Diversity in rat tissue accumulation of vitamin B12 supports a distinct role for the kidney in vitamin B12 homeostasis
Henrik Birn1,,
Ebba Nexø2,
Erik Ilsø Christensen1 and
Rikke Nielsen1
1 Department of Cell Biology, Institute of Anatomy, University of Aarhus and
2 Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Aarhus, Denmark
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Abstract
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Background. Vitamin B12 in plasma is complexed to the carrier proteins transcobalamin (TC) and haptocorrin. The TCB12 complex is filtered in the glomeruli and reabsorbed in the renal tubules by receptor-mediated endocytosis, providing a route for a significant renal accumulation of vitamin B12. The present study investigates the role of the rodent kidney in B12 homeostasis by examining the distribution of vitamin B12 in rats during vitamin B12 depletion or B12 load, and compares kidney accumulation with the vitamin distribution in other tissues including brain, liver, testes, intestine, spleen and plasma.
Methods. Fifteen rats were fed on a diet containing different concentrations of B12 supplemented with s.c. injections of B12. Twenty four hours prior to sacrifice, all animals were injected with [57Co]B12. The vitamin contents of kidneys, liver, spleen, brain, testis, intestine, skeletal muscle, serum and urine were analysed. Both total tissue vitamin B12 accumulation and [57Co]B12 were determined to compare steady-state B12 and the distribution of an acutely injected dose. In the kidney, free and protein-bound B12 was determined by gel filtration.
Results. The rat kidneys accumulated more B12 during normal and loaded conditions than any other tissue. A 110-fold increase in vitamin content was observed from the deficient to the loaded conditions in the kidney compared with a 3.5-fold increase in the liver. In contrast to all other organs, significantly smaller amounts of acutely injected B12 accumulated in the kidneys in the vitamin-deprived state compared with both the normal and the vitamin-loaded condition.
Conclusions. The present study suggests a significant role for the rodent kidney in vitamin B12 metabolism. We propose a model for rat tissue uptake consistent with the presence of two different TCB12 receptors and renal uptake following filtration of TCB12 in the glomeruli. The presented model allows for the reduced renal uptake and accumulation in vitamin-deprived conditions, thus reserving the vitamin for other tissues, including nerve tissue and bone marrow, which are more sensitive to vitamin B12 deficiency.
Keywords: cobalamin; megalin; renal physiology; vitamin metabolism
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Introduction
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Vitamin B12 acts as a cofactor in the production of succinyl-CoA and the essential amino acid methionine in the mitochondrial fraction and cytoplasm, respectively. Vitamin deficiency is associated with severe neurological and haematological symptoms, identifying the nervous tissue and the bone marrow as the organs most sensitive to vitamin deficiency. Because of the high vitamin B12 content in the human liver, this organ traditionally has been considered the major storage site for vitamin B12 [1]. However, a possible role for the rat kidney is suggested by the well known ability of this organ to accumulate the vitamin during states of vitamin surplus [24].
Absorbed B12 is transported to the tissues bound to transcobalamin (TC). Two receptors for the uptake of TCB12 have been reported in rats: (i) the multiligand, endocytic receptor megalin [4,5]; and (ii) a 62 kDa protein of as yet unknown structure [6,7]. Megalin (600 kDa) belongs to the low density lipoprotein (LDL) receptor family and is heavily expressed in kidney proximal tubules [8] and several other absorptive epithelia (reviewed in [9]). Plasma TCB12 is filtered in the renal glomeruli and reabsorbed by megalin in the mouse proximal tubule [5]. Thus, significant amounts of vitamin B12 are reabsorbed from the ultrafiltrate by the kidney [4].
To evaluate the role of the kidney in vitamin B12 homeostasis, we have compared rat renal accumulation of vitamin B12 with other tissues, including brain, liver, testes, intestine, spleen and plasma, during states of vitamin B12 depletion and vitamin B12 load. Our data suggest a unique role for the rat kidney allowing for the reduced renal uptake and accumulation in vitamin-deprived conditions, thus reserving the vitamin for other tissues. The results support a model of tissue vitamin uptake consistent with the presence of two different TCB12 receptors in different tissues.
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Subjects and methods
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Animals
Fifteen newly weaned, age-matched (4 weeks of age), male Wistar rats were divided into three groups: (i) vitamin B12-depleted rats (n=5); (ii) normal rats (n=5); and (iii) vitamin B12-loaded rats (n=5). Each group was treated as follows: (i) vitamin-depleted rats were fed a low vitamin B12 diet (0.003 mg/kg; Attromin, Germany) for 60 days; (ii) normal rats were fed a normal vitamin B12 diet (0.041 mg/kg; Attromin) for 60 days; and (iii) vitamin-loaded rats were fed a normal vitamin B12 diet (0.041 mg/kg; Attromin) for 60 days in addition to daily s.c. injections of cyanocobalamin (10 µg/day, SAD, Denmark) beginning at day 42 and excluding the last day before sacrifice. Twenty four hours prior to sacrifice, all rats were injected s.c with 2.5x105 c.p.m. [57Co]B12 (
0.01 mg=7 pmol, Amersham Biosciences, UK). At day 60, the animals were anaesthetized with halothane, and organs were removed, weighed and counted in a
-counter (Packard Cobra 5002
-counter, USA). The kidneys from each animal were split equally into two and all organs were stored at -80°C until further processing. The two kidney samples were weighed and homogenized in 1.0 or 1.5 ml of phosphate-buffered saline (PBS) for the determination of total vitamin B12 content and for estimating the size of the B12-binding proteins by gel filtration, respectively.
Determination of the vitamin B12 concentration in tissues, serum and urine
Tissues were homogenized in 1.0 or 1.5 ml of PBS, pH 7.4 followed by centrifugation at 4000 g for 15 min, 4°C. The amount of total B12 in the supernatant, in serum and in urine was analysed by an automated protein binding assay (ACS 180 Plus, CHIRON/Diagnostics).
The amount of total B12 and labelled B12 was calculated by multiplying the concentration (all B12/g or [57Co]B12/g) by the weight of the organ. The relative amount of labelled B12 was calculated by dividing the amount of [57Co]B12 in the organ by the total amount of [57Co]B12 injected. The tissue-specific activities of labelled vitamin (c.p.m./g) were converted to the corresponding vitamin concentrations (fmol/g tissue) by division by the specific activity of 3.7x105 c.p.m./pmol.
Gel filtration
The homogenized kidneys were examined by gel filtration on a SMART system (Amersham-Biosciences) employing a Superdex 200 column (10x300 mm) using 0.1 M Tris, 1 M sodium chloride, 0.02% sodium azide, 0.05% human albumin (Behringwerke) pH 8.0 as flow buffer at 0.4 ml/min. The OD at 280 nm was monitored continuously. Fractions of 0.4 ml were collected and counted in an automated
-counter (Wallac 1470 Wizard, USA). The column was calibrated employing dextran blue (Sigma, USA), void volume and 22Na (Amersham Biosciences), total volume. Normal rat serum saturated with [57Co]B12 was used to identify the elution peaks of the cobalamin-binding proteins.
Statistics
One-way ANOVA followed by the RyanEinotGabrielWelsch multiple range test was applied to test for differences among the three groups. Calculations were performed using SPSS. In some cases, the data were log transformed to ensure variance homogeneity. A P-value <0.05 is considered significant. Data represent mean±SD.
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Results
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All animals thrived throughout the study, revealing no symptoms relating to differences in vitamin intake.
Total recovered vitamin B12
A >10-fold difference in the content of vitamin B12 was observed amongst the three groups of animals. An average of 4500 pmol B12 was recovered from the B12-loaded animals compared with only 140 pmol from the depleted animals (Table 1
). The total, recovered amount of [57Co]B12 from the organs analysed ranged from 13.8±0.7% of the injected dose in the depleted animals to 47±3% in the loaded rats. This difference may be accounted for by redistribution of newly injected [57Co]B12 into skeletal muscle. To test this, we also counted the activity of [57Co]B12 in femoral quadriceps muscle biopsies (Table 1
). Skeletal muscle tissue may be assumed to constitute
50% of total body mass, enabling the calculation of the amount of [57Co]B12 accumulated. By including total [57Co]B12 in skeletal muscle, the recovered amount of [57Co]B12 increased to 46±2% of the injected dose in the depleted animals and to 62±3% in the loaded rats, confirming that much of the difference in recovered [57Co]B12 could be accounted for by a difference in skeletal muscle accumulation.
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Table 1. Total recovered vitamin B12 and total recovered [57Co]B12 as a percentage of that injected from all organs analysed
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The average, final weight of the rats was 326±8 g and not significantly dependent on the B12 supply. Also, the total weights of kidneys, liver, brain and spleen were not significantly influenced by the amount of B12 supplied (data not shown).
Steady-state distribution of vitamin B12
The B12 concentration in all investigated organs paralleled the B12 supply (Figure 1
). In all organs and in serum from the depleted group, significantly less vitamin B12 was identified compared with the normal and loaded groups (P<0.05). The kidney accumulated by far the highest amount of vitamin B12, demonstrating the kidney to be the major B12-accumulating organ. In the kidney, the loaded condition resulted in a >100-fold increase of B12 compared with the depleted condition, whereas the corresponding increase in the liver was <4-fold (Table 2
). The excretion of vitamin B12 in urine increased significantly with increasing vitamin supply (Figure 1B
).

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Fig. 1. Concentration of vitamin B12 in tissues of rats supplied with low (depleted), normal (normal) or high (loaded) levels of vitamin B12. The vitamin concentration is indicated as log fmol B12/g tissue in (A) and as log fmol B12/l serum and log fmol B12/mmol creatinine in serum and urine, respectively, in (B). Data are given as mean±SD. indicates a significant difference (P<0.05).
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Distribution of an acute dose of labelled vitamin B12
To examine tissue handling of B12 in vitamin-depleted, normal and loaded states, a single dose of [57Co]cyanocobalamin was injected subcutaneously into the animals 24 h before they were sacrificed. The distribution of injected [57Co]B12 was similar to the distribution of total B12 in most organs, suggesting a steady-state distribution of [57Co]B12. In contrast to all other organs, the kidney accumulated less [57Co]B12 in the vitamin-depleted state as compared with the normal or loaded condition. In other tissues except the intestine and testis, an increased concentration of labelled B12 was observed in the depleted group compared with the normal or loaded group (P<0.05, Figure 2A
). Only in the kidney and serum were significant differences in [57Co]B12 identified between the normal and the loaded states (P<0.05). Serum levels of [57Co]B12 were higher in vitamin-depleted than in normal or vitamin-loaded animals. As the half-life of TC in plasma is <2 h [10], almost all TC-bound, labelled B12 is expected to be cleared after 24 h. In contrast, the half-life of the other plasma B12 carrier, haptocorrin, is several days [11]. Thus, the increased levels of [57Co]B12 in depleted animals 24 h after injection conceivably represent increased binding of labelled B12 to haptocorrin due to higher levels of unsaturated carrier protein as a result of the vitamin-depleted state. This haptocorrin-bound [57Co]B12 is not readily available for tissue uptake except perhaps for the liver.

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Fig. 2. Concentration of [57Co]B12 in tissues of rats supplied with low (depleted), normal (normal) or high (loaded) levels of vitamin B12 24 h after injection of labelled vitamin B12. The concentration of [57Co]B12 is indicated as log fmol labelled B12/g tissue in (A) and as log fmol labelled B12/l serum and log fmol labeled B12/mmol creatinine in serum and urine, respectively, in (B). Data are given as mean±SD. indicates a significant difference (P<0.05).
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Intracellular protein binding of vitamin B12
In order to examine intracellular protein binding of vitamin B12 in the kidney, gel filtration was performed on kidney extracts (Figure 3
). In the normal and loaded conditions, most labelled vitamin B12 eluted as free vitamin (92 and 94%, respectively). In contrast, only a minor part of the labelled B12 in the depleted rat kidney was free (21%), indicating that vitamin B12 during states of vitamin surplus is stored in the kidney in a non-protein bound form.

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Fig. 3. Gel filtration of rat kidney extracts. The primary y-axis displays the c.p.m. detected in fractions 131 (blue line), which represents protein-bound vitamin, and the secondary y-axis displays the c.p.m. identified in fractions 3248 (red line), which represents free vitamin. The filled and open arrows indicate the localization of the void volume (dextran blue) and total volume (22Na), respectively.
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Discussion
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The present study establishes the rat kidney as a major vitamin B12-accumulating organ during conditions with sufficient vitamin supply. A >100-fold higher B12-concentration was observed in the kidneys of the vitamin-loaded animals compared with the depleted condition, whereas the corresponding difference in the liver was <4-fold (Table 2
). Even considering the difference in organ weight, most B12 accumulated in the kidneys of the vitamin-loaded animals.
Vitamin B12 is transported in plasma bound to two major transport proteins: TC and haptocorrin. Whereas TC is the major protein involved in transport and uptake of B12 in the tissues in general, haptocorrin only seems to be internalized into liver cells possibly by binding to the asialoglycoprotein receptor [12]. Animal studies have identified at least two different receptors, megalin and a 62 kDa TC receptor involved in the renal uptake of TCB12. Megalin is a 600 kDa multiligand member of the LDL receptor family identified in several absorptive epithelia and heavily expressed in the luminal membranes and endocytic apparatus of proximal tubule cells (for review, see Christensen and Birn [9]). It is involved in the tubular reabsorption of a variety of different vitamins complexed to carrier proteins, including the vitamin D-binding protein-vitamin D [13], retinol-binding proteinretinol [14] and TCB12 [4,5]. Recent evidence suggests that megalin is the major receptor protein involved in luminal uptake of TCB12 filtered in the glomeruli, and megalin deficiency is associated with decreased renal B12 concentration [4]. In addition to megalin, a 62 kDa (normally present as a 124 kDa dimer) TCB12-binding protein has been identified in the kidney, placenta, intestines and liver [6,7,15]. Injection of polyclonal antibodies against this 62 kDa receptor in rabbits caused failure to thrive and elevated levels of homocysteine and methylmalonic acid [15], suggesting that this receptor is more generally involved in TCB12 uptake. The expression of this receptor appears to be regulated [6]. The present observations support a two-receptor model for rat TCB12 uptake which includes a high capacity, probably unregulated luminal uptake of filtered TCB12 in the kidney proximal tubules and a potentially regulated, saturable uptake in most other tissues (Figure 4
). In this model, megalin-mediated kidney uptake of TCB12 is dependent on filtration of TCB12 and thus on the concentration of TCB12 in plasma. While the 62 kDa TCB12 receptor appear to be expressed in the basolateral membranes of the kidney tubules [6], its role in renal B12 uptake is unclear as megalin deficiency alone significantly reduces renal B12 accumulation [4], and the present data show that renal uptake in relation to vitamin status is different from that of other organs, indicating a different mechanism.

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Fig. 4. Hypothetical model of vitamin B12 uptake and transport in rat kidneys and other organs in states of vitamin deprivation and surplus. In this hypothetical model, renal uptake of B12 is dependent mainly on filtration of TCB12 in plasma followed by megalin-mediated, proximal tubule reuptake. The vitamin is accumulated and transported back to plasma coupled to newly synthesized binding proteins by an as yet unknown mechanism. Thus, in states of B12 deficiency and low plasma, TCB12 renal vitamin uptake is reduced. In other tissues, regulated uptake is mediated by the ubiquitous TCB12 receptor, which may be up-regulated during B12 deficiency, thus explaining the increased uptake of an acutely administered dose of [57Co]B12. A higher specific activity of labelled TCB12 in plasma in the vitamin-depleted state may increase uptake of [57Co]B12 further in tissues other than the kidney since the capacity of the ubiquitous TCB12 receptor-mediated uptake is likely to be smaller than the high capacity, megalin-mediated uptake.
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Megalin is also expressed in other absorptive epithelia including fetal tissue such as yolk sac and placenta. The role of megalin in these organs has not been established, but it may supplement the ubiquitous TCB12 receptor, enabling transport to the fetus even when this receptor is down-regulated.
As suggested by the present data, the kidney is the only rat organ accumulating an acutely administered dose of B12 in proportion to vitamin B12 status, thus accumulating large amounts of B12 in states of B12 load and reserving the vitamin for other organs in states of B12 deficiency. Whether the kidney retains accumulated vitamin and releases it during later vitamin deficiency or whether the vitamin is redistributed to other organs, i.e. the liver, for long-term storage remains to be established. The observation that in the vitamin-loaded and normal animals the relative distribution of the administered dose of [57Co]B12 paralleled the distribution of total, unlabelled B12 suggests that the kidney is able to maintain the large accumulated amount of B12, as a result either of storage or of a high flow of vitamin through this organ. Such potential high flow of vitamin may indicate a role for the kidney in vitamin metabolism involving the conversion between different forms of vitamin B12.
In kidneys from the normal and loaded group, most labelled vitamin was present as free vitamin B12, whereas in depleted animals the major part was protein bound. The free form is most probably localized in lysosomes [4,16]. This is in agreement with the lysosomal degradation of endocytosed TC followed by secretion together with newly synthesized binding proteins [17,18]. Thus, during conditions of vitamin load, the processing of free vitamin, most probably transport out of lysosomes, is the rate-limiting and perhaps regulated step in transtubular vitamin transport.
The present findings support a unique role for the rodent kidney in B12 turnover consistent with the presence of two different TCB12 receptors in different tissues enabling an accumulation of B12 in the kidney reflecting the overall vitamin B12 status. This may indicate an important role for the kidney in B12 homeostasis. At present, very limited data are available on the renal handling of vitamin B12 in humans. Increased excretion of TC in patients with tubular defects (Dent's disease) has been shown [4]. Although conflicting data have been published, some studies may indicate that elevated homocysteine in end-stage renal disease is responsive to B12 administration [19,20]; however, its possible relationship to the renal handling of vitamin B12 remains to be established.
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Acknowledgments
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The skilful technical assistance of Pia Nielsen, Hanne Sidelmann, Inger Kristoffersen and Anna Lisa Christensen is greatly appreciated. The work was supported in part by the Danish Medical Research Council, the University of Aarhus, the Birn Foundation, the NOVO-Nordisk Foundation and the Biomembrane Research Center. The study was presented in part at the ASN/ISN World Congress of Nephrology, San Francisco, USA, October 2001, and published in abstract form.
Conflict of interest statement. None declared.
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Notes
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Correspondence and offprint requests to: Henrik Birn, MD, PhD, Department of Cell Biology, Institute of Anatomy, University of Aarhus, Building 234, DK-8000 Aarhus C, Denmark. Email: hb{at}ana.au.dk
The term vitamin B12 is generally used for cyanocobalamin only. However, since the different forms of cobalamin may be converted into each other, the term vitamin B12 or B12 herein includes all forms of cobalamin identified by our assay, including also adenosylcobalamin, methylcobalamin and hydroxycobalamin. 
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Received for publication: 9. 9.02
Accepted in revised form: 8. 1.03