Retinoid Homeostatic Gene Expression in Liver, Lung and Kidney: Ontogeny and Response to Vitamin A-Retinoic Acid (VARA) Supplementation from Birth to Adult Age

Abstract

Vitamin A (VA, retinol) metabolism is homeostatically controlled, but little is known of its regulation in the postnatal period. Here, we determined the postnatal trajectory of VA storage and metabolism in major compartments of VA metabolism-plasma, liver, lung, and kidney from postnatal (P) day 1 to adulthood. We also investigated the response to supplementation with VARA, a combination of VA and 10% all-trans-retinoic acid that previously was shown to synergistically increase retinol uptake and storage in lung. Nursling pups of dams fed a VA-marginal diet received an oral dose of oil (placebo) or VARA on each of four neonatal days: P1, P4, P7, and P10; and again as adults. Tissues were collected 6 h after the final dosing on P1, P4, P10, and at adult age. Gene transcripts for Lrat and Rbp4 in liver and Raldh-1 and Raldh-3 in lung, did not differ in the neonatal period but were higher, P<0.05, in adults, while Cyp26B1, Stra6, megalin, and Raldh-2 in lung did not differ from perinatal to adult ages. VARA supplementation increased total retinol in plasma, liver and lung, with a dose-by-dose accumulation in neonatal liver and lung, while transcripts for Lrat in liver, megalin in kidney, Cyp26A1/B1 in liver and lung, respectively, and Stra6 in lung, were all increased, suggesting pathways of VA uptake, storage and RA oxidation were each augmented after VARA. VARA decreased hepatic expression of Rbp4, responsible for VA trafficking from liver to plasma, and, in lung, of Raldh-1 and Raldh-2, which function in RA production. Our results define retinoid homeostatic gene expression from neonatal and adult age and show that while supplementation with VARA acutely alters retinol content and retinoid homeostatic gene expression in neonatal and adult lung, liver and kidney, VARA supplementation of neonates increased adult-age VA content only in the liver.

Fig 1. Experimental design and total retinol concentrations in plasma, liver, and lung of neonatal rats to adult-age rats fed VAM diet supplemented with or without VARA.

Experimental design and treatment schedule (1A), and total retinol concentrations in plasma (B), liver (C), and lung (D) of control (oil-treated) neonates nursed by dams fed vitamin A marginal (VAM) diet, oil placebo) and VARA supplemented rats at postnatal day (P)1, P4, P10 and adult age. Sprague-Dawley offspring were generated by mating female and male rats; dams were fed marginal VA diet, which was maintained throughout gestation and postnatally, and after weaning in the offspring that reached adult age. Pups were randomly assigned to receive oil or VARA treatments at five ages. The adult group marked 0 received VARA at neonatal days P1-P10, but not at adult age, to assess any carry over effect of neonatal VARA supplementation at adult age. For all ages, the final treatment with oil or VARA was administered 6 h before euthanasia and tissue collection (A). Data in B, C, and D are shown as mean ± SEM, n ≥ 5/group. One-way ANOVA with posthoc analysis was used to determine differences between oil-treated groups, indicative of differences due to ontogeny; means without a common letter differ significantly, P < 0.05. For each age, VARA versus Oil groups were compared by t-test; * indicates P < 0.05. In C, linear trends with age were determined for the Oil and VARA groups separately. Abbreviations: P, postnatal day; VAM, vitamin A marginal; VARA, vitamin A with 10% retinoic acid.

Fig 2. Expression of retinoid homeostatic genes in liver of neonatal rats and adult-age rats fed VAM diet supplemented with or without VARA.

Relative levels of mRNA transcripts for Rbp4 (A), Lrat (B), and Cyp26a1 (C) are shown for liver, under experimental conditions described in Fig 1A. Developmental expression patterns in the control group are shown by black bars for Rbp4 (A), Lrat (B), and Cyp26A1 (C) and levels after VARA in blue bars. The group labeled “VARA Carryover” (red bars) received VARA on P1, P4, P7, and P10 as neonates, but were not treated with VARA as adults (see Fig 1A). For comparison of relative values, the average value for the control group (P1, oil) was set to 1.00 for each assay and fold change for each gene is shown as relative to this value. Data are shown as mean ± SEM, n ≥ 5/group. One-way ANOVA with post-hoc analysis was used to determine differences between oil-treated groups at different ages, indicative of differences due to ontogeny. Means without a common letter differ significantly, P < 0.05. For each age, VARA versus Oil groups were compared by t-test; * indicates P < 0.05. Abbreviations: P, postnatal day; VARA, vitamin A combined with 10% retinoic acid; Rbp4, retinol-binding protein 4; Lrat, lecithin:retinol acyltransferase; Cyp26A1, cytochrome P450 family 26 subfamily c polypeptide 1.

Fig 3. Expression of retinoid homeostatic genes in lungs of neonatal rats and adult-age rats fed VAM diet supplemented with or without VARA.

Relative levels of mRNA transcripts are shown for Stra6 (A), Lrat (B), and Cyp26B1 (C) in lung under experimental conditions described in Fig 1A. Data are shown as mean ± SEM, n ≥ 5/group. One-way ANOVA with post-hoc analysis was used to determine differences between oil-treated groups at different ages, indicative of differences due to ontogeny. Means without a common letter differ significantly, P < 0.05. For each age, VARA versus Oil groups were compared by t-test; * indicates P < 0.05. Abbreviations: P, postnatal day; VARA, vitamin A combined with 10% retinoic acid; Stra6, Stimulated by retinoic acid gene 6; Rbp4, retinol-binding protein 4; Lrat, lecithin:retinol acyltransferase; Cyp26B1, cytochrome P450 family 26 subfamily b polypeptide 1.

Fig 4. Expression of retinoid biosynthetic genes in lungs of neonatal rats and adult-age rats fed VAM diet and treated with or without VARA.

Relative levels of mRNA transcripts are shown for Raldh-1 (A), Raldh-2 (B) and Raldh-3 (C) in lung, under experimental conditions described in Fig 1A. Data are shown as mean ± SEM, n ≥ 5/group. One-way ANOVA with post-hoc analysis was used to determine differences between oil-treated groups at different ages, indicative of differences due to ontogeny. Means without a common letter differ significantly, P < 0.05. For each age, VARA versus Oil groups were compared by t-test; * indicates P < 0.05. Abbreviations: P, postnatal day; VARA, vitamin A combined with 10% retinoic acid; Raldh, retinaldehyde dehydrogenase.

Fig 5. Expression of megalin and Lrat in kidney of neonatal rats and adult-age rats fed VAM diet and treated with or without VARA.

Relative levels of mRNA transcripts are shown for megalin (A) and Lrat (B) in kidney. under experimental conditions described in Fig 1A. Asterisks (*) denote differences between oil and VARA groups in adult kidney, P < 0.05. Data are mean ± SEM, n ≥ 7/group. Abbreviations: P, postnatal day; VARA, vitamin A combined with 10% retinoic acid.