蛋白结合硫酸吲哚酚损伤红细胞膜

Albumin-bound indoxyl sulfate damages erythrocytes in vitro

目的研究蛋白结合硫酸吲哚酚对红细胞寿命的影响及作用机制。方法以4%白蛋白结合不同浓度的硫酸吲哚酚孵育0.4%红细胞悬液48 h,采用流式细胞仪检测红细胞体积,Annexin-V-FITC凋亡试剂盒检测红细胞表面磷脂酰丝氨酸的标记率,钙离子荧光探针(Fluo3/AM)检测红细胞内Ca2+浓度,酶标分析仪检测红细胞的溶血率,采用SPSS18.0统计软件进行统计分析。结果 4%白蛋白结合硫酸吲哚酚孵育0.4%红细胞悬液48 h后,随硫酸吲哚酚浓度逐渐增加,红细胞体积逐渐变小[(1603.33±8.50)vs(742.67±14.04),P<0.05],红细胞表面磷脂酰丝氨酸的标记率[(0.26±0.06)vs(10.32±1.64),P<0.05]、红细胞内Ca2+浓度以及溶血率逐渐增加,与对照组相比差异有统计学意义(P<0.05),呈剂量依赖性;当细胞外Ca2+缺乏时,硫酸吲哚酚对红细胞的这种损伤效应明显降低。结论 4%白蛋白结合硫酸吲哚酚通过增加钙内流而损伤红细胞,促进红细胞衰亡,是尿毒症患者贫血的发生机制之一,清除蛋白结合毒素有助于改善贫血。

Objective To investigate the effect of albumin-bound indoxyl sulfate on erythrocytes and related mechanism.Methods Different concentrations of indoxyl sulfate were bound to 4% albumin.Erythrocytes with 0.4% hematocrit were prepared from normal adults.Forward scatter was used to estimate erythrocyte volume.Phosphatidylserine-exposure from annexin V binding was used to check eryptosis.Erythrocyte intracellular free calcium concentration（[Ca^2 ＋]i） was detected by Fluo3-fluorescence and assayed by flow cytometry.Hemolysis from hemoglobin release was estimated with an enzyme-mark analyzer.Statistical analysis was made with SPSS18.0 software.Results Exposure to albumin-bound indoxyl sulfate for 48 hours significantly decreased erythrocyte volume（1603.33 ± 8.50 vs 742.67 ± 14.04,P 〈 0.05）,and increased eryptosis（0.26 ± 0.06 vs 10.32 ± 1.64,P 〈 0.05）,[Ca2 ＋]iand hemolysis ratio in a dose-dependent manner（P 〈 0.05）.The damage effect of albumin-bound indoxyl sulfate on erythrocytes was weakened in the absence of Ca^2 ＋in the supernatant.Conclusion Albumin-bound indoxyl sulfate induces eryptosis through calcium influx,which is an important mechanism of anemia in uremic patients.Eliminating albumin-bound indoxyl sulfate might alleviate renal anemia.