Vitamin C transporter Slc23a1 links renal reabsorption, vitamin C tissue accumulation, and perinatal survival in mice

Abstract

Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated Slc23a1-/- mice. Compared with wild-type mice, Slc23a1-/- mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to Slc23a1-/- dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of Slc23a1-/- pups born to Slc23a1-/- dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although Slc23a1-/- mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice.

Figure 1. Design, preparation, and confirmation of Slc23a1^–/– mice.

(A) Northern blot analysis of mouse Slc23a1 gene expression. Multitissue mouse Northern blot panel probed with [α³²P]-d-CTP–labeled mouse Slc23a1 cDNA (top), normalized to β-actin gene expression (bottom). (B) Genomic DNA PCR analyses. DNA obtained from Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– littermates was analyzed by PCR. The wild-type allele was predicted to be a 3-kb fragment; the deletion allele was predicted to be a 10-kb fragment. M, DNA marker standards. (C) RT-PCR analysis of Slc23a1 gene expression in progeny from heterozygous crosses. Gene expression was assessed in liver, kidney, and small intestine from Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– mice. A 366-bp fragment was amplified by RT-PCR in Slc23a1^+/+ and Slc23a1^+/– RNA, but not in RNA isolated from Slc23a1^–/– mice. As an internal control, gene expression of Gapdh was assessed by amplifying a 900-bp GAPDH PCR product in all tissues analyzed. bl, blank control; st, DNA standards. (D) Body weight as a function of time in Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– mice. Female (n = 3–19) and male (n = 4–29) littermates were weighed at weaning (3 weeks) and weekly thereafter. P = NS.

Figure 2. Role of Slc23a1 in ascorbate renal reabsorption and tight control.

(A) Urine ascorbate as a function of plasma ascorbate concentrations. Plasma and urine samples were obtained at the same time in female and male Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– mice (n = 6–18). Ascorbate concentrations were determined by HPLC as described in Methods. (B and C) Ascorbate fractional excretion and clearance. Plasma and urine was obtained at the same time from individual female (B) and male (C) mice (n = 6 per sex and genotype). Ascorbate, creatinine, and inulin were analyzed, and fractional excretion and clearance values were calculated as described in Methods. Differences between inulin clearances for genotypes of each sex were not statistically significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus respective control.

Figure 3. Ascorbate transport activity in intestine and liver of male and female Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– mice.

(A–C) Biodistribution of orally administered 6-bromo-6-deoxy-L-ascorbate. Male and female Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– mice received 0.3 mg 6-bromo-6-deoxy-L-ascorbate by gavage. After 30 minutes, urine was collected by bladder massage, and mice were immediately sacrificed. 6-Bromo-6-deoxy-L-ascorbate was measured in (A) intestinal mucosa (n = 5), (B) liver (n = 5), and (C) urine (n = 6). ND, no peak detected. **P ≤ 0.01, ***P ≤ 0.001 versus respective control.

Figure 4. Dam and pup ascorbate and perinatal survival.

(A) Perinatal mortality in offspring from Slc23a1^–/– dams compared with Slc23a1^+/– controls. Perinatal mortality was determined after crossing unsupplemented or supplemented mice. Numbers denote deceased pups relative to total pups. (B) Pregnancy and maternal ascorbate concentrations. Plasma and urine ascorbate concentrations in spot samples from female mice (n = 5–9). Samples obtained prior to pregnancy (pre-), within 72 hours before delivery (pregnancy), and 3 weeks postpartum (post-). *P ≤ 0.05, ***P ≤ 0.001 versus unsupplemented Slc23a1^–/–. (C) Ascorbate in newborn Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– pups. All samples were obtained less than 24 hours postpartum. Total ascorbate (n = 5–8) and plasma ascorbate concentrations (n = 6) were determined. Additionally, Slc23a1^–/– mice were crossed, with dams unsupplemented or supplemented, and plasma ascorbate values were determined (n = 5). (D) Ascorbate urine concentrations as a function of ascorbate plasma concentrations in Slc23a1^–/–, Slc23a1^–/– supplemented, and Slc23a1^+/+ (encircled) female mice. Shown are ascorbate levels in plasma and urine samples from B. Individual dams are represented by different colors, with symbols distinguishing prepregnant (triangle), pregnant (square), and postpartum (circle) levels. Left: r² = 0.86; x = –0.56 when y = 0 (renal threshold). Right: r² = 0.67; x = 0.77 when y = 0 (renal threshold).

Figure 5. SLC23A1 SNPs and ascorbate intake.

(A) Distribution of SLC23A1 in kidney nephron segments. Serial Analysis of Gene Expression (SAGE) libraries GSM10419 and GSM10423–GSM10429 (53) containing expression data from microdissected glomeruli and 6 different nephron segments were interrogated (54); values are expressed as tags per million. (B) Effect of SLC23A1 SNPs on ascorbate transport. X. laevis oocytes were microinjected with the following SLC23A1 cRNAs: common type; sham injected; human deletion construct; and SNPs A652G rs34521685, G790A rs33972313, A772G rs35817838, and C180T rs6886922. (C) Population prevalences of SLC23A1 polymorphisms. Shown are averaged minor allelic frequencies of SLC23A1 genotypes in African (n = 48), American-African (n = 438), and white (n = 1,874) individuals, using pooled genotype data (35, 55, 56). (D) Modeled effects of SLC23A1 polymorphisms on plasma ascorbate concentrations in humans. Values in healthy young women for common type SLC23A1 are measured (stars; ref. 11) and calculated fasting steady-state plasma ascorbate concentrations. For women with SNPs, values are calculated. (E) Percentiles of 19- to 30-year-old, pregnant, and lactating women in relation to range of ascorbate plasma concentrations and intake. Plasma concentrations as a function of intake were calculated based on dose concentration pharmacokinetics data (D and ref. 11). Percentiles of women with varying intakes used food intake data (8); the y axis is not continuous because of the sigmoid relationship between ascorbate intake and plasma concentration (D and ref. 11).

Figure 6. Role of Slc23a1 in ascorbate body content, urinary loss, tissue distribution, and upregulation of synthesis.

(A) Total body ascorbate content of male and female Slc23a1^+/+ and Slc23a1^–/– mice (n = 6). (B) Amount of ascorbate excreted in urine, as a function of age, by male and female Slc23a1^+/+ and Slc23a1^–/– littermates aged 7–28 weeks. For clarity, error bars are unidirectional. The slope of all lines except that for Slc23a1^+/+ females (P < 0.05) was not significantly different from 0. (C) Percent ascorbate body stores excreted in urine by male and female Slc23a1^+/+ and Slc23a1^–/– mice. Calculations based on data in A and B. (D and E) Ascorbate in tissues isolated from male and female Slc23a1^+/+, Slc23a1^+/–, and Slc23a1^–/– littermates. Tissues with the highest ascorbate concentrations are grouped in D; others are shown in E. (F) Urinary glucuronic acid excretion (n = 8). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus respective control.

Figure 7. Differential expression of genes in the ascorbic acid biosynthesis pathway in livers of male and female Slc23a1^–/– and Slc23a1^+/+ mice.

Differences were not statistically significant.