Abstract
Introduction
Urea transporter-B (UT-B), an integral membrane protein, was originally isolated from erythrocyte. UT-B has 10 transmembrane domains and 2 short helices in the soluble regions of the N- andC-termini in erythrocyte plasma membranes and mediates urea permeability at 1.1×10−5 cm/s in human erythrocyte [15] and at 3.3×10−5 cm/s in mouse erythrocyte [14]. UT-B is also permeable to urea analogues, such as formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with Ps > 5.0×10−6 cm/s [28] and water with Pf 2.9×10−3 cm/s [14, 26]. Erythrocyte lacking UT-B protein does not exhibit phloretin-sensitive facilitated urea and water transport [10]. However, the physiological roles of UT-B in erythrocyte are not clear.
The rapid urea transport across the erythrocyte membrane facilitated by UT-B is conventionally considered to ensure structural stability of erythrocyte. The structural features of erythrocyte membrane enable it to undergo large reversible deformations during its life-span of 120 days. With high elasticity, rapid respond to applied fluid stresses and strong structural resistance, the erythrocyte membrane is a dynamic and fluid structure comprised of a lipid bilayer that is studded with skeletal proteins, transmembrane proteins and linker proteins [6]. Some mutations of membrane and skeletal proteins including α- and β-spectrin, band 3, protein 4.2, protein 4.1, glycophorin C and ankyrin-1 have been found in hereditary erythrocyte membrane disorders, such as hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) syndromes [1, 11]. The mutations in membrane protein genes result in numerous symptoms including chronic haemolysis with anaemia, jaundice, splenomegaly, reticulocytosis, and spherocytes on peripheral blood smear, deteriorated hemorheology properties of erythrocyte [17]. Furthermore, impaired hemorheology is also strongly associated with numerous pathogenesis and development of other diseases [27], including cardiovascular disease, hypertension [7], sickle cell anemia [1], sepsis, septic shock [19], renal failure and chronic vascular complications of diabetes mellitus [2, 23].
In this study, we studied hemorheological properties of erythrocyte with UT-B knockout or functional inhibition. It was found that hemorheological parameters in UT-B null erythrocyte were improved compared to those in wild-type erythrocyte. Using UT-B inhibitor PU-14 to “chemically knockout” UT-B also resulted in the improvement in deformability of erythrocyte. Our data indicate that the knockout or functional inhibition of UT-B in erythrocyte may have a beneficial role in improving their hemorheological properties. UT-B might be a potential therapeutic target for treating abnormal erythrocyte in various diseases.
Materials and methods
Animals and blood samples
UT-B knockout mice were generated by targeted gene disruption [24]. All wild-type and knockout mice used in this study were in the C57BL/6J genetic background. All animal procedures in this study were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China Association for Laboratory Animal Science. All animal care protocols were approved by the Animal Care Committee of Peking University Health Science Center. All sacrifices were performed under pentobarbitone anesthesia. The anesthetized mice were kept on a heating block to maintain the body temperature. Every effort was made to minimize animal suffering.
The venous blood samples were collected from wild-type mice (male, 8∼10 weeks old) and UT-B knockout mice (male, 8∼10 weeks old) by orbital puncture.
Reagents
PVP (polyvinyl pyrrolidone)-K30, sucrose and sodium citrate were purchased from Beijing Chemical Reagent Company (China).
Measurements of erythrocyte deformation index (DI), small deformation index (DI) d ,and orientation index (DI) or
Forty μl of heparinized blood was suspended in 1 ml of 15% PVP buffer (w/v, pH 7.4, 290 mOsm/kg, and viscosity 15 mPa·s). The DIs at shear rates of 50 s−1 ∼1000 s−1 were meausred using an ektacytometer (Precil, Beijing, China) and the data were plotted. For (DI)d and (DI)or measurements, 20 μl of heparinized blood was suspended in 1 ml isotonic PBS (pH 7.4, 290 mOsm/kg, and viscosity 0.94 mPa·s). (DI)d and (DI)or at shear rate 100 s−1 were measured with the ektacytometer.
Effect of UT-B inhibitor PU-14 on the deformation index of erythrocyte
The erythrocyte of wild-type and UT-B knockout mice was treated by co-incubation with UT-B inhibitor PU-14 at 22°C for 20 min in vitro. The deformation indices were measured as described in 2.3.
Measurement of erythrocyte osmotic fragility
To observe the influence of UT-B deficiency on the erythrocyte osmotic fragility, the osmotic fragility tests were performed on the erythrocytes from wild-type and UT-B knockout mice. Fifty μl blood was washed three times with PBS and resuspended in hypotonic NaCl solutions with concentrations ranging from 0.7% to 0.35%. The mixtures were incubated at room temperature for 2 h and hemolysis occurred. After taking pictures, the mixtures were then centrifuged and the absorbance of supernatant was measured at 540 nm with a spectrophotometer (UNICO Instruments CO., Shanghai, China).
Measurement of erythrocyte electrophoretic rate
Five μl of blood samples was suspended in 1 ml 0.9% sucrose solution and then drawn into a rectangular pipette. The erythrocyte electrophoresis rate was measured in the cell electrophoresis apparatus (LIANG-100 Shanghai Medical Instrument Co., Shanghai, China).
Measurement of whole blood viscosity (WBV) and hematocrit (Hct)
The whole blood viscosities were measured in an automatic cone-plate viscometer (Precil, Beijing, China). The viscosities from high (200 s−1) to low (3 s−1) shear rates were recorded. The hematocrit (Hct) of the blood samples was measured by drawing the blood into micropipette and centrifuging at 12,000 rpm for 10 min in a 3F-2 microdosis super-speed hydroex-tractor (Beijing, China).
Erythrocyte size and morphology
The size and morphology of erythrocyte were recorded as described before [25].
Statistical analysis
SPSS 11.5 for Windows was used for statistical analysis. Data were presented as mean ±SEM. Multiple comparisons between groups were performed by one-way or two-way analysis of variance (ANOVA). For data presenting a nonnormal distribution, we used the Kruskal-Wallis test and Mann-Whitney U test. Significance was accepted at p < 0.05.
Results
Deformation index (DI), small deformation index (DI) d , and orientation index (DI) or were improved in UT-B null erythrocyte
DI indicates the ability of erythrocyte deformation. The higher the DI, the better the deformability is. Figure 1A shows that DI of UT-B null erythrocyte was significantly higher than that in the wild-type at low shear rate (50∼100 s−1), suggesting a higher erythrocyte deformability in UT-B null erythrocyte. At higher shear rate (>100 s−1), there was no significant difference between two groups. (DI)d and (DI)or were measured simultaneously. UT-B null erythrocyte had higher (DI)d and (DI)or than wild-type erythrocyte (Fig. 1B, C). Since the erythrocyte deformability is determined by its morphology, rigidity of membrane, and the viscosity of intracellular fluid [8, 21], the increased erythrocyte deformability might result from low intracellular viscosity caused by UT-B knockout. The (DI)d is positively correlated with fluidity of erythrocyte membrane. There data suggest that the erythrocyte membrane lipid fluidity increased greatly after UT-B knockout. The increased (DI)or might be the consequence of the improved membrane rigidity of erythrocyte.
UT-B inhibitor improved the deformation of erythrocyte
PU-14 is a novel small-molecule thienoquinolin UT-B specific inhibitor identified in 2013 [13]. We treated wild-type erythrocyte and UT-B null erythrocyte with PU-14 to identify the effect of chemical knockout of UT-B on the deformability of erythrocyte. The results showed that DI in wild-type erythrocyte treated by PU-14 in vitro was significantly higher than that in control ones at low shear rate (50∼100s−1) (P < 0.05) (Fig. 2A). However, DI in UT-B null erythrocyte treated by PU-14 did not change (Fig. 2B). The results indicate that erythrocyte deformability was concerned with the UT-B deletion and the UT-B inhibition lead to improved deformability.
The osmotic fragility was lower in UT-B null erythrocyte
In the osmotic fragility measurement, we unexpectedly found that, at 0.6% NaCl, hemolysis occurred in wild-type erythrocyte but not in UT-B null erythrocyte (Fig. 3A). The data for the absorbance measurements at 540 nm showed that the hemolysis rates of UT-B null erythrocyte were significantly lower than wild-type erythrocyte at 0.6%, 0.5%, and 0.45% NaCl (Fig. 3B). The results indicate that UT-B deletion could assist erythrocyte to resist the extracellular hyposmotic condition and decrease the osmotic fragility. Because osmotic fragility is determined by the membrane skeleton [18], this data suggest that UT-B knockout may cause change in membrane skeleton.
Electrophoretic rate increased in UT-B null erythrocyte
The electrophoretic mobility of UT-B null erythrocytes significantly increased as compared with wide-type ones (p < 0.05) as shown in Fig. 4. The erythrocyte electrophoresis rate is a parameter indicating the surface charge density of erythrocytes. We found that erythrocyte electrophoresis rate of UT-B null erythrocytes increased (Fig. 4). These data suggest that the surface charge density was increased by UT-B knockout. Higher surface charge density decreases the tendency of erythrocyte aggregation formation.
The whole blood viscosity and HCT did not change in UT-B knockout mice
As compared with wild-type mice, the whole blood viscosity of the UT-B knockout mice had a slight reduction at shear rates from 50 to 200 s−1 (p > 0.05) (Fig. 5A), which may result from the better deformability and higher electrophoresis rate in UT-B knockout mice. Additionally, HCT of UT-B knockout mice had no significant change (Fig. 5B).
Discussion
The primary feature of erythrocyte membrane is the composite structure consisting of a lipid bilayer studded with proteins including skeletal proteins (spectrin α- and β-chains, proteins 4.1, or 4.1R, and actin), transmembrane proteins (band 3, glycosylphosphatidylinositol-linked proteins, aquaporin 1 and UT-B) and linker proteins (ankyrin-1 and 4.1R) [6]. As the membrane protein is fundamentally important for the integrity and hemorheological properties of erythrocyte, abnormalities or deficiency of the erythrocyte membrane proteins result in various of clinical syndromes including HS, HE, hereditary ovalocytosis, and hereditary stomatocytosis [5, 11].
Deformability, electrophoretic mobility and osmotic fragility are the main hemorheological properties of erythrocyte. The deformability is believed to be a major determinant for proper blood microcirculation on account of that erythrocyte need to pass through various blood vessels such as the capillaries of the microcirculation, the thick endothelial slits in the red pulp of the spleen and the inner medullary vasa recta, in which erythrocyte deforms extensively to resist external force and to pass through the narrowest blood vessels [23]. Geometry and size of the microvessels, mechanical properties of the cell membrane and its cytoskeleton, and intracellular viscosity are the major determinants of the deformability [3, 20]. In addition, the shear stress is another factor that affects the deformability. The DIs at low shear stresses mainly reflects the mechanical property of cell membrane while DIs at high shear stresses mainly indicate that of whole cell. We noticed that the UT-B null erythrocyte and PU-14 treated erythrocyte showed better deformabilities than UT-B expressing erythrocyte only at low shear stress. This indicates that UT-B knockout or inhibition only affects the mechanical property of erythrocyte membrane, probably by changing the membrane fluidity or the structural stability of membrane skeleton.
Erythrocyte osmotic fragility gives an idea about membrane integrity that represent the erythrocyte tensile strength [22]. Altered erythrocyte fragility is also associated with changes in membrane fluidity and increased oxidative stress. The better osmotic fragility in UT-B null erythrocyte is in accordance with the better erythrocyte deformability at low shear stress. Erythrocyte electrophoretic rate is a parameter indicating the surface charge density of erythrocytes, which is a determinant of erythrocyte aggregation. The increased electrophoretic rate indicates that there would be less erythrocyte aggregates in the blood of UT-B knockout mice, which is beneficial to the microcirculation perfusion.
In this study, we firstly demonstrated the effect of UT-B on the biophysical properties of erythrocyte using models of UT-B genetic and chemical knockout. We found that UT-B knockout and functional inhibition could improve the erythrocyte deformability, membrane rigidity and increase the surface charge density. All data indicate that UT-B functional inhibition may reverse the defected hemorheological properties. In HE and HS treatment, there is no effectively therapeutic way besides splenectomy, which reduces the severity of anemia in patients by increasing the circulatory life span of fragmented erythrocyte [16]. But splenectomy may cause some disorders in cardiovascular and other systems [11]. Our study proposes UT-B as a potential therapeutic target to reverse the deteriorated hemorheological properties in hereditary erythrocyte diseases. UT-B specific inhibitor may be developed as potential drug to correct the impaired hemorheological properties in various diseases, such as HS, HE, cardiovascular disease, hypertension, sickle cell anemia, etc.
Footnotes
Acknowledgments
This work was supported by National Natural Science Foundation of China grants 31200869, 31570938, 81261160507, 81330074 and 81170632, and the 111 Project.