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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 19, 2003 as doi:10.1096/fj.02-0965fje. |
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,2
* Department of Biochemistry, School of Dentistry, Showa University, Shinagawa-ku, Tokyo, Japan;
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka, Japan;
Department of Orthopaedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; and
Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical School, Hidaka-shi, Saitama, Japan
2Correspondence: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
SPECIFIC AIMS
To elucidate the physiological roles of 24-hydroxylase (CYP24) in vivo, previously, we generated rats that constitutively express the CYP24 gene and showed the unexpected phenotypes such as low plasma levels of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]. The present study was designed to elucidate the mechanisms for inducing low plasma 24,25(OH)2D3 levels and bone loss in the transgenic (Tg) rats.
PRINCIPAL FINDINGS
1. Plasma 24,25(OH)2D3 and 25-hydroxyvitamin D3 [25(OH)D3] levels were decreased in Tg rats
The plasma 24,25(OH)2D3 level significantly decreased unexpectedly in Tg rats after 8 weeks of age [wild type (WT) 10.2±0.7 vs. Tg 4.7±0.5 ng/dl; P<0.05], despite constitutive expression of CYP24. No statistical differences of plasma 1
,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels were seen between WT and Tg rats. Also, plasma 25(OH)D3 decreased in Tg rats starting at 8 weeks of age (WT 18.0±0.9 vs. Tg 12.8±1.1 ng/dl; P<0.05). The expression of megalin, a multifunctional clearance receptor responsible for reuptake of vitamin D-binding protein (DBP)–25(OH)D3 complex from urine, was comparable between WT and Tg rats.
2. 25(OH)D3 and DBP are excreted into urine
Megalin has been reported to bind not only to DBP but also to other proteins including albumin; therefore, we examined urinary biochemical parameters. There was a significant excretion of protein and albumin in Tg rats soon after weaning (WT 1.3±0.1 vs. Tg 10.0±1.3 mg/mg creatinine; P<0.05) and increased remarkably with age. We assumed that excreted albumin competes with DBP by binding to megalin, and DBP–25(OH)D3 complex may be excreted. Eight-week-old Tg rats excreted a significant amount of 25(OH)D3, which increased markedly with age (Fig. 1
A). The excretion of 25(OH)D3 in Tg rats increased as albumin excretion increased, and these two parameters correlated positively (P<0.001; Fig. 1C
). The excretion of 1,25(OH)2D3, which binds to DBP as well, was apparent in Tg rats at 8 weeks of age and increased with age (Fig. 1B
). There was a strong, positive correlation between 1,25(OH)2D3 and albumin excretion (P<0.001; Fig. 1D
).
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Next, we examined the excretion of DBP by Western blot analysis. No DBP excretion was seen in WT rats, and in Tg rats, DBP was present in the urine as early as 4 weeks of age (Fig. 1G
), when urinary vitamin D metabolites were not yet detectable. An equivalent amount of albumin was detected near 67 kDa in Tg rats at each developmental stage by SDS-PAGE (Fig. 1E
), and DBP was also detected with the same intensity in Tg rats. Urinary protein from aged Tg rats (lanes 4 and 5 in Fig. 1E
) contained low molecular weight proteins smaller than albumin, and proteins smaller than albumin were barely detected in 4-week-old Tg rats, indicating that tubular function for protein reabsorption was impaired only in aged Tg rats.
3. Bone mass was reduced in Tg rats, which can be prevented by 25(OH)D3 supplementation
Next, we examined the influence of 25(OH)D3 loss on bone mineralization. There was a significant decrease in the bone mineral density (BMD) of Tg rats at 8 weeks of age, assessed by peripheral quantitative computed tomography (pQCT; Fig. 2
A). Tg rats, which were infused with 25(OH)D3 for 3 weeks right after weaning, were protected from loss of BMD when compared with the Tg rats infused with vehicle. The plasma level of 1,25(OH)2D3 did not change after 25(OH)D3 infusion to Tg rats. The osteiod tissue of trabecular bone increased in Tg rats infused with vehicle, compared with that of WT rats. The increase in osteiod was reduced to the level of the WT rats after supplementation with 25(OH)D3. The trabeculae were sparse, and the connection between them decreased in the vehicle-infused Tg rats, which were also improved with 25(OH)D3 infusion (Fig. 2B
).
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CONCLUSIONS AND SIGNIFICANCE
Megalin is a multifunctional clearance receptor that mediates the endocytosis of the DBP–25(OH)D3 complex from the lumen into the proximal tubular cells. It is reported that all DBP is filtered through the glomerulus once, even in normal animals, and reabsorbed completely in the proximal tubules, if tubular function is intact. Megalin can also bind proteins other than DBP, such as albumin. From these aspects and our results, we conclude that if an excessive amount of albumin is excreted, it competes with DBP by binding to megalin, even though albumin does not bind to megalin strongly, and DBP–25(OH)D3 is lost into urine (Fig. 3
). The decreased plasma 25(OH)D3 level is a result of decreased reuptake because of the excess albumin excretion. This marked decrease in the plasma 25(OH)D3 level leads to a decreased plasma 24,25(OH)2D3 level despite constitutive CYP24 expression.
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Most patients with renal failure are affected with bone disease at its end stages, termed "renal osteodystrophy". 1,25(OH)2D3 and its derivative, 1-hydroxyvitamin D3 [1(OH)D3], are widely used in patients with chronic renal failure to supply 1,25(OH)2D3, which is decreased by the reduction of the intact nephron that can synthesize it. However, some reports indicate that the 1,25(OH)2D3 level is normal or mildly elevated in early renal failure. Indeed, its level was maintained in Tg rats within a normal range, probably by up-regulation of CYP27B1. In these states, we supposed that 25(OH)D3 as well as 1,25(OH)2D3 has therapeutic potential to ameliorate bone disease. Supplying 25(OH)D3 to Tg rats prevented the mineralization defect, and the osteiod tissue decreased to a level similar to that of WT rats. The free form of DBP may be increased by the loss of 25(OH)D3 and may be involved in the trapping of 1,25(OH)2D3 in the circulation by binding to it, leading to the prevention of 1,25(OH)2D3 to permeate into target cells. Supplying 25(OH)D3 to Tg rats decreases the free form of DBP, leading to the correction of the intracellular deficiency of 1,25(OH)2D3. Although the effectiveness of 1,25-(OH)2D3 and 1(OH)D3 in ameliorating the skeletal lesions in a patient with less-advanced renal insufficiency has been reported, the results obtained in this study suggest that increasing the plasma 1,25(OH)2D3 level by its administration is not necessarily essential, and supplementation of excreted 25(OH)D3 is also efficacious for treating patients with an early form of renal osteodystrophy. How constitutive CYP24 expression induces nephritis must be elucidated by future studies.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0965fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0965fje 
3 Correspondence: Department of Biochemistry, School of Dentistry, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: shinki{at}dent.showa-u.ac.jp
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). There was a positive correlation between 25(OH)D3 and albumin (P<0.001; C) and 1
