从蛋白结合型尿毒素中筛选肾小管OATs-MRPs通道功能评价的特异性标志物

The renal excretion of drugs is the net result of the contribution from glomerular filtration and tubular secretion. Creatinine, a glomerular filtration biomarker, could not accurately predict the renal excretion and adjust dosage of all most drugs. Many vector transport pathways have been observed in renal tubular, and OATs-MRPs pathway plays a crucial role in the tubular excretion of anionic drugs. However, there are no endogenous biomarkers for evaluating the function of tubular OATs-MRPs pathway in clinical setting. Thus, the key is how to screen the biomarkers that are eliminated by the tubular OATs-MRPs pathway (<1kDa) and not filtered by the glomerulus (>50kDa). It has recently been shown that protein-bound compounds (>50kDa) are excreted by renal tubular transporters, but not filtered by the glomerulus. Our studies have found that there is a correlation between the changes of plasma concentrations of protein-bound uremic toxins and expression levels of OATs and/or MRPs. Therefore, this project aimed to screen the specific biomarkers of the function evaluation of OATs-MRPs pathway from protein-bound uremic toxins and to provide an index for predicting the OATs-MRPs pathway mediated excretion of drugs.

肾小球滤过和肾小管分泌是构成药物肾排泄的主要部分，而目前应用评估肾小球滤过功能的肌酐作为唯一调整用药剂量的指标已无法满足临床药物治疗的需求。肾小管上皮细胞上存在众多转运体组成的矢量转运通道，其中OATs-MRPs通道在有机阴离子药物的肾排泄过程中起到重要作用。然而，目前临床上缺乏评估肾小管OATs-MRPs通道功能的标志物。因此，如何筛选出不被肾小球滤过（>50kDa）又特异性经肾小管OATs-MRPs通道排泄（<1kDa）的标志物是本项目的关键。近年来研究显示，蛋白结合型化合物（>50kDa）主要经肾小管转运体介导排泄，而不被肾小球滤过。课题组前期发现，血浆中一些蛋白结合型尿毒素的浓度变化与肾小管OATs和/或MRPs的表达水平相关。基于此，课题组拟从蛋白结合型尿毒素中筛选肾小管OATs-MRPs通道功能评价的特异性标志物，为预测该通道排泄药物的能力提供依据。

肾脏是机体重要的排泄器官之一，其排泄过程主要包括肾小球滤过、肾小管分泌和重吸收过程，其中肾小管OATs-MRP4通道在众多药物的分泌过程中起到重要作用。然而，目前仍然缺乏评价该通道分泌能力的标志物。因此，筛选评价OATs-MRP4通道排泄能力的标志物对准确评价该通道药物排泄能力或用药剂量确定/调整至关重要。本项目1）通过尿毒素数据和文献获得了26种候选化合物，2）通过蛋白结合率和HEK293T-OAT1/-OAT3/-MRP4摄取评价获得了蛋白结合率超过90%，且同时经OAT1、OAT3和MRP4转运的kynurenic acid、CMPF 和N-(cinnamoyl)glycine作为候选标志物；3）通过对候选标志物的体外评价、矢量转运评价和体内评价（抑制剂和基因敲除动物）获得了潜在标志物；4）通过不同表达肾小管分泌或重吸收转运体细胞系摄取获得了特异性的潜在标志物kynurenic acid 和N-(cinnamoyl)glycine；4）通过终末代谢评价、半衰期测定和亲和常数测定最终获得了OATs-MRP4通道的特异性和适宜性良好的内源性标志物kynurenic acid；5）通过评价kynurenic acid与OATs-MRP4通道底物药物头孢美唑的相关性证实kynurenic acid能够作为评价OATs-MRP4通道底物药物排泄的标志物。本研究筛选获得了OATs-MRP4功能评价的标志物，为该通道药物排泄评价或用药剂量确定/调整提供了参考依据。

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