Vitamin B₁₂ deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a

Abstract

Vitamin B₁₂ deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B₁₂ deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B₁₂ deficiency suggests that vitamin B₁₂ plays an important role in Pi homeostasis.

Key words:

• bone disorder
• inorganic phosphorus
• Napi2a

Vitamin B₁₂ deficiency is associated with various symptoms such as abnormal organic acid metabolism, megaloblastic anemia, and neurological dysfunction. We previously reported that vitamin B₁₂ deficiency decreased the ratio of S-adenosyl methionine (SAM), a methyl donor for DNA methylation to S-adenosyl homocysteine (SAH) or SAM:SAH.¹⁾ Thus, vitamin B₁₂ has an important role in DNA methylation. Homocysteinemia, a known risk factor for heart, kidney, and brain diseases, is also a typical symptom of vitamin B₁₂ deficiency. Recent reports revealed that elevation of homocysteine affected an osteoblast differentiation by in vitro experiment.^2,3) However, homocysteine concentration in bone tissue is unchanged in vitamin B₁₂-deficient rats.⁴⁾ Moreover, a lower bone mineral density (BMD), another risk factor for disorders of bone, is more likely when vitamin B₁₂ concentrations are low.⁵⁾ However, the mechanisms by which a vitamin B₁₂ deficiency contributes to a lower BMD are not fully understood. In this study, we analyzed whether a vitamin B₁₂ deficiency may have an impact on bone disorders other than just homocysteine-dependent bone disorders.

We first examined the effect of a vitamin B₁₂ deficiency and supplementation on bone morphology in vitamin B₁₂-deficient and vitamin B₁₂-supplemented rats by using histomorphometric methods (OsteoMeasure, OsteoMetrics, USA). Data shown are represented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by the Tukey–Kramer test for multiple comparisons. In brief, Wistar rats were weaned from dams, fed a vitamin B₁₂-deficient diet during pregnancy, and lactation as we previously reported.¹⁾ Further, these animals were subsequently divided into three groups: control, vitamin B₁₂ deficient, and Cyano-vitamin B₁₂ (CN-B₁₂) supplemented (CN-B₁₂; 1 μg/day) for seven days before sacrifice. The bone morphology results showed that osteoclasts play an important role in bone resorption, and the osteoclast number (OcN) was a rising trend in vitamin B₁₂-deficient rats but rescued in CN-B₁₂-supplemented rats (Fig. 1(A)). On the other hand, osteoblast plays an important role in bone formation, and osteoblast numbers were not affected by vitamin B₁₂ status. (control vs. vitamin B₁₂ deficient; 4.241 ± 1.551 vs. 4.845 ± 1.270 number/mm. Values are means ± SD, control and vitamin B₁₂ deficient; n = 6, not significant.) These results indicated that the bone resorption was induced by elevation of OcN. Bone Volume Fraction (Bone Volume/Tissue Volume; BV/TV) in vitamin B₁₂-deficient rats showed a trend toward being comparable to control rats (p < 0.14), whereas CN-B₁₂-supplemented rats showed no such trend (Fig. 1(B)). Trabecular separation (TbSp) showed a trend toward increase in vitamin B₁₂-deficient rats compared with the controls (p < 0.052). CN-B₁₂-supplemented rats showed a decrease in TbSp compared to control and vitamin B₁₂-deficient rats (Fig. 1(C)). Thus, from the bone morphology results, an increasing tendency of TbSp was associated with stimulated osteoclastogenesis. Previous reports have shown that bone tissue concentration of homocysteine is no different in vitamin B₁₂-deficient rats.⁴⁾ Thus, these data suggest that a homocysteine (Hcy)-independent mechanism may play a role in bone disorders.

Fig. 1. Vitamin B₁₂ deficiency affects bone morphology.

Notes: The effect of bone morphology was measured using OsteoMeasure (A) Osteoclast Numbers/Bone Surface (N. Oc/BS), (B) Bone Volume/Tissue Volume (BV/TV), and (C) the trabecular separation shown in vitamin B₁₂ deficiency and supplementations. Values are means ± SD, control and vitamin B₁₂ deficient; n = 6, supplementation; n = 3 per group. Bars with different superscript letters indicate significant differences among groups with p < 0.05.

Inorganic phosphorus (Pi) has several important roles in the body and is a necessary component of bone, cell membranes, and nucleic acids.⁵⁾ Serum Pi concentrations are maintained by intestinal phosphorus absorption, the renal resorption system, bone, and intracellular fluid. Several hundred milligrams of Pi are absorbed daily in the intestine, and an equivalent amount is excreted in urine to maintain Pi homeostasis.⁶⁾ To analyze the effects of vitamin B₁₂ on the serum Pi and calcium (Ca) homeostasis, serum Pi and Ca concentrations were measured using enzyme assays (Mitsubishi Chemical Medicine, Tokyo, Japan). Serum Pi was significantly increased in vitamin B₁₂-deficient rats compared with controls. Supplementation with CN-B₁₂ rescued the serum concentration of Pi which was not different from that in the control group (Fig. 2(A)). In contrast, serum Ca concentrations were not different among groups (Fig. 2(B)). Parathyroid hormone (PTH) is a Pi-regulatory hormone and controls Pi metabolism. Serum PTH concentration was measured using an electro-chemiluminescence immunoassays (Mitsubishi Chemical Medicine, Tokyo, Japan). However, the concentration of PTH was no different in vitamin B₁₂-deficient rats compared with control (Fig. 2(C)). We confirmed that other Pi-regulating FGF23 hormones also remained unchanged (control vs. vitamin B₁₂ deficient; 48.44 ± 7.01 vs. 48.44 ± 4.68 μg/ml. Values are means ± SD, control and vitamin B₁₂ deficient; n = 4, not significant.). These results suggested that bone disorders occurring in the presence of a vitamin B₁₂ deficiency could be caused by an elevated concentration of serum Pi.

Fig. 2. Serum Pi levels change in vitamin B₁₂-deficient rats but not Ca and PTH levels.

Notes: Serum Pi, Ca, and PTH levels in vitamin B₁₂-deficient and supplemented rats are shown in (A), (B), and (C), respectively. Values are means ± SD. n = 4 per group. Bars with different superscript letters indicate significant differences among groups with p < 0.05.

As described above, the renal resorption of Pi plays an important role in its retention in serum. Previous reports have shown that abnormal renal resorption, including in states of chronic kidney disease, induces bone disorders.⁷⁾ Therefore, renal metabolism is tightly involved in bone metabolism. Renal uptake occurs as serum Pi is filtered by the glomerulus and thereafter resorbed in the nephron. This resorption occurs in the proximal tubule by the sodium-dependent phosphate cotransporter (Napi2a). Napi2a is a major Pi cotransporter, which is localized in the apical brush border membrane of the proximal tubule, and is implicated in more than 70% of the renal uptake of Pi.^8,9) We assumed that the accumulation of serum Pi occurred as a result of Napi2a dysfunction perturbing the renal regulation of Pi resorption. Western blot and qPCR were performed to examine whether B₁₂ concentrations affect Napi2a expression. mRNA levels were quantified using a Roche LightCycler real-time PCR machine. The following primer sequences were used: Napi2a forward 5′-TCCTCGTCAAGATGCTCAAC-3′; Napi2a reverse 5′-GAACACGGAACTGCTCTGG-3′; β-actin forward 5′-CTGACAGACTACCTCATGAAGATCC-3′; and β-actin reverse 5′-TAGCACAGCTTCTCTTTAATGTC-3′. RNA data are presented as relative mean vs. β-actin ± SD. mRNA levels of Napi2a were not observed to be different between vitamin B₁₂-deficient rats and control (Fig. 3(A)). Napi2a antibodies were generated by immunize oligo peptide (CRPEPRSPQLPPRVFLEE) against rabbit. Napi2a protein levels were determined by western blotting. Protein levels of Napi2a significantly elevated in the vitamin B₁₂-deficient rats compared with control rats. This elevation was rescued by supplementation with CN-B₁₂ (Fig. 3(B) and (C)). To examine the effect of vitamin B₁₂ on the localization of Napi2a, we performed light microscopic immunohistochemistry of Napi2a. The studies were performed as described previously.¹⁰⁾ Control rats showed a weak staining intensity of Napi2a in the renal proximal tubule (Fig. 3(D)). As indicated in Fig. 3(A) and (B), vitamin B₁₂ deficiency showed a strong staining intensity of Napi2a and supplementation with CN-B₁₂ decreased the intensity of Napi2a as well as control (Fig. 3(D)). These results suggest that the accumulation of serum Pi occurred as a result of Napi2a translocation inhibition in a B₁₂-deficient state.

Fig. 3. Napi2a translocation is inhibited during vitamin B₁₂ deficiency.

Notes: (A) Quantitative PCR analysis of Napi2a gene expression. Bar graphs show arbitrary units (means ± SD) of expression relative to β-actin expression. n = 4 per group. (B) Western blotting data of Napi2a in the kidney of vitamin B₁₂-deficient rats. (C) Quantification of intensity of (B) is shown. Bars with different superscript letters indicate significant differences among groups with p < 0.01. (D) The Napi2a immunostaining was performed using control, vitamin B₁₂ deficiency, and supplementation. (E) Schematic model of this study.

In Fig. 2, we also showed that serum concentration of Pi but not Ca accumulated according to vitamin B₁₂ status (Fig. 2(A) and (B)). An increase in serum Pi concentration induced bone disorders in vitamin B₁₂-deficient rats (Fig. 1). Other reports have also indicated that vitamin B₁₂ deficiency-induced bone disorders, such as osteopenia and osteoporosis, can increase fracture risk.^5,11) Thus, we suggest that an accumulation of excessive Pi in serum can cause bone disorders in a vitamin B₁₂ deficiency state. Interestingly, the primary Pi-regulatory hormones including PTH, FGF23, and 1–25(OH)₂D₃, as well as dietary Pi, did not show any difference in response to vitamin B₁₂ level (Fig. 2(C)). We consider that other mechanisms affect Pi dyshomeostasis during a vitamin B₁₂ deficiency.

Renal resorption of Pi via Napi2a is important for Pi homeostasis.¹²⁾ Our immunohistochemistry data showed that apical expression of Napi2a was arrested by a vitamin B₁₂ deficiency (Fig. 3). The mRNA was not affected, but protein levels were elevated (Fig. 3). These data suggest that Napi2a translocation is inhibited by a vitamin B₁₂ deficiency. On the other hand, Napi2c is also known to exist on the renal plasma membrane since Napi2a has a similar topological structure to Napi2a. We hypothesize that Napi2c also accumulates on the plasma membrane in vitamin B₁₂-deficient rats.¹³⁾ Translocation promotes interaction with endocytic receptors such as megalin that enables cellular internalization of nutrients.^14,15) Indeed, our previous studies indicated that intracellular trafficking of megalin was inhibited in the kidney by vitamin B₁₂ deficiency.¹⁰⁾ It seems that trafficking of Napi2a was also arrested in the cell membrane with megalin. We found a novel pathway by which the inhibition of Napi2a translocation induced bone disorders in a state of vitamin B₁₂ deficiency (Fig. 3(E)). Further studies are needed to examine Napi2a translocation mechanism. Here, we presented evidence of the role of vitamin B₁₂ as a Pi-regulatory nutrient.

Authors’ contributions

Takahiro Shiga planned the experiments and wrote the draft of the article. Yoshifumi Kimira and Hiroshi Mano analyzed the data. Tetsunori Kawata contributed material tools. Tadahiro Tadokoro, Tsukasa Suzuki, and Yuji Yamamoto designed the research and approved the final version of the article.

Disclosure statement

No potential conflict of interest was reported by the authors.