Disorders of Erythrocyte Volume Homeostasis

Abstract

Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneity characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis.

Erythrocyte Volume Homeostasis

Maintenance of cellular volume homeostasis is essential for survival of the erythrocyte (reviewed in ^1-4).Perturbation of this homeostasis, a feature of several inherited anemias, leads to abnormal erythrocytes. Complex interactions between solutes, their net charge, conductance of the membrane to monovalent anions and cations, membrane surface area, and ion transport pathways determine erythrocyte hydration status, including ion content and hemoglobin concentration.⁵ Several pathways mediate water and solute homeostasis in normal red cells, where cellular volume is primarily controlled via regulation of monovalent cation content.^{1; 3; 4; 6; 7} The sodium potassium ATPase pump (Na⁺K⁺ATPase) maintains the intracellular low sodium, high potassium composition of the erythrocyte by actively transporting sodium out of and potassium into the cell. Several passive cation leak pathways allow outward potassium and inward sodium gradients. Cation leak pathways have limited capacity to respond to alterations in monovalent cation leak, and, when the cation leak exceeds this capacity, cellular volume changes in parallel with the change in total intracellular cation content. Erythrocytes swell when the inward sodium leak exceeds the potassium leak out and shrink when the potassium leak out exceeds the inward sodium leak. Alterations in membrane permeability are detected by the analysis of intracellular sodium, potassium, total cation content, and altered indices of erythrocyte hydration, e.g. increased or decreased mean corpuscular hemoglobin concentration (MCHC).^{4; 8}

Disorders of Erythrocyte Volume Homeostasis

Disorders of erythrocyte volume homeostasis are a heterogeneous group of inherited disorders.⁹ These disorders are classified by the degree of abnormal membrane permeability to sodium and potassium, resulting in concomitant alteration of intracellular water. The phenotypes observed range from dehydrated to overhydrated erythrocytes. Depending on the degree of perturbation of water and solute homeostasis and its effect on the erythrocyte, hemolytic anemia of varying severity may occur (Table 1). Another classification scheme utilizes the temperature dependence of the cation leak after inhibition of the Na⁺K⁺ATPase and Na⁺K⁺2Cl⁻ co-transporters.⁸ Historically, these disorders were called the stomatocytosis syndromes due to the variable numbers of stomatocytes sometimes observed on peripheral blood smear. However, stomatocytosis is rare in some of these disorders and it is not a sine qua non for diagnosis. With these previous observations in mind, it is not surprising that genetic bases of the erythrocyte volume homeostasis disorders are proving to be heterogeneous, as they parallel the marked variability in clinical, laboratory and physiologic manifestations observed from patient to patient.¹⁰

Table 1.

Feature of Disorders of Erythrocyte Volume Homeostasis

	Stomatocytosis (Hydrocytosis)		Intermediate Syndromes
	Severe Hemolysis	Mild Hemolysis	Cryohydrocytosis	Stomatocytic Xerocytosis	Xerocytosis with High PC	Familial Pseudohyper- kalemia	Xerocytosis
Hemolysis	Severe	Mild to moderate	Moderate	Mild	Moderate	None	Moderate
Anemia	Severe	Mild to moderate	Mild to moderate	None	Mild	None	Moderate
Blood smear	Stomatocytes	Stomatocytes	Stomatocytes or nl	Stomatocytes	Targets	Rare target or stomatocyte	Targets, echinocytes
MCV (80–100 μ3)a	110–150	95–130	90–105	91–98	84–92	82-104	100–110
MCHC (32– 36g/dl)	24–30	26–29	34–40	33–39	34–38	33-39	34–38
Unincubated osmotic fragility	Very increased	Increased	Normal	Decreased	Very decreased	Slightly decreased	Very decreased
RBC Na+ (5– 12 mEq/LRBC)	60–100	30–60	40–50	10–20	10–15	10-25	10–20
RBC K+ (90– 103 mEq/LRBC)	20–55	40–85	55–65	75–85	75–90	75-100	60–80
RBC Na+ +K+ (95–110 mEq/LRBC)	110–140	115–145	100–105	87–103	93–99	87-109	75–90
Phosphatidylc holine content	Normal	±Increased	Normal	Normal	Increased	Normal	Normal
Cold autohemolysis	No	No	Yes	No	No	No	?
Effect of splenectomy	Good	Good	Fair	?	?	?	?Poor
Genetics	AD, ?AR	AD	AD	AD	AD	AD	AD

^aValues in parentheses are the normal range.

^bSplenectomy is contraindicated in these disorders.

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; LRBC, liter of red blood cells; PC, phosphatidylcholine.

Source: Modified with permission from Walensky LD, Narla M, Lux SE. Disorders of the red blood cell membrane. In: Handin RI, Lux SE, Stossel TO, eds. Blood: principles and practice of hematology. 2nd ed. Philadelphia: Lippincott Williams & Wilkins, 2003:1818.

Disorders of erythrocyte volume homeostasis have been classified as primary, due to inherent disorders of volume regulation, and secondary, due to other disorders affecting the erythrocyte that also influence cell volume.¹¹ Primary disorders with erythrocyte dehydration are the hereditary xerocytosis syndromes, while secondary erythrocyte dehydration is associated with spherocytosis, thalassemia, sickle cell disease, hemoglobin C disease, Southeast Asian ovalocytosis, and malaria invasion. In these disorders, the secondary erythrocyte dehydration is frequently a factor in disease pathobiology. Primary disorders with erythrocyte over hydration are the hydrocytosis or stomatocytosis syndromes. Alterations in the MCHC may be seen in both primary and secondary disorders of erythrocyte volume homeostasis, e.g. the MCHC is elevated in both hereditary xerocytosis (primary) and hereditary xerocytosis (secondary).

Hereditary Xerocytosis (HX)

The hereditary xerocytosis syndromes are the most common disorder of erythrocyte volume homeostasis and they are the most clinically heterogeneous. Anemia is variable and some patients do not come to medical attention until late in life. The HX syndromes are also pleiotropic. Beyond the hematological findings, some patients suffer from transient perinatal edema and nonimmune hydrops fetalis that spontaneously resolves. Some HX patients suffer from pseudohyperkalemia. Iron overload may be a significant finding in adult HX patients, even to the degree of needing chelation.^12-14 The basis for the iron overload in HX is unknown.

HX patients typically exhibit mild to moderate, well compensated hemolytic anemia.¹⁵ The MCHC is increased and the erythrocyte osmotic fragility is decreased, both reflecting cellular dehydration. HX erythrocytes are macrocytic, attributed in part to an artifact of cellular stiffness. In electronic cell counters, the conversion of pulse height to cellular volume is dependent on cell shape. Xerocytes do not deform to the same degree as normal erythrocytes, causing the electronically measured MCV to be estimated ~10% too high. Reticulocytosis may also contribute to the elevated MCV. Erythrocyte morphology on peripheral blood smear is relatively normal, with a few targets, dessicytes-cells with their hemoglobin puddled to the side, and rare stomatocytes seen (Figure 1).¹⁶ Osmotic gradient ektacytometry shows a leftward shift of the minimum in the deformability index (O_min) at low osmolarities, as well as decrease in DI_max, whereas in hereditary spherocytosis, the O_min is shifted to the right and the DI_max is normal.¹⁷

Figure 1.

Peripheral blood smears from patients with abnormalities of erythrocyte volume homeostasis. (A) A Wright-stained peripheral blood smear from a patient with hereditary hydrocytosis is shown. Numerous stomatocytes (arrow), erythrocytes with a central mouth-like ‘stoma’ are seen. (B) A Wright-stained peripheral blood smear from a patient with hereditary xerocytosis due to a PIEZO1 mutation showing rare stomatocytes, occasional dessicytes–dense, abnormal erythrocyte forms where hemoglobin appears puddled at the periphery, and rare target cells (arrow).

Dominantly inherited missense mutations in PIEZO1 have been identified in HX patients, primarily located in the highly conserved COOH-terminus of the protein.^18-20 Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells.²¹ Functional studies of HX-associated PIEZO1 mutations demonstrate a partial gain-of-function phenotype with many mutants demonstrating delayed inactivation, ^{19; 22; 23} suggesting increased cation permeability leads to HX erythrocyte dehydration. Because the channel may homo-tetramerize, this delayed inactivation may be due to a dominant negative effect. In some PIEZO1 HX-associated variants, the mechanism of cellular dehydration is unknown. In other HX patients, no mutations are found in PIEZO1, indicating that there are other genetic loci associated with HX.

Overhydrated Stomatocytosis (OHSt, Hydrocytosis)

Overhydrated stomatocytosis or hydrocytosis refers to a very rare, heterogeneous group of disorders associated with moderate to severe hemolytic anemia and marked sodium permeability of the red cell membrane.²⁴ A significant increase in sodium permeability, anywhere from 10 to 40 times normal, leads to increased intracellular sodium and a lesser decrease in intracellular potassium, resulting in increased total monovalent cation content and increased intracellular water. There is frequently a marked compensatory increase in erythrocyte Na⁺ K⁺ ATPase activity; this increased activity of the pump is unable to fully compensate for the significantly increased inward sodium leak.

Hydrocytosis patients typically exhibit moderate to severe, partially compensated hemolytic anemia, while a few patients experiencing a less severe hematologic picture. Excessive intracellular cations elevate cell water, leading to large, macrocytic erythrocytes (110 to 150 fL) that are osmotically fragile with a low MCHC (24 to 30g/dl). Peripheral blood smear demonstrates prominent stomatocytes, erythrocytes with a mouth-shaped (stoma) area of central pallor (Figure 1). In most patients, hemolytic anemia is improved after splenectomy. However, splenectomy is contraindicated in most cases due to the high risk of postsplenectomy thromboembolic disease and chronic pulmonary hypertension. This has been attributed to increased erythrocyte-endothelial cell adhesion resulting from increased phosphatidylserine exposure of overhydrated erythrocytes. Fortunately, most patients have at least partially compensated hemolysis and splenectomy is not required.

Cryohydrocytosis

Cryohydrocytosis is a hydrocytosis variant characterized by overhydrated erythrocytes that exhibit abnormal cation transport at lower temperatures.²⁴ Cryohydrocytosis erythrocytes exhibit a minimal to modest cation leak at 37°C, but exhibit a marked cation leak due to altered cation permeability at 5°C.

Band 3 (Anion exchanger 1, AE1, SLC4A1)

Heterozygous missense mutations in band 3, the anion exchanger (AE1, SLC4A1), associated with increased erythrocyte cation permeability have been found in patients with variants of stomatocytosis and spherocytosis.^25-29 With a few exceptions, these mutations are located in the region between membrane spanning domain 8 and the final two membrane spanning domains, the region involved in anion translocation. In vitro, mutations in this region inactivate band 3-mediated anion exchange.²⁴ While still expressed at the membrane, these mutations may induce the mutant band 3 protein to produce an abnormal cation leak, a hypothesis supported by studies performed in Xenopus oocytes.^{25; 30} Another hypothesis is that mutant band 3 mediates a cation leak by activating other endogenous cation leak pathways.^{27; 31} Erythrocytes from patients with southeast Asian ovalocytosis, a disorder associated with an in-frame 9 amino acid deletion in band 3, is indistinguishable from that observed in cryohydrocytosis.³²

Abnormalities in the Rh-associated glycoprotein (RhAG) have been identified in a subset of hydrocytosis patients.³³ Erythrocytes from these patients exhibit a significant cation leak at physiologic temperatures. Erythrocyte membranes have a decrease or absence of the protein stomatin. In affected patients, heterozygous missense mutations, either Ile61Arg or Ser65Phe, located in the second predicted transmembrane domain of the RhAG protein, have been found. RhAG is a member of the band 3 protein macromolecular complex, which contains RHCE, RHD, CD47, LW, glycophorin B, and other proteins. In this complex, RhAG may function as an ammonium transporter or a gas channel. It has been suggested that the hydrocytosis-associated RhAG mutations widen the transport pore, thereby permitting cations to flow through.²⁴

GLUT1 (Glucose transporter 1, SLC2A1)

In a few patients, abnormalities of erythrocyte volume homeostasis have been associated with abnormalities of GLUT1, the glucose transporter. GLUT1 facilitates transmembrane glucose transport in mammalian cells.^34-36 Patients with GLUT1 deficiency syndromes exhibit a variety of manifestations with typical patients exhibiting severe seizures, complex movement disorders, motor and mental developmental delay, microcephaly, and relative hypoglycorrhachia.³⁷ Treatment with a ketogenic diet has been shown to improve seizures and ameliorate movement abnormalities.

Erythrocyte abnormalities were first reported in a patient with paroxysmal exertion-induced dyskinesia, a mild variant of GLUT1 deficiency.³⁶ This patient’s erythrocytes exhibited ecchinocytic morphology and a cation leak. Subsequently, two patients with a rare variant of OHSt associated with GLUT1 mutations were described.³⁵ Erythrocytes from these patients exhibited a significant low temperature cation leak, similar to cryohydrocytosis, and stomatin deficiency, similar to OHSt. Beyond hemolytic anemia, affected patients suffered from cataracts, seizures, developmental delay, and a movement disorder. Previous studies had shown that stomatin interacts with and influences GLUT1 activity, switching its preferred substrate from glucose to dehydroascorbic acid, an oxidized form of vitamin C.³⁸ This led to the hypothesis that by reducing membrane stomatin, cryohydrocytosis erythrocytes can maximize their glucose uptake to meet the increased demands of the metabolically stressed cell.³³ A fourth kindred with similar symptomatology due to an in-frame amino acid deletion in GLUT1 has been described.³⁴

Intermediate Syndromes

Hereditary xerocytosis and hereditary hydrocytosis represent the extremes of a spectrum of red cell permeability defects.⁹ Several kindreds with features of both conditions have been reported.³⁹ Some patients with severe permeability defects exhibit little or no hemolysis. Others have increased of phosphatidylcholine in their erythrocyte membranes.

Familial pseudohyperkalemia (FP) is a dominantly inherited disorder associated with in vitro loss of erythrocyte potassium into the plasma after cooling of samples to <37°C.^{12; 40} Patients with this temperature-dependent defect in erythrocyte permeability, primarily associated with mutations in ABCB6, do not suffer from hematologic abnormalities.⁴¹ Pseudohyperkalemia has been observed in PIEZO1-associated HX and GLUT1-associated erythrocyte abnormalities.

Advances in cell biology, physiology, and genetics are contributing to our knowledge of normal and abnormal erythrocyte volume homeostasis. Understanding the hydration pathways altered in HX and OHSt will shed light on the perturbations of these pathways in sickle cell disease, hereditary spherocytosis, and other inherited hemolytic disorders where erythrocyte dehydration is a critical contributor to the disease pathobiology.⁵ Translation of these advances into therapeutic targets, patient-specific interventions, and other tools to prevent, ameliorate, or cure the clinical consequences of perturbation of erythrocyte hydration in patients with inherited hematologic diseases.