Localization of broadly selective equilibrative and concentrative nucleoside transporters, hENT1 and hCNT3, in human kidney

• Published in: American journal of physiology. Renal physiology 2007-07, Vol.293 (1), p.F200-F211
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Adenosine; Antibodies, Monoclonal; Antibody Specificity; Biochemistry; Biomarkers - metabolism; Blotting, Western; Cells, Cultured; Equilibrative Nucleoside Transporter 1 - antagonists & inhibitors; Equilibrative Nucleoside Transporter 1 - metabolism; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Kidney - drug effects; Kidney - metabolism; Kidney Tubules, Collecting - metabolism; Kidney Tubules, Proximal - metabolism; Kidneys; Kinetics; Loop of Henle - metabolism; Membrane Transport Proteins - metabolism; Membranes - drug effects; Membranes - metabolism; Nephrology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; RNA, Messenger - isolation & purification; Thioinosine - analogs & derivatives; Thioinosine - pharmacology; Uridine - metabolism
• ISSN: 1931-857X; 1522-1466
• DOI: 10.1152/ajprenal.00007.2007

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.