Damaraju Vijaya L, Elwi Adam N, Hunter Charlene, Carpenter Pat, Santos Cheryl, Barron Gerry M, Sun Xuejun, Baldwin Stephen A, Young James D, Mackey John R, Sawyer Michael B, Cass Carol E
Membrane Protein Research Group, University of Alberta, Edmonton, Alberta, Canada.
Am J Physiol Renal Physiol. 2007 Jul;293(1):F200-11. doi: 10.1152/ajprenal.00007.2007. Epub 2007 Apr 4.
Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays. hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [(3)H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [(3)H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs.
肾脏中的核苷转运体介导核苷的肾脏重吸收和分泌。我们利用逆转录聚合酶链反应（RT-PCR）证实，皮质和髓质中均存在编码人等效核苷转运体1（hENT1）、人等效核苷转运体2（hENT2）、人钠依赖型核苷转运体1（hCNT1）、人钠依赖型核苷转运体2（hCNT2）和人钠依赖型核苷转运体3（hCNT3）的信使核糖核酸（mRNA）。用粗制膜制剂进行免疫印迹分析显示，皮质和髓质中均有大量的hENT1和hCNT3，而hENT2、hCNT1或hCNT2即便有也极少，这表明后三者要么不存在，要么低于免疫测定的检测限。在近端小管的顶端表面以及髓袢升支粗段和集合管的顶端及基底表面均观察到hENT1免疫染色。除了一些细胞质染色外，但在近端小管的顶端表面以及髓袢升支粗段还观察到显著的hCNT3免疫染色。hENT1的高亲和力抑制剂[³H]硝基苄基巯基嘌呤核糖核苷（NBMPR）与皮质刷状缘膜囊泡的平衡结合证实了近端小管顶端表面存在hENT1。极化的肾近端小管细胞对[³H]尿苷的摄取表现出一个受胸苷和肌苷抑制的钠依赖成分，以及一个受NBMPR部分抑制、受地拉卓完全抑制的钠非依赖成分，这表明hENT1和hCNT3水平较高，而hENT2活性水平较低。1）人肾脏中存在hENT1/2和hCNT1/2/3的转录本以及hENT1和hCNT3蛋白，2）培养的近端小管细胞中存在hENT1、hENT2和hCNT3活性，这表明hENT1、hCNT3，可能还有hENT2参与了肾脏对核苷和核苷类药物的处理。
