Hemodialysis alters tyrosine phosphorylation in band 3 protein of erythrocytes

Band 3 is an integral membrane glycoprotein of the red blood cell. The N-terminal segment (cdb3) of Band 3 protrudes into the cytosol and anchors the membrane to the cytoskeleton. Changes in cell membrane stability, e.g. caused by high environmental osmolality, are associated with phosphorylation of tyrosine (Tyr) residues in the cdb3 domain. Tyr phosphorylation in Band 3 is controlled by cytoplasmic Tyr-kinases and Tyr-phosphatases, and is affected by the redox balance of the cell. Since during hemodialysis erythrocytes may undergo oxidative stress, we studied Tyr phosphorylation of cdb3 in patients on dialysis (HD). 5 patients on standard HD with cellulosic membrane were studied. Blood was collected from HD patients anf healthy controls and filtered on cellulose. Erythrocytes (10%) were incubated in isotonic PBS, then in hypertonic PBS (600 mosm/kg) to stimulate Tyr phosphorylation, and finally reincubated in isotonic PBS. The phosphotyrosine content of Band 3 was assayed after SDS/PAGE through immunoblotting with monoclonal anti-phosphotyrosine antibody. Erythrocytes of HD patients displayed a modest constitutive cdb3 Tyr phosphorylation; hypertonic treatment raised similarly Tyr phosphorylation in red blood cells of HD and control. However, after the restoration of isotonic medium Tyr phosphorylaiton was no longer detectable in erythrocytes of control, while it persisted in those of HD patients. These results indicate an imbalance in Tyr-kinase and Tyr-phosphatase activity in erythrocytes of HD patients, resulting in a potential alteration of Band 3 phosphorylation. This alteration may impair the deformability of erythrocytes and shorten their life span.