DECREASED AQUAPORIN 1 (AQP1) EXPRESSION AND LOW SERUM OSMOLALITY IN HEREDITARY SPHEROCYTOSIS (HS)

CRISP, R; SOLARI, L; VITTORI, D; GAMMELA, D; SCHVARTZMAN, G; GARCÍA, E; RAPETTI, C; ALFONSO, G; DONATO, H

Amsterdam

Congreso; 17th Congress of the European Hematology Association; 2012

European Hematology Association

Background: AQP1 is the membrane water channel responsible for fast changes of red cells volume as response to tonicity of medium. The red blood cell membrane is extensively remodeled during erythropoiesis, conditioning the expression of membrane proteins. Throughout the process of enucleation, proteins are distributed between the extruded nuclei and the remaining reticulocyte. As the aberrant distribution of proteins in HS generates deficiencies of proteins other than those codified by the mutated gen, we postulated that AQP1expression might be impaired in HS erythrocytes. Moreover, since AQP1 expression in the mature red cell is modulated by serum osmolality, we also measured serum osmolality of HS patients Aim: To evaluate AQP1 expression and impact of serum osmolality on AQP1 expression in patients with HS Methods: AQP1 expression was evaluated through flow cytometry in 6 normal controls (NC), 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers (SC). Fixed and permeabilized red cells were stained with rabbit anti-AQP1 antibody followed by FITC or Phycoerythrin conjugated anti-rabbit IgG. The number of AQP1 molecules per erythrocyte was cuantified. The effect M) was evaluatedmM and Cl2Cu 400 mof AQP1 inhibitors (Cl2Hg 0.1-40  through water flow-based tests: osmotic fragility (OF) and hypertonic cryohemolysis (CH). Serum osmolality was measured using a pressure vapor osmometer in 20 NC and 13 HS (4 mild, 4 moderate, 2 severe, and 3 splenectomized). HS and SC individuals presented single or combined deficiencies of ankyrin, spectrin, protein 4.1, and protein 4.2 Results: AQP1 expression was decreased, compared to simultaneously performed NC, in all of the HS patients and SC.  The decrease was greater as HS was more severe: significant correlation between AQP1 expression and hemoglobin level was found (rho Spearman: 0.6510; p; 0.0086). Negative correlationship between AQP1 expression and CH was observed (n=16; r2=-0,607; p:0,006). AQP1 inhibitors increased CH but did not modify OF. The CH increase induced by Hg++ showed a direct positive relationship with the level of AQP1 expression (n=4, r2= 0,916). Osmolality was significantly lower in HS patients compared to NC (270.5±1.58 vs. 275.9±1.50, respectively; p: 0.015) Conclusion: Decreased AQP1 expression reveals a deficiency common to all patients with HS, a disorder based on alterations of membrane proteins and cytoskeleton. It is likely that an abnormal AQP1 secretion occurs during the enucleation process. As reticulocytes of HS patients are immersed within plasma with lower osmolality than normal, the loss of AQP1 might be increased during the subsequent reticulocyte maturation process. We suggest that these two mechanisms could help to explain these observations. Results of assays with inhibitors suggest that AQP1 decrease influences processes involving water efflux rather than water influx. No study concerning these findings has been published in the literature up to date