Sickle Cell Dehydration Research Articles

Magnesium for treating sickle cell disease.

Sickle cell disease is an autosomal recessive inherited haemoglobinopathy which causes painful vaso-occlusive crises due to sickle red blood cell dehydration. Vaso-occlusive crises are common painful events responsible for a variety of clinical complications; overall mortality is increased and life expectancy decreased compared to the general population. Experimental studies suggest that intravenous magnesium has proven to be well-tolerated in individuals hospitalised for the immediate relief of acute (sudden onset) painful crisis and has the potential to decrease the length of hospital stay. Some in vitro studies and open studies of long-term oral magnesium showed promising effect on pain relief but failed to show its efficacy. The studies show that oral magnesium therapy may prevent sickle red blood cell dehydration and prevent recurrent painful episodes. There is a need to access evidence for the impact of oral and intravenous magnesium effect on frequency of pain, length of hospital stay and quality of life. This is an updated version of the review. To evaluate the effects of short-term intravenous magnesium on the length of hospital stay and quality of life in children and adults with sickle cell disease. To determine the effects of long-term oral magnesium therapy on the frequency of painful crises and the quality of life in children and adults with sickle cell disease. We searched the Cochrane Haemoglobinopathies Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books.Date of last search of the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register: 03 February 2019.Date of last search of other resources (clinical trials registries): 04 April 2019. We searched for published and unpublished randomized controlled studies of oral or intravenous magnesium compared to placebo or no magnesium. Authors independently assessed the study quality and extracted the data using standard Cochrane methodologies. We included five randomized placebo-controlled studies with a total of 386 participants (aged three to 53 years). Of these, two shorter parallel studies (n = 306) compared intravenous magnesium sulphate to placebo (normal saline) for admission to hospital due to a vaso-occlusive crisis, for which we were able to analyse data. The quality of evidence was moderate for studies in this comparison, mainly due to limitations due to risk of bias and imprecision. Two of the three longer-term studies comparing oral magnesium pidolate to placebo had a cross-over design. The third was a parallel factorial study which compared hydroxyurea and oral magnesium to each other and to placebo over a longer period of time; we only present the comparison of oral magnesium to placebo from this study. The quality of evidence was very low with uncertainty of the estimation.The eight-hourly dose levels in the two studies of intravenous magnesium were different; one used 100 mg/kg while the second used 40 mg/kg. Only one of these studies (n = 104) reported the mean daily pain score while hospitalised (a non-significant difference between groups, moderate quality evidence). The second study (n = 202) reported a number of child- and parent-reported quality of life scores. None of the scores showed any difference between treatment groups (low quality evidence). Data from one study (n = 106) showed no difference in length of stay in hospital between groups (low quality evidence). Both studies reported on adverse events, but not defined by severity as we had planned. One study showed significantly more participants receiving intravenous magnesium experienced warmth at infusion site compared to placebo; there were no differences between groups for other adverse events (low quality evidence).Three studies (n = 80) compared oral magnesium pidolate to placebo. None of them reported data which we were able to analyse. One study (n = 24) reported on the number of painful days and stated there was no difference between two groups (low quality evidence). None of the studies reported on quality of life or length of hospital stay. Two studies (n = 68) reported there were no differences in levels of magnesium in either plasma or red blood cells (moderate quality evidence). Two studies (n = 56) reported adverse events. One reported episodes of mild diarrhoea and headache, all of which resolved without stopping treatment. The second study reported adverse events as gastrointestinal disorders, headache or migraine, upper respiratory infections and rash; which were all evenly distributed across treatment groups (moderate quality evidence). Moderate to low quality evidence showed neither intravenous magnesium and oral magnesium therapy has an effect on reducing painful crisis, length of hospital stay and changing quality of life in treating sickle cell disease. Therefore, no definitive conclusions can be made regarding its clinical benefit. Further randomized controlled studies, perhaps multicentre, are necessary to establish whether intravenous and oral magnesium therapies have any effect on improving the health of people with sickle cell disease.

Total Serum Magnesium Levels and Calcium-To-Magnesium Ratio in Sickle Cell Disease.

Background and Objectives: Imbalance of calcium/magnesium ratio could lead to clinical complications in sickle cell disease (SCD). Low levels of magnesium have been associated with sickling, increased polymerization and vaso-occlusion (VOC) in sickle cell due to cell dehydration. The K-Cl cotransport plays a very important role in sickle cell dehydration and is inhibited by significantly increasing levels of magnesium. The study evaluated total serum magnesium levels and computed calcium/magnesium ratio in SCD patients and “healthy” controls. Materials and Methods: The study was a case-control cross-sectional one, involving 120 SCD patients (79 Haemoglobin SS (HbSS)and 41 Haemoglobin SC (HbSC)) at the steady state and 48 “healthy” controls. Sera were prepared from whole blood samples (n = 168) and total magnesium and calcium measured using a Flame Atomic Absorption Spectrometer (Variant 240FS manufactured by VARIAN Australia Pty Ltd., Melbourne, VIC, Australia). Calcium/magnesium ratios were calculated in patients and the controls. Results: The prevalence of hypomagnesemia and hypocalcaemia among the SCD patients was observed to be 39.17% and 52.50% respectively, higher than the controls (4.17% and 22.92%, for hypomagnesemia and hypocalcaemia, respectively). Level of magnesium was significantly lower in the SCD patients compared to their healthy counterparts (p = 0.002). The magnesium level was further reduced in the HbSS patients but not significantly different from the HbSC patients (p = 0.584). calcium/magnesium ratio was significantly higher in the SCD patients (p = 0.031). Although calcium/magnesium ratio was higher in the HbSC patients compared to those with the HbSS genotype, the difference was not significant (p = 0.101). Conclusion: The study shows that magnesium homeostasis are altered in SCD patients, and their levels are lower in HbSS patients. Although calcium/magnesium ratio is significantly higher in SCD patients compared with controls, there is no significant difference between patients with HbSS and HbSC genotypes. Magnesium supplementation may be required in sickle cell patients.

Combined genetic disruption of K-Cl cotransporters and Gardos channel KCNN4 rescues erythrocyte dehydration in the SAD mouse model of sickle cell disease

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.

New Therapeutic Options for the Treatment of Sickle Cell Disease.

Sickle cell disease (SCD; ORPHA232; OMIM # 603903) is a chronic and invalidating disorder distributed worldwide, with high morbidity and mortality. Given the disease complexity and the multiplicity of pathophysiological targets, development of new therapeutic options is critical, despite the positive effects of hydroxyurea (HU), for many years the only approved drug for SCD.New therapeutic strategies might be divided into (1) pathophysiology-related novel therapies and (2) innovations in curative therapeutic options such as hematopoietic stem cell transplantation and gene therapy. The pathophysiology related novel therapies are: a) Agents which reduce sickling or prevent sickle red cell dehydration; b) Agents targeting SCD vasculopathy and sickle cell-endothelial adhesive events; c) Anti-oxidant agents.This review highlights new therapeutic strategies in SCD and discusses future developments, research implications, and possible innovative clinical trials.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Magnesium for treating sickle cell disease.

Sickle cell disease is an autosomal recessive inherited haemoglobinopathy which causes painful vaso-occlusive crises due to sickle red blood cell dehydration. Vaso-occlusive crises are common painful events responsible for a variety of clinical complications; overall mortality is increased and life expectancy decreased compared to the general population. Experimental studies suggest that intravenous magnesium has proven to be well-tolerated in individuals hospitalised for the immediate relief of acute (sudden onset) painful crisis and has the potential to decrease the length of hospital stay. Some in vitro studies and open studies of long-term oral magnesium showed promising effect on pain relief but failed to show its efficacy. The studies show that oral magnesium therapy may prevent sickle red blood cell dehydration and prevent recurrent painful episodes. There is a need to access evidence for the impact of oral and intravenous magnesium effect on frequency of pain, length of hospital stay and quality of life. To evaluate the effects of short-term intravenous magnesium on the length of hospital stay and quality of life in children and adults with sickle cell disease. To determine the effects of long-term oral magnesium therapy on the frequency of painful crises and the quality of life in children and adults with sickle cell disease. We searched the Cochrane Haemoglobinopathies Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books.Date of last search of the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register: 01 December 2016.Date of last search of other resources (clinical trials registries): 29 March 2017. We searched for published and unpublished randomized controlled studies of oral or intravenous magnesium compared to placebo or no magnesium. Authors independently assessed the study quality and extracted the data using standard Cochrane methodologies. We included five randomized placebo-controlled studies with a total of 386 participants (aged three to 53 years). Two shorter parallel studies (n = 306) compared intravenous magnesium sulphate to placebo (normal saline) for admission to hospital due to a vaso-occlusive crisis, for which we were able to analyse data. The quality of evidence was moderate for studies presenting this comparison mainly due to limitations due to risk of bias and imprecision. Two of the three longer-term studies comparing oral magnesium pidolate to placebo had a cross-over design. The third was a parallel factorial study which compared hydroxyurea and oral magnesium to each other and to placebo over a longer period of time; we only present the comparison of oral magnesium to placebo from this study. The quality of evidence was very low with uncertainty of the estimation.The eight-hourly dose levels in the two studies of intravenous magnesium were different; one used 100 mg/kg while the second used 40 mg/kg. Only one of these studies (n = 104) reported the mean daily pain score while hospitalised (a non-significant difference between groups, moderate quality evidence). The second study (n = 202) reported a number of child- and parent-reported quality of life scores. None of the scores showed any difference between treatment groups (low quality evidence). Data from one study (n = 106) showed no difference in length of stay in hospital between groups (low quality evidence). Both studies reported on adverse events, but not defined by severity as we had planned. One study showed significantly more participants receiving intravenous magnesium experienced warmth at infusion site compared to placebo; there were no differences between groups for other adverse events (low quality evidence).Three studies (n = 80) compared oral magnesium pidolate to placebo. None of them reported data which we were able to analyse. One study (n = 24) reported on the number of painful days and stated there was no difference between two groups (low quality evidence). None of the studies reported on quality of life or length of hospital stay. Two studies (n = 68) reported there were no differences in levels of magnesium in either plasma or red blood cells (moderate quality evidence). Two studies (n = 56) reported adverse events. One reported episodes of mild diarrhoea and headache, all of which resolved without stopping treatment. The second study reported adverse events as gastrointestinal disorders, headache or migraine, upper respiratory infections and rash; which were all evenly distributed across treatment groups (moderate quality evidence). Moderate to low quality evidence showed neither intravenous magnesium and oral magnesium therapy has an effect on reducing painful crisis, length of hospital stay and changing quality of life in treating sickle cell disease. Therefore, no definitive conclusions can be made regarding its clinical benefit. Further randomized controlled studies, perhaps multicentre, are necessary to establish whether intravenous and oral magnesium therapies have any effect on improving the health of people with sickle cell disease.

Rheological effect of activation of the KCl-cotransport pathway in normal and sickle erythrocytes

Activation of the KCl-cotransport pathway in vivo is a potentially important cause of dehydration and rheological impairment of sickle cells. Its activation in vitro is a useful new method for investigating the rheological effects of putative anti-sickling compounds, including specific inhibitors of the KCl-cotransporter

Protein Disulfide Isomerase in Sickle Cell Disease

Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] DisclosuresNo relevant conflicts of interest to declare.

The Novel PIEZO1 Mutation p.L2023V Is Causal for Hereditary Xerocytosis Resulting in Delayed Channel Inactivation and a Dehydrated Red Blood Cell Phenotype

The regulation of cell volume is important for the maintenance of integrity in all cells and is especially critical for the highly specialized red blood cell (RBC) which must withstand pressure changes within the vasculature and remain deformable to traverse small vessels. Disorders that interfere with volume homeostasis result in the premature destruction of RBCs. One protein that appears to play a prominent role in RBC hydration is the recently described nonselective cation channel PIEZO1 which is involved in mechanotransduction. Mutations of PIEZO1 have been associated with an autosomal dominant form of hereditary hemolytic anemia (HHA) characterized by RBC dehydration known as hereditary xerocytosis (HX) (Zarychanski et al., Blood 2012;120:1908). There is evidence that PIEZO1 may also be responsible for a channel activity that participates in the dehydration of sickle cells which exacerbates sickling and vaso-occlusive events in patients with Sickle Cell Disease (reviewed in Gallagher, Curr Opin Hematol 2013, 20:201).Using a Next-Generation sequencing panel containing 27 hemolytic anemia associated genes, we identified and characterized a novel PIEZO1 mutation p.L2023V which results in delayed channel inactivation and a dehydrated RBC phenotype. This single amino acid substitution at a highly conserved site was detected in the heterozygous state in a 15 year old Caucasian young man (CQ15) with chronic hemolysis well compensated with reticulocytosis, along with heterozygosity for the SLC4A1 p.E40K mutation known as band 3 Montefiore. Hematologic characteristics included macrocytosis, elevated MCHC, and a smear showing occasional stomatocytes. Several family members also had hemolysis and jaundice and were given a diagnosis of hereditary pyropoikilocytosis prior to our evaluation. The L2023V mutation was considered possibly damaging by the PolyPhen-2 algorithm with a score of 0.777. The mother was heterozygous for the PIEZO1 L2023V mutation while the father carried the band 3 Montefiore mutation. Ektacytometry was used to evaluate RBC deformability; the mother’s RBCs had a profile of classic xerocytosis with decreased Omin and Ohyp, the father had a very mild spherocytosis profile, while the patient’s RBCs had mixed ektacytometry characteristics with decreased Ohyp and an intermediate Omin (Figure 1A). Intracellular cation values determined by flame emission spectroscopy for CQ15 and his mother demonstrated K+ loss. The mutation p.L2023V is located at a site predicted to be at the border between membrane and cytoplasm in the carboxy terminal part of the protein, similar to the previously described p.R2456H mutation in a kindred with HX (Zarychanski et al., 2012).To enable detailed physiologic study of the L2023V mutation, we prepared an HEK293 cell line with a stable, single copy integrant of the variant with an inducible promoter. Whole cell patch clamp studies were performed on HEK293 cells expressing wild type PIEZO1 and PIEZO1 L2023V. Traces of mechanically activated currents were recorded from the cells and were normalized to peak current (Figure 1B). The inactivation time constant tau (in ms) was determined from mono-exponential fits for wild type and mutant PIEZO1 channels. The difference in average inactivation time between wild type PIEZO1 and PIEZO1 L2023V was highly significant (Student’s t-test; p<0.0001) and is predicted to lead to cellular dehydration.Patients often present with a complex clinical picture and laboratory results and may have combinations of potentially damaging genetic variants identified by Next-Generation sequencing. The examination of clinical, laboratory, and genetic data from family members and, in some cases, in vitro studies are required to clarify the relative contribution of these variants and to arrive at an accurate diagnosis. The data presented here further our understanding of the role of PIEZO1 in RBCs and its potential pathological contribution in HHAs with associated cellular dehydration and may facilitate the future development of therapeutic targets for treatment of these conditions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Senicapoc, a Gardos Channel Inhibitor Developed to Treat Sickle Cell Disease, Exhibits Antimalarial Activity

▪ BACKGROUNDThe inhibitor of the erythrocyte Gardos channel (KCNN-4, IK-1), senicapoc, was developed for treatment of sickle cell disease. Activation of the Gardos channel constitutes a major potassium leak pathway contributing to cellular dehydration. Administration of senicapoc to patients with sickle cell disease was well tolerated and reduced hemolysis through attenuation of sickle red cell dehydration, but failed to reduce the frequency of vaso-occlusive pain crises (Ataga KI et al. Br J Haematol 2011;153:92-104). Red cell volume regulation is critical both to the pathology of sickle cell disease and to the growth of Plasmodium, the parasites that cause malaria, since Plasmodium depends upon ion flow across the host membrane for growth (Glushakova S et al. Curr Biol 2010; 20:1117-1121). We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits intraerythrocytic growth and development of malarial parasites. METHODSBlood type O+ erythrocytes were infected with P. falciparum strains 3D7, W2mef, and 7G8, and P. knowlesi strains H-1 and YH-1. Parasites were cultured in the presence of senicapoc and in parallel, with drugs with known antimalarial properties (e.g. mefloquine, chloroquine, clotrimazole, and dihydroartemisinin). Parasites were also cultured in the presence of each of eight senicapoc analogs with variable inhibitory potency for the Gardos channel. To define interactions between these drugs, parasites were cultured in fixed-ratio combinations of senicapoc and senicapoc analogs using the fixed-ratio isobologram method. Parasite growth and maturation were determined by microscopy and flow cytometry. The effect of senicapoc in vivo was determined using wild-type C57Bl/6 mice along with IK-1+/+ and IK-1-/- pups on a C57Bl/6 background. Mice were infected with the murine parasite, P. yoelii and treated with senicapoc or vehicle control (methylcellulose 0.5%). RESULTSWe observed that senicapoc blocked the intraerythrocyte growth of P. falciparum 3D7 with an IC50 value of 7 uM and P. knowlesi H1 with an IC50 value of 18 uM. Senicapoc inhibited growth leading to death of intracellular parasites at and beyond the late trophozoite stage, demonstrating minimal effect on ring stage parasites, and an intermediate effect on mature schizonts. Analogs with IC50 values for inhibition of parasite growth similar to senicapoc were potent inhibitors of the Gardos channel. These analogs also shared common elements of the molecular structure. Combined treatment with senicapoc and analogs with potent inhibition of the Gardos channel demonstrated an additive effect on inhibition of parasite growth. Both additive and synergistic interactions were demonstrated when parasites were cultured with senicapoc and analogs without potent inhibition of the Gardos channel. In the murine model, C57Bl/6 mice treated with senicapoc exhibited suppression in parasite growth, confirming the efficacy of senicapoc against Plasmodia in vivo. Parasite growth was suppressed in IK+/+ mice treated with senicapoc, though the effect was not sustained through the period of treatment. Surprisingly, parasite growth was also suppressed in IK-/-mice treated with senicapoc, though the response was delayed. The kinetics of parasite growth differed between mice with and without the Gardos channel. CONCLUSIONSSenicapoc demonstrates antimalarial activity against P. falciparum and P. knowlesiin vitro and in vivo against P. yoelii in mice. Our in vivo studies suggest a mechanism for inhibition of parasite growth in mice that includes both Gardos-dependent and independent components. The excellent safety profile of senicapoc and its long half-life in humans demonstrated in clinical trials suggest its possible utility in antimalarial development either as a lead compound or in combination with other antimalarials. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1

Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR. DisclosuresNo relevant conflicts of interest to declare.

Angiogenic growth factors augment K-Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α.

The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease. Am. J. Hematol. 89:273–281, 2014. © 2013 Wiley Periodicals, Inc.

N-ethylmaleimide activates a Cl−-independent component of K+ flux in mouse erythrocytes

The K-Cl cotransporters (KCCs) of mouse erythrocytes exhibit higher basal activity than those of human erythrocytes, but are similarly activated by cell swelling, by hypertonic urea, and by staurosporine. However, the dramatic stimulation of human erythroid KCCs by N-ethylmaleimide (NEM) is obscured in mouse erythrocytes by a prominent NEM-stimulated K(+) efflux that lacks Cl(-)-dependence. The NEM-sensitivity of Cl(-)-independent K(+) efflux of mouse erythrocytes is lower than that of KCC. The genetically engineered absence of the K-Cl cotransporters KCC3 and KCC1 from mouse erythrocytes does not modify Cl(-)-independent K(+) efflux. Mouse erythrocytes genetically devoid of the Gardos channel KCNN4 show increased NEM-sensitivity of both Cl(-)-independent K(+) efflux and K-Cl cotransport. The increased NEM-sensitivity and stimulation magnitude of Cl(-)-independent K(+) efflux in mouse erythrocytes expressing transgenic hypersickling human hemoglobin SAD (HbSAD) are independent of the presence of KCC3 and KCC1, but absence of KCNN4 reduces the stimulatory effect of HbSAD. NEM-stimulated Cl(-)-independent K(+) efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride. The NEM-stimulated activity is modestly reduced at pH6.0 but not significantly altered at pH8.0, and is abolished at 0°C. Although the molecular identity of this little-studied K(+) efflux pathway of mouse erythrocytes remains unknown, its potential role in the pathophysiology of sickle red cell dehydration will be important for the extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Thrombophilia and Pediatric Stroke

Thrombophilia refers to a systemic predisposition to thrombosis, including stroke. Thrombophilia may be genetic (eg, factor V Leiden) or acquired (eg, antiphospholipid antibodies). Approximately 60% of thrombosis risk is attributable to genetic factors.1 Thrombosis is a disease for which there is a relatively well-defined management strategy in adults but less so in children. Thrombophilia is not a disease but a susceptibility to thrombosis, and its management strategy is less well defined in adults and not at all defined in children. Currently, the role of thrombophilia in pediatric stroke is poorly characterized. Article see p 1838 The extensive published literature on stroke in adult patients cannot be assumed to apply to children because common underlying mechanisms of adult stroke, such as atherosclerosis, hypertension, smoking, atrial fibrillation, and diabetes mellitus, are infrequently involved in pediatric stroke. Known underlying mechanisms of pediatric stroke include perinatal placental thromboembolic events through a physiologically patent foramen ovale, congenital heart disease, sickle cell disease, arterial dissection, trauma, infection, inflammation, immobilization, dehydration, metabolic syndromes, moyamoya, and vasculitis. An increasing number of large pediatric centers now have dedicated multidisciplinary stroke services, which include neurology, hematology, neuroradiology, interventional radiology, neurosurgery, and physical, occupational, and speech therapists. Stroke as a diagnosis should be restricted to patients with cerebral infarction (see Figure, top). Multiple additional conditions with predisposition to stroke or cerebral vasculopathy may also be seen by these multidisciplinary groups, including cerebral sinovenous thrombosis (CSVT) (see Figure, bottom), moyamoya, vasculitis, transient ischemic attacks, complex migraine, and nontraumatic intracranial hemorrhage. Figure. AIS (top) and CSVT (bottom). Top left, Magnetic resonance angiogram. Top right, Axial T2 magnetic resonance image. Bottom left, Sagittal T1 magnetic resonance image. Bottom right, Time-of-flight magnetic resonance venography. Arrows highlight abnormalities. The incidence of pediatric arterial ischemic stroke (AIS) …

In vitro Inhibitory Effects of Disodium Cromoglycate on Ionic Transports Involved in Sickle Cell Dehydration

Background: The antiallergic and antiasthmatic drug disodium cromoglycate (DSCG) has also demonstrated an activity against sickle cell disease, but the mechanism of this action still remains unknown. Methods: Na⁺ and K⁺ fluxes were studied in red cells obtained from 9 patients affected with sickle cell disease in the absence or in the presence of 1 mM of DSCG and deoxygenated under an N₂ flow during up to 24 h. Results: A significant inhibiting effect of DSCG on the intracellular K⁺ exit and the Na⁺ entry was observed. Conclusion: These results demonstrate that DSCG partially inhibits the abnormal K⁺ loss which is implicated in the dehydration of the sickle cell and the stimulation of sickling.

The effects of transport perturbations on the homeostasis of erythrocytes.

The control of erythrocyte volume, pH, membrane potential and ion content results from the interaction of many passive and active transport systems, cytoplasmic buffers, and from the charge and osmotic properties of haemoglobin and other impermeant solutes. The complexity of the system is such that the understanding of cell responses to experimental, physiological and pathophysiological challenges is beyond intuitive grasp. Mathematical models of erythrocyte and reticulocyte homeostasis have delivered a wealth of novel and unexpected predictions that have been confirmed experimentally. Those concerning effects of Ca(2)+ and K+ permeabilization on cell volume, pH and osmolality have helped solve long-standing issues on the pathophysiology of sickle-cell dehydration and will be briefly reviewed here. To study the effects of parasite growth and of new permeation pathways (NPP) on host cell homeostasis, we have developed a model of a Plasmodium falciparum- infected erythrocyte. Modelling NPP to fit reported changes in both Na+/K+ fluxes and gradients predicted large variations in host cell haemoglobin concentration, [Hb]. However, preliminary estimates seem to indicate that host cell [Hb] is conserved throughout the parasite's asexual cycle, suggesting that the properties of the NPP vary in subtle, stage-dependent ways.

Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells.

The Ca2+-sensitive K+ channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca2+ loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age–activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K+ permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age–density distribution pattern during dehydration. However, when Ca2+ loads were used to induce maximal K+ fluxes via Gardos channels in all RBCs (F max), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F max was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age–activity relation revealed a monotonic decline in F max with cell age, with a broad quasi-Gaussian F max distribution among the RBCs.

The Dual Endothelin Receptor Antagonist Bosentan Prevents the Acute Sickle Cell-Related Hypoxic Lung and Kidney Injury in Transgenic SAD Mice.

Vaso-occlusive (VOC) crisis is a major manifestation of sickle cell disease (SCD) leading to acute organ damages. Increased levels of endothelin-1 (ET-1), a potent vasoconstrictor peptide, have been reported in SCD patients. In addition, ETB receptor has been identified in red blood cells (RBCs) membrane and in vitro, ET-1 increases the activity of Gardos channel in sickle RBCs, which is important in generation of dehydrated sickle RBCs. First, we evaluated the effects of the dual-endothelin-receptor (ETR) antagonist bosentan in vitro on mouse RBCs from transgenic SAD mice and in vivo on hypoxic model of acute sickle VOCs. ET-1 (500 nM) significantly increased Gardos channel activity in SAD mouse RBCs, this was prevented by bosentan (10 μM). We next evaluated the in vivo effects of bosentan (100 mg/kg/d by gavage for 14 days) in both control and SAD mice. Under normoxic condition, bosentan did not modify either RBCs density profiles or RBCs K+ content in SAD mice, suggesting that ET-1 may not contribute to sickle cell dehydration in the steady state. The bosentan-treated mice were then exposed to hypoxia (8%, 18 hrs). We first evaluated the cardiac output and renal artery blood flow (RBF) by ultrasound color imaging. In SAD mice only, hypoxia induced a marked reduction in RBF (− 40%; p<0.01, n=10) and in the time-average mean blood velocities (p<0.01) compared to basal values. Such changes were not explained by changes in cardiac output. Infusion of bosentan reversed the increase in renal vascular resistance by 50% (p<0.01) within 5 minutes, suggesting an ET-1-dependent vasoconstriction on the SCD renal vasculature. In a third set of experiments, we evaluated the effect of bosentan on experimental lung and kidney VOC-induced damages. Normal and SAD mice were exposed to 46 hrs hypoxia (8%) followed by 2 hrs recovery under normoxia. Mice were treated with either vehicle or bosentan (100 mg/kg/d, starting one week before hypoxia). We evaluated RBF and cardiac output, complete blood counts, RBCs density profiles and cation content, bronchoalveolar fluid leukocyte counts, kidney and lung histopathology. In SAD mice exposed to hypoxia, bosentan amelioratedRBCs dehydration;the reduction in RBF;congestion of the renal microcirculation and injury;lung injury and pulmonary vessel constriction;renal and pulmonary leukocytic infiltrates.In conclusion, ETR blockade was beneficial by preventing RBC dehydration in vivo with protection from kidney and lung injury. In addition, we provide the first evidence that VOC is due not only to cell entrapment within the microvasculature but also to marked ET-dependent vasoconstriction. These data provide new insights into the possible use of bosentan in the treatment of acute VOCs in SCD.

Ascorbic acid does not prevent sickle cell dehydration induced by calcium-activated pottasium

Ascorbic acid does not prevent sickle cell dehydration induced by calcium-activated pottasium