratio. Platelet-rich plasma was then prepared by centrifugation of the specimen at room temperature for 7 min at 700 g, followed by centrifugation at 400 g for 20 min to sediment the platelets. The sedimented platelets were then gently re-suspended in HEPES-buffered saline (HBS) with the following composition (mMol/l): 145 NaCl, 5.0 KCl, 0.8 Na2HPO4, 0.2 KH2PO4, 1.0 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. This resulted in a platelet suspension containing 2x108 cells/ml. Cytosolic calcium concentration ([Ca2+]i) was determined with the fluorescent calcium indicator fura 2-AM. The suspended platelets were loaded with 4 µM fura 2-AM in the presence of 0.02% pluronic F-127 to facilitate entry of the indicator into the cells. In addition, 2 mM probenecid was added to minimize leakage of fura 2 out of the platelets [15]. Cells were incubated for 60 min at 37°C and then centrifuged at 400 g for 20 min. The supernatant was decanted and an equal volume of HBS was added. The cells were incubated at 37°C for 1 h to allow complete hydrolysis of the AM group. The dye-loaded cells were suspended in HBS containing 2 mM CaCl2 and were kept under constant magnetic stirring in a thermostatically controlled cuvette of a spectrofluorometer (DMX 1000; SLM Instruments, Urbana, IL, USA). Alternating excitation wavelengths of 340 and 380 nm were used, with an emission wavelength of 510 nm. Ratios of fluorescence (R=340/380 nm) were measured every 1 s, automatically corrected for autofluorescence, and plotted graphically for each sample analysed. The number of platelets in each cuvette of spectrofluorometer was kept constant (
1x107) in all groups. Throughout the study, the platelet pellet was easily resuspended, indicating negligible contact activation due to careful centrifugation procedure. Values of autofluorescence were <5% of the fluorescence of the dye-loaded cells and were measured for every experiment.
[Ca2+]i was calculated as described previously [15] using the following formula: [Ca2+]i=(Kd)(B)(R-Rmin)/(Rmax-R), where B is the ratio of 380 nm fluorescence in the absence and presence of a saturating concentration of calcium, and Kd is the dissociation constant for fura 2, assumed to be 225 nM. The cells were lysed with Triton (0.05%) to obtain the maximal value (Rmax) in the presence of 2 mMol calcium, and the minimum value (Rmin) was obtained by the addition of 10 mMol EGTA and sufficient NaOH to raise the pH to 8.5.
To discern the short-term effect of Epo on [Ca2+]i, platelet [Ca2+]i from control rats was measured before and after the addition of Epo at a final concentration of 200 U/ml. The choice of this concentration was based on our earlier observations that Epo at 200 U/ml caused significant vascular muscle contraction in vitro [15]. In parallel experiments, Epo-free vehicle was used for comparison. In another set of experiments, the effect of Epo on the thrombin-mediated rise in platelet [Ca2+]i was examined. To this end, platelets were incubated for 30 min in a buffer containing either Epo (200 U/ml) or the Epo-free vehicle (at room temperature). [Ca2+]i was then continuously monitored prior to and after platelet activation with 0.1 U/ml thrombin. To discern the effect of Epo on calcium uptake versus release from intracellular stores, in a separate set of experiments platelets were incubated in the presence of Epo or Epo-free vehicle for 30 min, after which extracellular calcium was chelated by EGTA (5 mM) at pH 7.4. The platelets were then activated by the addition of thrombin 30 s later, and [Ca2+]i was measured.
Data presentation and analysis
Data are presented as means±SE. One-way analysis of variance (ANOVA) and Duncan's range test were used in analysis of the data. P values 0.05 were considered statistically significant.
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Results |
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To determine the effect of Epo on intracellular calcium stores, platelets were first incubated in a calcium-containing buffer supplemented with either Epo or the vehicle alone for 30 min to allow calcium uptake. The extracellular Ca2+ was then chelated by EGTA, and [Ca2+]i was measured after activation with thrombin. The results showed a significantly higher [Ca2+]i surge in Epo-treated platelets than in those exposed to the vehicle alone (142±23 vs 76±14 nM; n=8, P<0.05).
Long-term effect of Epo on [Ca2+]i (Figures 1 and 2
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Resting platelet [Ca2+]i was elevated in all CRF groups compared with the normal control group. Comparison of the placebo-treated with Epo-treated groups showed significantly higher resting [Ca2+]i values in the Epo-treated animals (P<0.05, ANOVA). As expected, activation with thrombin led to a marked rise in platelet [Ca2+]i in the normal controls. Thrombin-induced rise in platelet [Ca2+]i was markedly attenuated in the placebo-treated CRF animals compared with the normal control animals. However, platelets from both the iron-sufficient and -deficient Epo-treated CRF groups showed a nearly complete restoration of [Ca2+]i response to thrombin activation.
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Discussion |
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In an attempt to test the hypothesis that the improved platelet function after Epo therapy is due to the correction of decreased stimulated [Ca2+]i in uraemic platelets, we studied three different groups of CRF animals treated with Epo or placebo and in the presence and absence of anaemia. We found a significant increase in [Ca2+]i at baseline in all CRF groups compared with the normal control group. Comparison of the Epo- and placebo-treated CRF animals revealed significantly higher resting [Ca2+]i values in the former group. The expected thrombin-induced rise in [Ca2+]i was significantly attenuated in the placebo-treated CRF animals compared with that observed in the normal control group. Interestingly, the Epo-treated CRF groups showed restoration of [Ca2+]i response to thrombin whether the haematocrit was allowed to rise or was prevented from rising by diet-induced iron deficiency. These findings are consistent with clinical studies reporting improvement or normalization of bleeding time and platelet function with short-term Epo therapy in CRF patients exhibiting no change in haematocrit [6,7]. This phenomenon could be explained by the observations in iron-deficient CRF animals, where a thrombin-induced rise in platelet [Ca2+]i was normalized despite persistent anaemia. Thus, the present study revealed a severe platelet Ca2+ signalling defect in the untreated CRF rats. Epo therapy completely normalized the attenuation of the thrombin-induced surge in uraemic platelets [Ca2+]i. The CRF-induced defective agonist-stimulated surge in [Ca2+]i, which is the final pathway of platelet activation, and its correction with chronic Epo therapy provides the biochemical basis of impaired platelet function and prolonged bleeding time in uraemia and the improvements thereof with Epo therapy.
It is well known that hypertension is associated with dysregulation of [Ca2+]i [18]. To discern the effect of hypertension, a subgroup of Epo-treated CRF animals was simultaneously treated with felodipine. Concurrent administration of felodipine abrogated the Epo-induced hypertension and normalized both basal and thrombin-stimulated [Ca2+]i. These observations clearly demonstrate that improved Ca2+ signalling with Epo was unrelated to blood pressure. The latter conclusion is based on the fact that animals in the Epo-treated CRF group and the Epo/felodipine co-treated CRF group had a comparable degree of renal insufficiency and stimulated [Ca2+]i, but markedly different blood pressures.
In an attempt to explore the mechanisms by which Epo affects cytosolic calcium, we examined the effect of Epo on [Ca2+]i in normal platelets in vitro. The results showed that the addition of Epo at high concentrations significantly raised resting platelet [Ca2+]i. In order to discern the effect of Epo on calcium uptake and intracellular stores, the platelets were first incubated in a calcium-containing buffer in the presence and absence of Epo. At the end of this period, Ca2+ was removed from the extracellular medium by chelation with EGTA, and platelets were activated with thrombin to raise [Ca2+]i. We found that the magnitude of the thrombin-induced rise in [Ca2+]i was significantly increased by preincubation with Epo. This observation suggests that Epo augments calcium entry into the cells during the incubation period, leading to an expanded intracellular calcium store. The latter was, in turn, assumed to be partially responsible for the augmented rise in [Ca2+]i with thrombin. These observations are consistent with the conclusions of Tepel et al. [18]. In addition, recent studies have revealed that Epo binding to its receptors leads to the activation and coupling of cytoplasmic tyrosine kinase, which results in phosphorylation (activation) of phospholipase C-1 [19]. This in turn promotes generation of inositol triphosphate (IP3) from phosphatidyl inositol diphosphate (PIP2), which triggers an initial Ca2+ release from intracellular stores followed by an extended influx of Ca2+ from the extracellular medium [19]. In fact, it has been shown that Epo therapy corrects the impaired tyrosine phosphorylation associated with uraemic platelets in humans [20].
It should be noted that the results of the present study may be consistent with the conclusions of Noris and Remuzzi [1], who proposed that uraemic platelet dysfunction may be due to increased platelet and vascular NO production. This is because through a cGMP-mediated process, NO lowers [Ca2+]i. By virtue of its ability to raise [Ca2+]i and augment intracellular Ca2+ stores, Epo can counteract the Ca2+-lowering action of NO and thus help to restore normal Ca2+ signalling in uraemic platelets.
In summary, uraemic platelets exhibited a profound attenuation of agonist-stimulated surge in [Ca2+]i, which is the final pathway of platelet activation. Long-term Epo administration led to normalization of the thrombin-induced surge in platelet [Ca2+]i in the CRF animals, which is independent of its effects on haematocrit and blood pressure. Further studies revealed that improved Ca2+ signalling with Epo is associated with increased Ca2+ uptake and expanded Ca2+ stores in platelets. The defective Ca2+ signalling in uraemic animals and its improvement with chronic Epo therapy provides the biochemical basis of the previously reported platelet dysfunction and prolonged bleeding time in uraemia, and their amelioration with chronic Epo therapy.
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Acknowledgments |
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Notes |
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References |
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