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

Abstract

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.

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

“…More widely distributed in cells and tissues than paralogs hCNT1 or hCNT2 (10), and with a central role in renal nucleoside and nucleoside drug transport (37,40), the multifunctional capability of hCNT3 and robust heterologous expression in Xenopus oocytes makes it the protein of choice for molecular characterization by site-directed mutagenesis and other approaches. Here we report an investigation of hCNT3 glutamate and aspartate residues.…”

“…For immunoblotting, solubilized proteins from two oocytes/lane were resolved on 12% SDS-polyacrylamide gels (36). The electrophoresed proteins were transferred to polyvinylidene difluoride membranes and probed with affinity-purified antihCNT3-(45-69) polyclonal antibodies (37). Blots were then incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (GE Healthcare) and developed with enhanced chemiluminescence reagents (GE Healthcare).…”

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3C602R) Showing Altered Sodium-Binding Capacity

“…CNT3 has also been shown to transport gemcitabine and the antiviral drugs azidothymidine and ribavirin (Hu et al, 2006;Yamamoto et al, 2007). Its location at the apical membrane of a polarized cell model (Mangravite et al, 2003;Errasti-Murugarren et al, 2007) together with its presence along the rat and human renal tubule (Rodríguez-Mulero et al, 2005;Damaraju et al, 2007) and human intestine (Ritzel et al, 2001) suggest that CNT3 plays an important role in the absorption and disposition of nucleosides and synthetic nucleoside analogs.…”

Tubuloglomerular feedback and renal function in mice with targeted deletion of the type 1 equilibrative nucleoside transporter

“…Facilitation of transport by ENT proteins is a downhill movement according to prevailing concentration gradients thereby permitting equilibration between intra-and extracellular levels of nucleosides (1). The main equilibrative nucleoside transporter ENT1 is widely expressed across tissues and species, and it is found in the kidney in both tubular and extratubular locations including glomeruli and vascular smooth muscle and endothelial cells (5,9). The present studies show some prevalence of medullary compared with cortical ENT1 expression, and this may be due to ENT 1 expression in collecting ducts and thick ascending limbs as previously reported (5).…”

“…The main equilibrative nucleoside transporter ENT1 is widely expressed across tissues and species, and it is found in the kidney in both tubular and extratubular locations including glomeruli and vascular smooth muscle and endothelial cells (5,9). The present studies show some prevalence of medullary compared with cortical ENT1 expression, and this may be due to ENT 1 expression in collecting ducts and thick ascending limbs as previously reported (5). ENT2 expression in the kidney was found to be rather low comparatively, and its abundance was not affected by deletion of ENT1.…”