Diversity in rat tissue accumulation of vitamin B 12 supports a distinct role for the kidney in vitamin B 12 homeostasis

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1 Nephrol Dial Transplant (2003) 18: DOI: /ndt/gfg089 Original Article Diversity in rat tissue accumulation of vitamin B 12 supports a distinct role for the kidney in vitamin B 12 homeostasis Henrik Birn 1, Ebba Nexø 2, Erik Ilsø Christensen 1 and Rikke Nielsen 1 1 Department of Cell Biology, Institute of Anatomy, University of Aarhus and 2 Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Aarhus, Denmark Abstract Background. Vitamin B 12 in plasma is complexed to the carrier proteins transcobalamin (TC) and haptocorrin. The TC B 12 complex is filtered in the glomeruli and reabsorbed in the renal tubules by receptormediated endocytosis, providing a route for a significant renal accumulation of vitamin B 12. The present study investigates the role of the rodent kidney in B 12 homeostasis by examining the distribution of vitamin B 12 in rats during vitamin B 12 depletion or B 12 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 B 12 supplemented with s.c. injections of B 12. Twenty four hours prior to sacrifice, all animals were injected with [ 57 Co]B 12. The vitamin contents of kidneys, liver, spleen, brain, testis, intestine, skeletal muscle, serum and urine were analysed. Both total tissue vitamin B 12 accumulation and [ 57 Co]B 12 were determined to compare steady-state B 12 and the distribution of an acutely injected dose. In the kidney, free and protein-bound B 12 was determined by gel filtration. Results. The rat kidneys accumulated more B 12 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 B 12 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 B 12 metabolism. We propose a model for rat tissue uptake 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. hb@ana.au.dk consistent with the presence of two different TC B 12 receptors and renal uptake following filtration of TC B 12 in the glomeruli. The presented model allows for the reduced renal uptake and accumulation in vitamindeprived conditions, thus reserving the vitamin for other tissues, including nerve tissue and bone marrow, which are more sensitive to vitamin B 12 deficiency. Keywords: cobalamin; megalin; renal physiology; vitamin metabolism Introduction Vitamin B 12 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 B 12 content in the human liver, this organ traditionally has been considered the major storage site for vitamin B 12 [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 [2 4]. Absorbed B 12 is transported to the tissues bound to transcobalamin (TC). Two receptors for the uptake of TC B 12 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 The term vitamin B 12 is generally used for cyanocobalamin only. However, since the different forms of cobalamin may be converted into each other, the term vitamin B 12 or B 12 herein includes all forms of cobalamin identified by our assay, including also adenosylcobalamin, methylcobalamin and hydroxycobalamin. # 2003 European Renal Association European Dialysis and Transplant Association

2 1096 H. Birn et al. epithelia (reviewed in [9]). Plasma TC B 12 is filtered in the renal glomeruli and reabsorbed by megalin in the mouse proximal tubule [5]. Thus, significant amounts of vitamin B 12 are reabsorbed from the ultrafiltrate by the kidney [4]. To evaluate the role of the kidney in vitamin B 12 homeostasis, we have compared rat renal accumulation of vitamin B 12 with other tissues, including brain, liver, testes, intestine, spleen and plasma, during states of vitamin B 12 depletion and vitamin B 12 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 TC B 12 receptors in different tissues. Subjects and methods Animals Fifteen newly weaned, age-matched (4 weeks of age), male Wistar rats were divided into three groups: (i) vitamin B 12 - depleted rats (ns5); (ii) normal rats (ns5); and (iii) vitamin B 12 -loaded rats (ns5). Each group was treated as follows: (i) vitamin-depleted rats were fed a low vitamin B 12 diet (0.003 mgukg; Attromin, Germany) for 60 days; (ii) normal rats were fed a normal vitamin B 12 diet (0.041 mgukg; Attromin) for 60 days; and (iii) vitamin-loaded rats were fed a normal vitamin B 12 diet (0.041 mgukg; Attromin) for 60 days in addition to daily s.c. injections of cyanocobalamin (10 mguday, 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 c.p.m. [ 57 Co]B 12 (;0.01 mgs7 pmol, Amersham Biosciences, UK). At day 60, the animals were anaesthetized with halothane, and organs were removed, weighed and counted in a c-counter (Packard Cobra 5002 c-counter, USA). The kidneys from each animal were split equally into two and all organs were stored at 808C 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 B 12 content and for estimating the size of the B 12 -binding proteins by gel filtration, respectively. Determination of the vitamin B 12 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, 48C. The amount of total B 12 in the supernatant, in serum and in urine was analysed by an automated protein binding assay (ACS 180 Plus, CHIRONuDiagnostics). The amount of total B 12 and labelled B 12 was calculated by multiplying the concentration (all B 12 ugor[ 57 Co]B 12 ug) by the weight of the organ. The relative amount of labelled B 12 was calculated by dividing the amount of [ 57 Co]B 12 in the organ by the total amount of [ 57 Co]B 12 injected. The tissue-specific activities of labelled vitamin (c.p.m.ug) were converted to the corresponding vitamin concentrations (fmolug tissue) by division by the specific activity of c.p.m.upmol. Gel filtration The homogenized kidneys were examined by gel filtration on a SMART system (Amersham-Biosciences) employing a Superdex 200 column ( 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 mlumin. The OD at 280 nm was monitored continuously. Fractions of 0.4 ml were collected and counted in an automated c-counter (Wallac 1470 Wizard, USA). The column was calibrated employing dextran blue (Sigma, USA), void volume and 22 Na (Amersham Biosciences), total volume. Normal rat serum saturated with [ 57 Co]B 12 was used to identify the elution peaks of the cobalamin-binding proteins. Statistics One-way ANOVA followed by the Ryan Einot Gabriel Welsch 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 is considered significant. Data represent mean"sd. Results All animals thrived throughout the study, revealing no symptoms relating to differences in vitamin intake. Total recovered vitamin B 12 A )10-fold difference in the content of vitamin B 12 was observed amongst the three groups of animals. An average of 4500 pmol B 12 was recovered from the B 12 - loaded animals compared with only 140 pmol from the depleted animals (Table 1). The total, recovered amount of [ 57 Co]B 12 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 [ 57 Co]B 12 into skeletal muscle. To test this, we also counted the activity of [ 57 Co]B 12 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 [ 57 Co]B 12 accumulated. By including total [ 57 Co]B 12 in skeletal muscle, the recovered amount of [ 57 Co]B 12 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 [ 57 Co]B 12 could be accounted for by a difference in skeletal muscle accumulation. The average, final weight of the rats was 326"8 g and not significantly dependent on the B 12 supply. Also, the total weights of kidneys, liver, brain and spleen were not significantly influenced by the amount of B 12 supplied (data not shown). Steady-state distribution of vitamin B 12 The B 12 concentration in all investigated organs paralleled the B 12 supply (Figure 1). In all organs and in

3 The kidney in vitamin B 12 homeostasis Table 1. Total recovered vitamin B 12 and total recovered [ 57 Co]B 12 as a percentage of that injected from all organs analysed 1097 Total B 12 recovered from analysed organs (pmol) Activity of [ 57 Co]B 12 in analysed organs (% of injected) Activity of [ 57 Co]B 12 in analysed organs qskeletal muscle tissue (% of injected) Vitamin-depleted rats 140"30* 13.8"0.7* 46"2* Normal rats 1800"600* 38"4* 57"4* Vitamin-loaded rats 4500"800* 47"3* 62"3* The activity of [ 57 Co]B 12 in the femoral quadriceps muscle was determined, and total activity in skeletal muscle tissue was estimated assuming that this comprises ;50% of rat body weight. Data represent mean"sd of ns5 animals in each group. *P-0.05 between all groups (depleted, normal and loaded groups, ANOVA, followed by post hoc test). Analysed organs were the kidneys, liver, brain, testes, spleen and serum. It is assumed that the rats contain a total of 20 ml of blood corresponding to 12 ml of serum. Fig. 1. Concentration of vitamin B 12 in tissues of rats supplied with low (depleted), normal (normal) or high (loaded) levels of vitamin B 12. The vitamin concentration is indicated as log fmol B 12 ug tissue in (A) and as log fmol B 12 ul serum and log fmol B 12 ummol creatinine in serum and urine, respectively, in (B). Data are given as mean"sd. u indicates a significant difference (P-0.05). Table 2. Accumulation of vitamin B 12 in kidney and liver Kidney B 12 concentration (pmolug tissue) Total B 12 (pmol) Liver B 12 concentration (pmolug tissue) Total B 12 (pmol) Vitamin-depleted rats 19"8* 50"20* 6"1* 80"20* Normal rats 710"260* 1600"600* 15"2* 170"30* Vitamin-loaded rats 2100"200* 4200"800* 21"2* 220"40* Data represent mean"sd of ns5 animals in each group. *P-0.05 between all groups (depleted, normal and loaded groups, ANOVA, followed by post hoc test). serum from the depleted group, significantly less vitamin B 12 was identified compared with the normal and loaded groups (P-0.05). The kidney accumulated by far the highest amount of vitamin B 12, demonstrating the kidney to be the major B 12 -accumulating organ. In the kidney, the loaded condition resulted in a )100-fold increase of B 12 compared with the depleted condition, whereas the corresponding increase in the liver was -4-fold (Table 2). The excretion of vitamin B 12 in urine increased significantly with increasing vitamin supply (Figure 1B). Distribution of an acute dose of labelled vitamin B 12 To examine tissue handling of B 12 in vitamindepleted, normal and loaded states, a single dose of

4 1098 H. Birn et al. [ 57 Co]cyanocobalamin was injected subcutaneously into the animals 24 h before they were sacrificed. The distribution of injected [ 57 Co]B 12 was similar to the distribution of total B 12 in most organs, suggesting a steady-state distribution of [ 57 Co]B 12. In contrast to all other organs, the kidney accumulated less [ 57 Co]B 12 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 B 12 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 [ 57 Co]B 12 identified between the normal and the loaded states (P-0.05). Serum levels of [ 57 Co]B 12 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 B 12 is expected to be cleared after 24 h. In contrast, the half-life of the other plasma B 12 carrier, haptocorrin, is several days [11]. Thus, the increased levels of [ 57 Co]B 12 in depleted animals 24 h after injection conceivably represent increased binding of labelled B 12 to haptocorrin due to higher levels of unsaturated carrier protein as a result of the vitamin-depleted state. This haptocorrin-bound [ 57 Co]B 12 is not readily available for tissue uptake except perhaps for the liver. Intracellular protein binding of vitamin B 12 In order to examine intracellular protein binding of vitamin B 12 in the kidney, gel filtration was performed on kidney extracts (Figure 3). In the normal and loaded conditions, most labelled vitamin B 12 eluted as free vitamin (92 and 94%, respectively). In contrast, only a minor part of the labelled B 12 in the depleted rat kidney was free (21%), indicating that vitamin B 12 during states of vitamin surplus is stored in the kidney in a non-protein bound form. Discussion The present study establishes the rat kidney as a major vitamin B 12 -accumulating organ during conditions with sufficient vitamin supply. A )100-fold higher B 12 -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 B 12 accumulated in the kidneys of the vitamin-loaded animals. Vitamin B 12 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 B 12 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 TC B 12. 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 Fig. 2. Concentration of [ 57 Co]B 12 in tissues of rats supplied with low (depleted), normal (normal) or high (loaded) levels of vitamin B h after injection of labelled vitamin B 12. The concentration of [ 57 Co]B 12 is indicated as log fmol labelled B 12 ug tissue in (A) and as log fmol labelled B 12 ul serum and log fmol labeled B 12 ummol creatinine in serum and urine, respectively, in (B). Data are given as mean"sd. u indicates a significant difference (P-0.05).

5 The kidney in vitamin B 12 homeostasis 1099 Fig. 3. Gel filtration of rat kidney extracts. The primary y-axis displays the c.p.m. detected in fractions 1 31 (blue line), which represents protein-bound vitamin, and the secondary y-axis displays the c.p.m. identified in fractions (red line), which represents free vitamin. The filled and open arrows indicate the localization of the void volume (dextran blue) and total volume ( 22 Na), respectively. proteins, including the vitamin D-binding proteinvitamin D [13], retinol-binding protein retinol [14] and TC B 12 [4,5]. Recent evidence suggests that megalin is the major receptor protein involved in luminal uptake of TC B 12 filtered in the glomeruli, and megalin deficiency is associated with decreased renal B 12 concentration [4]. In addition to megalin, a 62 kda (normally present as a 124 kda dimer) TC B 12 -binding protein has been identified in the kidney, placenta, intestines and liver [6,7,15]. Injection of polyclonal antibodies Fig. 4. Hypothetical model of vitamin B 12 uptake and transport in rat kidneys and other organs in states of vitamin deprivation and surplus. In this hypothetical model, renal uptake of B 12 is dependent mainly on filtration of TC B 12 in plasma followed by megalinmediated, 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 B 12 deficiency and low plasma, TC B 12 renal vitamin uptake is reduced. In other tissues, regulated uptake is mediated by the ubiquitous TC B 12 receptor, which may be up-regulated during B 12 deficiency, thus explaining the increased uptake of an acutely administered dose of [ 57 Co]B 12. A higher specific activity of labelled TC B 12 in plasma in the vitamin-depleted state may increase uptake of [ 57 Co]B 12 further in tissues other than the kidney since the capacity of the ubiquitous TC B 12 receptor-mediated uptake is likely to be smaller than the high capacity, megalin-mediated uptake. 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 TC B 12 uptake. The expression of this receptor appears to be regulated [6]. The present observations support a two-receptor model for rat TC B 12 uptake which includes a high capacity, probably unregulated luminal uptake of filtered TC B 12 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 TC B 12 is dependent on filtration of TC B 12 and thus on the concentration of TC B 12 in plasma. While the 62 kda TC B 12 receptor appear to be expressed in the basolateral membranes of the kidney tubules [6], its role in renal B 12 uptake is unclear as megalin deficiency alone significantly reduces renal B 12 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. 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 TC B 12 receptor, enabling transport to the fetus even when this receptor is down-regulated.

6 1100 H. Birn et al. As suggested by the present data, the kidney is the only rat organ accumulating an acutely administered dose of B 12 in proportion to vitamin B 12 status, thus accumulating large amounts of B 12 in states of B 12 load and reserving the vitamin for other organs in states of B 12 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 [ 57 Co]B 12 paralleled the distribution of total, unlabelled B 12 suggests that the kidney is able to maintain the large accumulated amount of B 12, 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 B 12. In kidneys from the normal and loaded group, most labelled vitamin was present as free vitamin B 12, 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 B 12 turnover consistent with the presence of two different TC B 12 receptors in different tissues enabling an accumulation of B 12 in the kidney reflecting the overall vitamin B 12 status. This may indicate an important role for the kidney in B 12 homeostasis. At present, very limited data are available on the renal handling of vitamin B 12 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 B 12 administration [19,20]; however, its possible relationship to the renal handling of vitamin B 12 remains to be established. Acknowledgements. 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. 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Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells. Biochem Biophys Res Commun 1992; 182: Nielsen R, Sørensen BS, Birn H, Christensen EI, Nexø E. Transcellular transport of vitamin B(12) in LLC-PK1 renal proximal tubule cells. J Am Soc Nephrol 2001; 12: Dierkes J, Domrose U, Ambrosch A et al. Supplementation with vitamin B12 decreases homocysteine and methylmalonic acid but also serum folate in patients with end-stage renal disease. Metabolism 1999; 48: Kaplan LN, Mamer OA, Hoffer LJ. Parenteral vitamin B12 reduces hyperhomocysteinemia in end-stage renal disease. Clin Invest Med 2001; 24: 5 11 Received for publication: Accepted in revised form: