Transporting down the road to dehydration

In this issue of Blood, Brown and colleagues provide strong evidence that erythrocyte hydration is an important modifier of disease severity in sickle cell disease (SCD).¹ 

Erythrocyte dehydration is a critical component of disease pathogenesis in SCD because sickle hemoglobin polymerization is exquisitely dependent on its cellular concentration.²  Complex interactions between the activity of several water and solute transport systems lead to cellular dehydration, forming dehydrated sickle erythrocytes, known as dense cells. Dense cells are much more prone to hemoglobin S polymerization, sickling, and vaso-occlusion. They also exhibit decreased deformability, increased fragility, and increased adhesion to endothelial cells, leukocytes, and sickle erythrocytes, exacerbating endothelial damage and facilitating vaso-occlusion.

Erythrocyte dehydration is a critical step in the SCD hemolytic pathway, with numbers of circulating dense cells positively correlated with severity of hemolysis in SCD patients.³  Hemolysis and the hemolytic rate contribute to significant complications of SCD including pulmonary hypertension, stroke, priapism, and leg ulcers.

Three pathways mediating cation loss and cellular dehydration in sickle erythrocytes have been identified: (1) a sickling-induced, deoxy-dependent nonselective pathway called P^sickle that mediates an increase in permeability to calcium and other ions at the initiation of the dehydration cascade; (2) a calcium-activated potassium channel, the Gardos channel,⁴  which when activated is the primary mediator of potassium and water loss in SCD; and (3) a K-Cl cotransport (KCC) pathway.

KCC cotransport is the primary volume-sensitive cation transport pathway in erythrocytes.⁵  KCC activation leads to loss of potassium, chloride, and water, reducing cell volume and increasing cellular hemoglobin concentration. KCC activity mediates volume reduction during reticulocyte maturation, decreasing to negligible levels in mature erythrocytes. At steady state, erythrocyte KCCs are phosphorylated. Activation under hypotonic conditions is mediated by dephosphorylation of a pair of highly phosphorylated threonines in KCC3 associated with increased transport activity.⁶ 

The contribution of KCC to erythrocyte dehydration in SCD is mediated via complex and variable factors. Although numerous in vitro activators of KCC activity have been described, including cell swelling, acid pH, urea, and decreased cellular magnesium, the specific relative contributions of activators in vivo are less clear. The set point for KCC-mediated volume regulation is abnormally elevated in sickle erythrocytes, resulting in decreased cellular volume and increased hemoglobin concentration. This dysregulation is complex, varying quantitatively and temporally in SCD patients. Its causes are unknown, with numerous contributing etiologies postulated including abnormal redox state, erythrocyte magnesium depletion, and combinations of environmental and genetic factors.

KCC activity is mediated by members of the cation-chloride cotransporter (SLC12) family. In human erythrocytes, 3 KCCs have been observed: KCC1, KCC3, and KCC4.⁷  The specific contributions of each of these transporters to KCC activity in erythrocytes are unknown. They may interact, regulating cotransport activity, as shown in vitro with full-length and truncated KCCs. Differences in expression of KCC proteins and their isoforms between wild-type and sickle erythrocytes could lead to altered KCC activity. Genetic variation could contribute to altered KCC structure, activity, and/or isoform composition.

In murine erythrocytes, knockout of Kcc1 has minimal effect on Kcc activity or cell volume. Knockout of Kcc3 leads to reduced Kcc activity in spite of increased Kcc1 expression. Knockout of both Kcc1 and Kcc3 abolishes Kcc activity,⁸  which when bred onto the sickle Antilles model, moderates the erythrocyte dehydration phenotype. The report in this issue by Brown and colleagues provides strong evidence for altered KCC as an important modifier of disease severity in SCD. In their study, N-ethyl-N-nitrosourea mutagenesis screening identified mice with microcytic, hypochromic erythrocytes with increased density due to a missense mutation in Kcc1. This mutation was associated with increased Kcc activity linked to altered phosphorylation of 2 nearby threonines, homologous to the threonines in KCC3 associated with hypotonic responses in human erythrocytes. When bred onto a humanized sickle cell background, heterozygous mutant Kcc1 sickle mice exhibited worse anemia, erythrocytes had increased density and increased Kcc activity, and worse pulmonary, hepatic, and renal pathology. Homozygous Kcc1 sickle mice rarely survived beyond the neonatal period.

SCD is a prototypic complex genetic disease, a common disorder with wide variation in phenotypic severity and numerous clinical manifestations.⁹  Phenotypic variability has been attributed to modifier genes, environmental factors, and interactions of the two. Identification of modifier genes that worsen or ameliorate SCD severity could facilitate prevention, diagnosis, and treatment of SCD complications.

Genome-wide association studies have demonstrated that a significant component of erythrocyte hydration is genetically determined. In addition, in many complex diseases, common variants explain only a small fraction of genetic risk, as expected from the effects of natural selection. Instead, rare, independent mutations frequently contribute to phenotypic variation. Thus, identification of common and rare hydration-associated genetic variants is predicted to provide information on the pathways regulating erythrocyte volume homeostasis.

The report by Brown et al indicates that variants in KCCs influence erythrocyte hydration and alter clinical manifestations in SCD in vivo. It becomes apparent that all of the proteins involved in maintaining erythrocyte volume homeostasis, from the transporters and channels to their regulatory kinases and phosphatases, are candidate modifiers in SCD, as well as in other disorders with primary or secondary alterations in erythrocyte hydration such as the xerocytosis syndromes, β-thalassemia, and hereditary spherocytosis.

Because hemoglobin S polymerization is a concentration-dependent process, a number of therapeutic strategies aimed at reducing polymerization by modifying the intracellular concentration of hemoglobin S have been attempted.¹⁰  Results of these approaches have been disappointing, complicated by incomplete or variable responses or impractical treatment regimens. To bring these strategies to fruition, a better understanding of erythrocyte volume homeostasis is needed. This could allow identification of novel therapeutic agents to prevent or treat hydration-related complications in SCD and other disorders and, ultimately, to identify a pharmacogenomic signature to predict which patients will or will not benefit from these treatment strategies.