Determination of Tacrolimus Concentration and Protein Expression of P-Glycoprotein in Single Human Renal Core Biopsies
Determination of Tacrolimus Concentration and Protein Expression of P-Glycoprotein in Single Human Renal Core Biopsies
复制标题
DOI：
10.1097/ftd.0000000000000510
复制
发表时间：
2018-06-01
影响因子：
2.5
通讯作者：
Christensen, Hege
中科院分区：
文献类型：
--
作者：
Krogstad, Veronica;Vethe, Nils T.;Christensen, Hege
背景：他克莫司（TAC）是目前肾移植受者免疫抑制方案的基石。尽管进行了治疗性全血监测，但TAC仍与肾毒性相关，并且已假设涉及TAC和/或其代谢物的肾内蓄积。由于TAC是P-糖蛋白（P-gp）的底物，该外排转运蛋白的表达和活性可影响肾组织中TAC的水平。本研究的主要目的是开发和验证一种用于定量来自单个人肾芯活检组织匀浆中TAC的方法。第二个目的是提供措施的P-gp的表达和TAC的脱甲基代谢产物在相同的renal biopsies.Methods：人肾组织，有和没有临床TAC暴露，用于方法的开发和验证。用珠磨法制备匀浆，蛋白沉淀后用液相色谱串联质谱法分析TAC及其去甲基代谢产物的浓度。免疫印迹法用于半定量的P-gp表达的匀浆。结果：TAC法的平均准确度为99.7%~ 107%，变异系数为10.7%~ 10.7%。
Background: Tacrolimus (TAC) is currently the cornerstone of immunosuppressive protocols for renal transplant recipients. Despite therapeutic whole blood monitoring, TAC is associated with nephrotoxicity, and it has been hypothesized that intrarenal accumulation of TAC and/or its metabolites are involved. As TAC is a substrate of P-glycoprotein (P-gp), the expression and activity of this efflux transporter could influence the levels of TAC in renal tissue. The primary aim of this study was to develop and validate a method for quantification of TAC in tissue homogenates from single human renal core biopsies. The secondary aim was to provide measures of P-gp expression and of the demethylated metabolites of TAC in the same renal biopsy.Methods: Human renal tissue, with and without clinical TAC exposure, was used for method development and validation. Homogenates were prepared with bead-beating, and concentrations of TAC and its demethylated metabolites were analyzed with liquid chromatography tandem mass spectrometry after protein precipitation. A Western blot method was used for semiquantification of P-gp expression in the homogenates. The final methods were applied to renal core biopsies from 2 transplant patients.Results: The TAC assay showed within- and between-nun mean accuracy between 99.7% and 107% and coefficients of variation