Eck P et al. (2013), The human sodium-dependent ascorbic acid transp...

XB-ART-48340

Physiol Rep 2013 Nov 01;16:e00136. doi: 10.1002/phy2.136.

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The human sodium-dependent ascorbic acid transporters SLC23A1 and SLC23A2 do not mediate ascorbic acid release in the proximal renal epithelial cell.

Eck P , Kwon O , Chen S , Mian O , Levine M .

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Sodium-dependent ascorbic acid membrane transporters SLC23A1 and SLC23A2 mediate ascorbic acid (vitamin C) transport into cells. However, it is unknown how ascorbic acid undergoes cellular release, or efflux. We hypothesized that SLC23A1 and SLC23A2 could serve a dual role, mediating ascorbic acid cellular efflux as well as uptake. Renal reabsorption is required for maintaining systemic vitamin C concentrations. Because efflux from nephron cells is necessary for reabsorption, we studied whether SLC23A1 and SLC23A2 mediate efflux of ascorbic acid in the human renal nephron. We found high gene expression of SLC23A1 but no expression of SLC23A2 in the proximal convoluted and straight tubules of humans. These data rule out SLC23A2 as the ascorbic acid release protein in the renal proximal tubular epithelia cell. We utilized a novel dual transporter-based Xenopus laevis oocyte system to investigate the function of the SLC23A1 protein, and found that no ascorbate release was mediated by SLC23A1. These findings were confirmed in mammalian cells overexpressing SLC23A1. Taken together, the data for SLC23A1 show that it too does not have a role in cellular release of ascorbic acid across the basolateral membrane of the proximal tubular epithelial cell, and that SLC23A1 alone is responsible for ascorbic acid uptake across the apical membrane. These findings reiterate the physiological importance of proper functioning of SLC23A1 in maintaining vitamin C levels for health and disease prevention. The ascorbate efflux mechanism in the proximal tubule of the kidney remains to be characterized.

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Species referenced: Xenopus laevis
Genes referenced: slc23a1 slc23a2 slc2a1

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	Figure 1. Sites of expression of SLC23A1 and SLC23A2 in microdissected segments of the human nephron: SLC23A1 expression is defined to the proximal convoluted and proximal straight tubule. SLC23A2 is found in the medullary and cortical thick ascending limb of Henle's loop. Data are depicted as SAGE tags/million tags.
	Figure 2. Dual-transporter Xenopus laevis oocyte expression system to determine ascorbic acid release. Glucose transporter isoform 1 (GLUT1) and SLC23A1 as a putative protein mediating cellular efflux (Effluxer) are coexpressed in a X. laevis oocyte. As a result, dehydroascorbic acid (DHA) is transported into the oocytes using facilitated diffusion via GLUT1, and once intracellular, it is reduced to ascorbic acid (AA). Dehydroascorbic acid is not a substrate of the putative Effluxer, which can utilize the intracellular ascorbic acid as a substrate and release it into the medium.
	Figure 3. SLC23A1 does not mediate ascorbic acid efflux in Xenopus laevis oocytes: Oocytes expressing GLUT1 (∇) only or coexpressing SLC23A1 + GLUT1 (▼) were incubated with 1 mmol/L dehydroascorbic acid, resulting in intracellular ascorbic acid concentrations of up to 2.5 mmol/L/oocyte. Sham-injected oocytes did not transport dehydroascorbic acid and therefore did not accumulate intracellular ascorbic acid (•). Inset right bottom: Ascorbic acid concentration in incubation medium of oocytes expressing GLUT1 (•) or coexpressing SLC23A1 + GLUT1 (○). Inset left top: relative ascorbic acid release in% of oocytes expressing GLUT1 (▪) or coexpressing SLC23A1 + GLUT1 (□). Intracellular [14C]ascorbic acid was determined based on internalized radioactivity and/or HPLC coupled with electrochemical detection.
	Figure 4. Concentration-dependent ascorbic acid uptake in SLC23A1 injected oocytes. SLC23A1 expressing (•) and sham-injected oocytes (○) were incubated with [14C]ascorbic acid (AA) in concentrations from 0 to 800 μmol/L and elevated uptake in the SLC23A1 expressing (•) oocytes demonstrates the presence of the functional protein. Intracellular [14C]ascorbic acid was determined based on internalized radioactivity, for oocytes incubated for 10 min.
	Figure 5. SLC23A1 does not mediate ascorbic acid efflux in Chinese Hamsters Ovarian (CHO) cells. CHO cells expressing SLC23A1 (▼) or vector only (▽) were incubated with 250 μmol/L dehydroascorbic acid, resulting in rapid elevations of intracellular ascorbic acid up to a concentration of 7 mmol/L. CHO cells do not contain any detectable ascorbic acid (○). Inset right bottom: ascorbic acid concentration in incubation medium of SLC23A1 overexpressing CHO cells (•) and vector alone containing CHO cells (○). Inset centre bottom: relative ascorbic acid release in% of SLC23A1 overexpressing CHO cells (▪) and vector alone containing CHO cells (□). Ascorbic acid was determined using HPLC coupled with electrochemical detection.
	Figure 6. Concentration-dependent ascorbic acid uptake in SLC23A1-transfected CHO cells. SLC23A1 overexpressing CHO cells (•) and vector alone containing CHO cells (○) were incubated with ascorbic acid 0–400 μmol/L. Elevated uptake in the SLC23A1 expressing (•) oocytes demonstrates the presence of the functional protein. Ascorbic acid was determined using HPLC coupled with electrochemical detection, for cells incubated for 10 min.

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Barrett, NCBI GEO: mining millions of expression profiles--database and tools. 2005, Pubmed