Protein-bound Indoxyl Sulfate Destroys Erythrocyte

BackgroundRenal anemia is caused by chronic renal failure(CRF).The mechanisms of renal anemia are very complex,include erythropoietin(EPO) reduction,uremic toxins(UTox) cause bone marrow to produce red blood cells disorders and excessive destruction,loss blood, inadequate intake of nutrients,indigestion and reduce the absorption caused by gastrointestinal symptoms,bone marrow lesions which are caused by secondary hyperparathyroidism.Renal anemia is one of the serious complications of renal failure in patients, which lead to ischemia and hypoxia of each system.Anemia increase cardiac stress,induce angina,severe anemia can lead to decrese myocardial contractility and ejection fraction,even congestive heart failure.The latter will promote deterioration of renal function and increase anemia.Renal failure,anemia and heart failure create a vicious cycle of three states.It seriously affects the life and prognosis of patients.Therefore,it is particularly important to improve the quality of life of hemodialysis patients,reduce complications and improve survival rates by effectively correct anemia.Lang proposed the concept of eryptosis in 2005,many factors could destroy erythrocytes, induced erythrocytes suicide or eryptosis,which was similar to apoptosis of nucleated cells.Erythrocytes did not have cell nucleus,important organelles and the mitochondria in the machinery executing apoptosis,therefore,erythrocytes lacked some typical performances of apoptosis include mitochondrial depolarization and condensation of nuclei.However,there were other characteristics of apoptosis(such as:cell shrinkage,size smaller,cell membrane vesicles),it reflected phosphatidylserine surface level at the cell surface.Previous studies were associated with CRF patients,there were some toxic substances with different molecular weights in plasma that could inhibit bone marrow to produce erythrocytes,damaged erythrocytes and caused anemia.Normal erythrocytes showed an increase in exposed phosphatidylserine surface level when resuspended in uremicplasma,whereas life span of erythrocytes was normal when resuspended in normal plasma.These findings confirmed that uremic toxins in CRF patients accounted for a very important role in the pathogenesis of renal anemia.Certain factors in plasma were associated with erythrocytes surface exposed PS in patients of CRF.Mario B’ research indicated that no difference was found in annexin-positive cells between undialyzed patients with CRF and hemodialysis patients.It suggested that these toxins could not be removed by dialysis.Further studies showed that these factors might not be heat-resistant macromolecule or protein,however,it might also be a low molecular weight substance that dued to high protein binding.Although various blood purification technology alleviated direct threat to life from a variety of lethal factor,such as uremic toxins, imbalances of water and acid-base,but could not significantly improve anemia.The reason might be that the current conventional dialysis do not effectively remove protein-bound toxins.Indoxyl sulphate(IS) is representative of highly protein-bound uremic toxins,it’coefficient is more than 90% in plasma,which is difficult to remove by general dialysis.Serum indoxyl sulphate concentration of Chronic kidney disease(CKD) 3-5 patients is 50-90 times as normal.Indoxyl sulfate of protein-bound uremic toxins recently get a lot of attention,whether is correlated with renal anemia in patients of CRF and its complications.ObjectiveWhether protein-bound uremic toxins indoxyl sulphate contribute to the renal anemia in CRF patients.MethodWe used the erythrocytes from the non-smoking healthy adult volunteers,indoxyl sulfate bound to albumin with 4% concentration.To investigate the effects of 4% protein-bound indoxyl sulfate on erythrocytes.After treating for 48 h,the erythrocytes with 0.4% hematocrit were harvested for these experiments.1. The PS and FSC on erythrocytes were determined by flow cytometry.2. The hemolysis from hemoglobin release were determined by enzyme-mark analyzer.3. The erythrocyte cytosolic Ca2+-activity were detected by flow cytometry.Results1. The erythrocytes exposure to protein-bound indoxyl sulfate(62.5,125, 250,500,1000μmol/L) for 48 hours,FSC of erythrocytes respectively were 1517.33±15.63 、1489.66±8.02、1361.00±13.75、877.33±23.02、742.67±14.04, compared with 4% albumin group,the volume of erythrocytes decreased with increasing the concentration of indoxyl sulphate,and dose-effect relationship(P <0.05).when absence of Ca2+ in supernatant,this effect on erythrocytes with increasing concentration of indoxyl sulphate became weak.2. The erythrocytes exposure to protein-bound indoxyl sulfate(62.5,125, 250,500, 1000μmol/L) for 48 hours,the PS exposure rate of erythrocytes respectively were 1.51±0.27%、1.73±0.23%、2.99±0.51%、4.78±0.36%、10.32±1.64%,compared with 4% albumin group, the PS exposure of erythrocytes increased with increasing the concentration of indoxyl sulphate, and dose-effect relationship(P <0.05).when absence of Ca2+ in supernatant,this effect on erythrocytes with increasing concentration of indoxyl sulphate became weak.3. The erythrocytes exposure to protein-bound indoxyl sulfate(62.5,125,250,500, 1000μmol/L) for 48 hours,the hemolysis ratio of erythrocytes respectively were 1.28±0.14%、2.55±0.29%、3.50±0.11%、4.70±0.23%、6.77±0.28%,compared with 4% albumin group,the hemolysis ratio of erythrocytes increased with increasing the concentration of indoxyl sulphate, and dose-effect relationship(P <0.05).when absence of Ca2+ in supernatant,this effect on erythrocytes with increasing concentration of indoxyl sulphate became weak.4. The erythrocytes exposure to protein-bound indoxyl sulfate(62.5,125,250,500, 1000μmol/L)for 48 hours, the Ca2+ fluorescence intensity of erythrocytes respectively were 6.04±0.70、6.87±0.77、8.72±0.55、9.94±0.47、12.03±0.50,compared with 4% albumin group,the Ca2+ fluorescence intensity of erythrocytes increased with increasing the concentration of indoxyl sulphate,and dose-effect relationship(P <0.05).ConclusionProtein-bound indoxyl sulfate promoted eryptosis by cell shrinkage, increased PS exposure and calcium influx, and dose-effect relationship.Remove protein-bound uremic toxins would help to improve renal anemia.