Sickle Erythrocyte Dehydration Research Articles

Ionophore-mediated swelling of erythrocytes as a therapeutic mechanism in sickle cell disease.

Sickle cell disease (SCD) is characterized by sickle hemoglobin (HbS) which polymerizes under deoxygenated conditions to form a stiff, sickled erythrocyte. The dehydration of sickle erythrocytes increases intracellular HbS concentration and the propensity of erythrocyte sickling. Prevention of this mechanism may provide a target for potential SCD therapy investigation. Ionophores such as monensin can increase erythrocyte sodium permeability by facilitating its transmembrane transport, leading to osmotic swelling of the erythrocyte and decreased hemoglobin concentration. In this study, we treated 13 blood samples from patients with SCD with 10 nM of monensin ex vivo. We measured changes in cell volume and hemoglobin concentration in response to monensin treatment, and we perfused treated blood samples through a microfluidic device that permits quantification of blood flow under controlled hypoxia. Monensin treatment led to increases in cell volume and reductions in hemoglobin concentration in most blood samples, though the degree of response varied across samples. Monensin-treated samples also demonstrated reduced blood flow impairment under hypoxic conditions relative to untreated controls. Moreover, there was a significant correlation between the improvement in blood flow and the decrease in hemoglobin concentration. Thus, our results demonstrate that a reduction in intracellular HbS concentration by osmotic swelling improves blood flow under hypoxic conditions. Although the toxicity of monensin will likely prevent it from being a viable clinical treatment, these results suggest that osmotic swelling should be investigated further as a potential mechanism for SCD therapy.

Endothelin-1 Receptor Antagonists Regulate Cell Surface-Associated Protein Disulfide Isomerase In Sickle Cell Disease

AbstractAbstract 265Erythrocyte hydration status and endothelial cell activation have been proposed as important contributors to vaso-occlusion and impaired blood flow in the pathophysiology of sickle cell disease (SCD). However, the physiological mechanism(s) that mediate the interplay between erythrocytes hydration status and the endothelium in SCD are unclear. We have recently reported a role for dual endothelin-1 receptor antagonists in improving sickle erythrocyte hydration status and K+ transport in vivo via modulation of Gardos channel activity (Rivera A., 2008, Amer J Physiol). The Gardos channel is an important contributor to sickle erythrocyte dehydration that maybe modulated by protein disulfide isomerase (PDI). PDI in leukocytes has been reported to catalyze disulfide interchange reactions, mediate redox modifications and has been observed to be up-regulated under hypoxic conditions. We report the detection of PDI by western blot analyses in membranes from both human and mouse sickle erythrocytes. We observed greater levels of cell surface associated PDI in sickle vs Hb A-containing erythrocytes. We also quantified PDI activity and observed a significant correlation between Gardos channel activity and cell surface associated PDI activity in human sickle erythrocytes and Hb A-containing cells (n=40, r2=0.3046, p=0.0002). In fact, closer examination revealed that sickle erythrocyte membranes had higher PDI activity than Hb A-containing erythrocyte membranes (5.07±0.4 vs 1.30±0.1%, n=22 and 18, respectively p<0.0001). Similar results were observed in membrane preparations of erythrocytes isolated from the BERK sickle transgenic mouse model when compared with wild-type controls. Consistent with a functional role for PDI in Gardos channel activation, we also observed that sickle erythrocytes incubated in cycles of oxygenation/de-oxygenation for 3 hr in the presence of PDI antibodies were associated with reduced sickle dense cell formation. Similar results were observed with bacitracin, another PDI inhibitor. We then treated BERK mice with dual ET-1 receptor antagonists (BQ123/BQ788) for 14 days and measured erythrocyte surface associated PDI activity. We observed that as with Gardos channel activity, cell surface associated PDI activity was significantly reduced following treatment with BQ123/BQ788 (8.80±0.5 to 6.4±0.6%, n=3 P<0.02). These changes were associated with an increase in erythrocyte MCV (31.3±1.63 to 40.4±0.35 fL, n=3, p<0.002) and a decrease in MCHC (40.4±0.8 to 27.4±3 g/dL, n=3, p<0.003). We then studied the direct effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of BERK mouse aortic endothelial cells (BMAEC). Using quantitative RT-PCR with Taqman chemistries and GAPDH and beta-actin as endogenous controls, we observed that stimulation of EA cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in BMAEC isolated and cultured for 10 days then incubated with 100 nM ET-1 for 4 hr. Thus, our results strongly implicate cell surface associated PDI in cellular hydration status and its regulation by ET-1. We posit that aberrant regulation of PDI activity and/or its expression and secretion from either erythrocytes or endothelial cells represent a novel target aimed at ameliorating the complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures:No relevant conflicts of interest to declare.

Regulation of Erythrocyte Volume by Cell Surface Associated Protein Disulfide Isomerase.

Acute and chronic clinical manifestations of sickle cell disease (SCD) are based on vaso-occlusion and impaired blood flow. Dense erythrocytes are believed to be important contributors to the vaso-occlusive manifestations of SCD. However, the physiological regulation of erythrocyte hydration status in SCD is not entirely clear. The Gardos channel and K/Cl cotransport are major contributors to sickle erythrocyte dehydration. Protein disulfide isomerase (PDI) on the cell surface catalyzes disulfide formation, causes redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report the detection of PDI in red cells. Western blot analysis revealed a prominent band, migrating at 55 kDa, in ghost preparation from both sickle and normal erythrocytes. To evaluate the role of PDI in Gardos channel activation, we measured charybdotoxin-sensitive K+ influx in the presence of bacitracin, a well-known blocker of PDI activity. When sickle erythrocytes are exposed to bacitracin, Gardos channel activity is significantly reduced (1.4 ±0.2 to 0.8± 0.05 mmol/L cell x min, n=6, P<0.01). We also observed that Gardos channel activity was attenuated by 0.25 mM bacitracin and was maximally inhibited by 3 mM in sickle and normal erythrocytes. Similar results were observed with phenyl arsine oxide and acetyl-thyroxin, two other well-known inhibitors of cell surface PDI activity. We then studied the effects of PDI inhibition on red cell density profiles of sickle and normal human red cells. Analysis of the baseline density profile indicates that erythrocytes have a median density of 1.10 g/mL. This value significantly increased to 1.125 g/mL (n=2) after 3 h of oxygenation/de-oxygenation cycles. However, in the presence of 3 mM bacitracin, the cellular density profile markedly shifted to the left (1.098 g/mL) following deoxygenation/oxygenation. We also investigated the reductive capacity of freshly isolated human erythrocytes by the ferrocyanide-production method. The reductive capacity of normal Hb A red cells was significantly lower than in Hb S containing cells (2.8 ± 1.1 vs 5.1 ± 1.3 mmol/L cell x h, n=18, p<0.0001). Similar results were observed in Santillies and NYKO1, two transgenic mouse models of sickle cell disease, when compared to C57 mice. These results strongly implicate cell surface associated PDI in cellular dehydration and formation of dense sickle erythrocytes by activating K+ efflux via the Gardos channel and suggest that aberrant regulation of PDI activity and/or its expression may contribute to the pathophysiology of Sickle Cell Disease.

Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.kg(-1).day(-1) intraperitoneally for 14 days), an ET-1 receptor B (ET(B)) antagonist, induced a significant decrease in Gardos channel activity (1.7 +/- 0.1 to 1.0 +/- 0.4 mmol.10(13) cell(-1).h(-1), n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D(50); n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET(A)) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET(B)/ET(A) antagonists but not by ET(A) antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET(B) receptor, leading to Gardos channel modulation in SCD.

Sickle Cell Disease: From Membrane Pathophysiology to Novel Therapies for Prevention of Erythrocyte Dehydration

Sickle cell anemia is characterized by the presence of dense dehydrated erythrocytes that have lost most of their K content. Due to the unique dependence of Hb S polymerization on intracellular Hb S concentration, preventing this dehydration should markedly reduce polymerization. The erythrocyte intermediate conductance Ca-activated K channel (hSK4 or KCNN4), first described by Gardos, has been shown to be a major pathway for sickle cell dehydration. Studies with the imidazole antimycotic clotrimazole have shown reduction of sickle cell dehydration in vivo in a small number of patients with sickle cell disease; dose-limiting gastrointestinal and liver toxicities were observed. Based on the chemical structure of clotrimazole metabolites, a novel Gardos channel inhibitor, ICA-17043, has been developed. It has shown substantial activity both in vitro and in vivo in transgenic sickle mice. ICA-17043 is currently in phase 2 human trials. Another potential therapeutic target is the K-Cl cotransport. When sickle erythrocytes are exposed to relatively acidic conditions, they undergo cell shrinkage via activation of this pathway. K-Cl cotransport can be blocked by increasing the abnormally low erythrocyte Mg content of sickle erythrocytes. Oral Mg supplementation has been shown to reduce sickle cell dehydration in vivo in transgenic sickle mice and in patients in two separate clinical trials. Oral Mg pidolate is being tested in clinical trials in homozygous sickle cell disease and in Hb S/HbC (SC) disease, either as a single agent or in combination with hydroxyurea. The ongoing trials will determine the clinical effectiveness of therapies aimed at preventing sickle erythrocyte dehydration.

Therapeutic Strategies for Prevention of Sickle Cell Dehydration

ABSTRACTThe intracellular concentration of Hb S is an important determinant of the kinetic of polymer formation and cell sickling. A variable fraction of dense, dehydrated erythrocytes with high Hb S concentration is seen in the blood of patients with sickle cell disease; these dense cells play an important role in the pathophysiology of the vasoocclusive events of sickle cell disease, due to their higher tendency to polymerize and sickle. Sickle cell dehydration is due to loss of K+, Cl−, and water: the two major determinant pathways of dehydration of sickle erythrocytes are the Ca2+-activated K+ channel (IK1 or Gardos channel) and the K–Cl cotransport (KCC). Specific inhibitors of these pathways being tested in patients with sickle cell disease are Mg2+ pidolate, which inhibits KCC by increasing the sickle cell content of Mg2+, and clotrimazole and derivatives of clotrimazole metabolites, which specifically block the Gardos channel. An inhibitor of Cl− conductance has been shown to reduce dehydration in a transgenic mouse model of sickle cell disease but has not been tested in humans. If clinical efficacy and benefit are demonstrated, an inhibitor of cell dehydration could be used in patients as a single agent or in combination with existing therapies, such as hydroxyurea.

Novel therapies for prevention of erythrocyte dehydration in sickle cell anemia.

Sickle cell anemia is a genetic disorder characterized by mutant hemoglobin (Hb) polymerization and resultant cell deformation (sickling) under conditions of reduced oxygen tension. The disease is caused by mutation of wild-type Glu to Val in position 6 of the beta-chain of hemoglobin, yielding hemoglobin S (HbS). The sickling process is markedly accelerated when the intracellular concentration of HbS is increased. A variable fraction of dehydrated erythrocytes is seen in the majority of patients, and these cells are believed to play an important role in the pathophysiology of the vasoocclusive events of sickle cell disease. Therapy of sickle cell disease is extremely limited in range and efficacy. Many patients still receive treatment only for symptomatic relief of sickle crises, painful episodes due to vasoocclusion by sickled cells. The last 15 years, however, have seen the identification of the principal transport pathways that mediate sickle erythrocyte dehydration, and the last 6 years have witnessed promising clinical tests of specific inhibitors of these pathways, with the intent of reducing cell sickling via inhibition of red cell dehydration. This review discusses the pathophysiology of sickle cell dehydration and explores current and future treatment options for in vivo prevention of sickle cell dehydration.

N‐acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1‐chloro‐2,4‐dinitrobenzene

Clotrimazole, a specific inhibitor of the Ca2+ activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K+ can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K+ efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity. Am. J. Hematol. 62:19–24, 1999. © 1999 Wiley-Liss, Inc.

N-acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1-chloro-2,4-dinitrobenzene

Clotrimazole, a specific inhibitor of the Ca(2+) activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K(+) can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K(+) efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity.

Potential therapeutic approaches for the treatment of vaso-occlusion in sickle cell disease.

Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The pathogenesis of vaso-occlusion likely involves many complex steps related to both the primary event of deoxyhemoglobin S polymerization and the many resultant pathologic changes in both the sickle erythrocyte and the vascular endothelium. Several therapeutic approaches have been designed to reduce the extent of deoxyhemoglobin S polymerization. Although direct chemical inhibition of deoxyhemoglobin S polymerization has proven difficult, strategies to increase levels of fetal hemoglobin or reverse sickle erythrocyte dehydration have been more successful. Although gene therapy is actively being pursued, the ability to cure sickle cell disease is currently limited to bone marrow transplantation with its attendant toxicities and limitations. Because the pathophysiology of vaso-occlusion in sickle cell disease is complex, its treatment will likely be optimized using a multifactorial therapeutic approach.

Erythrocyte membrane transport physiology.

Several transport pathways are involved in the regulation of cell volume and ion content in the human erythrocyte. Studies of these pathways have shown that K-Cl contransport and the Ca-gated K channel (Gardos channel) play an important role in the dehydration of sickle erythrocytes. Therapeutic strategies based on the specific blockade of these pathways have been tested in transgenic sickle mice and in patients with sickle cell disease, using oral Mg pidolate to inhibit K-Cl cotransport and oral clotrimazole to inhibit the Gardos channel. Studies on the erythrocyte Na-H (Na-Li) exchanger have uncovered an important role for insulin and blood pressure in mediating the activity of this pathway. These findings explain the reports of abnormal Na-Li exchange in diabetic nephropathy and essential hypertension. Targeted mutagenesis of the anion exchanger protein band 3 in mice has demonstrated that absence of this protein is compatible with life and leads to severe hemolytic anemia, normal assembly of the cytoskeleton, and reduced mechanical stability of the erythrocyte. Identification of the molecular identity of all the major erythrocyte transporters is imminent and will provide the bases for future studies of these proteins in the normal and abnormal erythrocyte.

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.