PS1526 EVALUATION OF THE EFFECTS OF ANGIOTENSIN II ON NORMAL AND SICKLE CELLS

Background: Sickle cell anemia is an inherited anemia that is related to a mutation in a beta‐globin gene, causing a substitution of the amino acid glutamic acid for valine at its sixth position of the β chain, leading to the formation of HbS.Hemoglobin S when deoxygenated assumes an elongated and rigid shape, known as a sickle shape. A study by Saraiva et al. (2011) demonstrated the presence of Angiotensin receptors (rAT1, rAT2) on the erythrocyte membrane. However, there are no studies demonstrating the role of Angiotensin II and its receptors on the red blood cell membrane. Aims: The objective is to evaluate the effects of Angiotensin II on healthy erythrocyte and sickle cell erythrocyte. Methods: Two methods were performed to evaluate whether angiotensin II, in different concentrations (10 –12 ,10 –12 ,10 –8 ,10 –6 ), is able to modulate the erythrocyte response: Osmotic fragility test (OFT) and polymerization inhibition test (PDF). To perform OFT, Erythrocytes of normal patients were washed three times with PBS (phosphate‐buffered saline). The leukocyte and platelet layer was removed and the pure erythrocytes were submitted to increasing concentrations of sodium chloride (0.1%–0.9%) to evaluate the percentage of hemolysis. The test was performed in triplicate using 3 μL of blood, 187 μL of saline and 10 μL of Angiotensin II for each tube. The tubes were left in a water bath for one hour at 37 ° C and centrifuged at 5000 rpm for 10 minutes. Reading was performed in a spectrophotometer at 540 nm absorbance (n = 4). To perform PDF, erythrocytes of sickle cell patients were washed three times with PBS. The leukocyte and platelet layer was removed and the pure erythrocytes were subjected to different concentrations of angiotensin, or Hydroxyurea (positive standard, 6.57 mM) and subsequently, 2% sodium metabisulfite was added and the plate was read immediately at 700 nm absorbance (n = 2). To verify differences among test and control concentrations ANOVA and post Tukey analysis were conducted. Results: Angiotensin 10 –12 demonstrated statistically significant results of protection against hemolysis at osmotic stress concentrations (0.4%) as well as in 0.6 and 0.9% NaCl. Angiotensin 10 –10 in addition to showing protection at 0.4%, 0.6% and 0.9% NaCl concentrations, also reduced hemolysis in 0.8% NaCl. The concentration of Ang 10 –8 showed a strong protection against hemolysis in 0.4% NaCl and to a lesser extent in 0.7%, 0.8% and 0.9% NaCl. The highest concentration of angiotensin, 10 –6 , showed greater protection in osmotic stress situations, decreasing the percentage of hemolysis by 0.3% and 0.4% NaCl, as well as 0.7%, 0.8 %, and 0.9%. Through the PDF test, we observed a dose‐dependent and significant effect when comparing the different concentrations of angiotensin II to the control. After the non‐linear regression of the polymerization rate, it was possible to observe that the two higher concentrations of angiotensin respond differently from the two lower concentrations. Concentrations 10 –12 and 10 –10 initiate the polymerization rate linearly and after approximately 20 minutes begin to decline while the 12 –8 and 10 –6 concentrations increase the polymerization rate within the first 10 minutes and tend to remain constant the rest of the time. Summary/Conclusion: In situations of osmotic stress and in situations close to the physiological concentration of NaCl, angiotensin II presented a protective effect, reducing the hemolysis of red blood cells HbAA. There was a decrease in the polymerization of sickle cell erythrocytes when submitted to different concentrations of angiotensin II.