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Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect¹

JÖRG R. LEHESTE^*, FLEMMING MELSEN^**, MAREN WELLNER, PERNILLE JANSEN, UWE SCHLICHTING^|, INGRID RENNER-MÜLLER^$, TROELS T. ANDREASSEN, ECKEHARD WOLF^$, SEBASTIAN BACHMANN^|, ANDERS NYKJAER and THOMAS E. WILLNOW^*,¶2

^* Max-Delbrueck-Center for Molecular Medicine and
^¶ Medical Faculty of the Free University of Berlin,
^| Institute for Anatomy and
Franz-Volhard-Clinic, Humboldt University of Berlin, Berlin;
^$ Gene Center, Ludwig-Maximilians-University, Munich, Germany; and Departments of
Medical Biochemistry and
Connective Tissue Biology, University of Aarhus; and
^** Department of Pathology, Aarhus University Hospital, Aarhus, Denmark

²Correspondence: Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Strasse 10, D-13125 Berlin, Germany. E-mail: willnow{at}mdc-berlin.de

SPECIFIC AIMS

Megalin is an endocytic receptor in proximal tubular cells (PTC) that mediates cellular uptake of 25-OH vitamin D₃ complexed with the vitamin D binding protein (DBP). The receptor is responsible for the renal uptake and activation of 25-OH vitamin D₃ to 1,25-(OH)₂ vitamin D₃, a central step in vitamin D metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded in-depth analysis of the significance of this receptor pathway for calcium homeostasis and bone turnover in vivo. We have generated a new mouse model with kidney-specific megalin gene defect with which to test the contribution of this receptor to renal physiology and bone metabolism.

PRINCIPAL FINDINGS

1. Generation of a mouse model with kidney-specific megalin gene defect
We introduced lox P recombination sites into the murine megalin gene and generated mice carrying the modified receptor gene through their germ line. Animals homozygous for the floxed megalin gene (megalin^lox/lox) exhibited normal development and unimpaired viability. In parallel, we established a mouse model with renal expression of Cre recombinase (Cre) using a fragment of the human apolipoprotein (apo) E promoter to drive the Cre transgene (apoE^Cre). We produced mice doubly transgenic for the floxed megalin gene and the Cre gene (megalin^lox/lox; apoE^Cre) by breeding of the individual lines. Cre-mediated inactivation of the megalin gene in (megalin^lox/lox; apoE^Cre) mice resulted in a 90% reduction in renal megalin expression as shown by Western blot and immunohistology. No decrease in megalin levels was observed in other tissues expressing the receptor.

2. Urinary excretion of DBP in (megalin^lox/lox; apoE^Cre) mice
Consistent with the loss of renal megalin expression, kidneys from (megalin^lox/lox; apoE^Cre) mice were unable to retrieve plasma proteins from the glomerular filtrate and excreted increased amounts of low molecular weight proteins into the urine (Fig. 1 A, lanes 4 and 5). Excreted proteins included ligands for the receptor such as DBP (Fig. 1B ). In contrast, urine samples from megalin^lox/lox (lanes 6 and 7) and apoE^Cre animals (lane 3) were indistinguishable from wild-type samples (lane 1).

View larger version (82K): [in this window] [in a new window]   Figure 1. Urinary excretion of DBP in (megalinlox/lox; apoECre) mice. 15 µL of urine from mice of the indicated genotypes was subjected to SDS-PAGE and staining with Coomassie (A) or immunochemical detection of DBP (B). The position of migration of marker proteins (in kDa) is shown. Arrowheads indicate the protein band corresponding to DBP. Excretion of DBP can be observed in (megalinlox/lox; apoECre) mice (lanes 4 and 5) but not in megalinlox/lox (lanes 6 and 7) or apoECre animals (lane 3). Urine samples from wild-type (lane 1) and megalin knockout mice (lane 2) were included as controls.

3. Impaired vitamin D metabolism in (megalin^lox/lox; apoE^Cre) mice
When ¹²⁵I-DBP was injected intravenously into megalin^lox/lox and (megalin^lox/lox; apoE^Cre) animals, no difference in the plasma turnover of the carrier was observed. However, (megalin^lox/lox; apoE^Cre) animals exhibited a fivefold increase in the urinary excretion of DBP vs. controls. A similar finding was obtained when the turnover of ³H-25-OH vitamin D₃/DBP complexes was analyzed. Approximately sixfold higher levels of the labeled vitamin were found in the urine of the receptor-deficient mice. These findings demonstrated that megalin does not affect plasma turnover of DBP but is essential to retrieve the fraction of 25-OH vitamin D₃/DBP complexes that have filtered through the glomerulus. Consistent with this hypothesis, (megalin^lox/lox; apoE^Cre) mice excreted significant amounts of endogenous 25-OH vitamin D₃ into their urine (10.9 nM/mM creatinine). Urinary loss of the metabolitecoincided with a 50% reduction in the plasma levels of 25-OH vitamin D₃ and 1,25-(OH)₂ vitamin D₃. On a vitamin D-enriched diet, serum calcium levels were not significantly altered; on a vitamin D-deplete diet, however, the mice exhibited hypocalcemia.

4. (Megalin^lox/lox; apoE^Cre) mice suffer from true severe osteomalacia
Finally, we analyzed the consequences of renal megalin deficiency on bone metabolism. Some alterations in vertebral bodies were detected when megalin^lox/lox and (megalin^lox/lox; apoE^Cre) mice on a normal diet were compared (Fig. 2 A, C, E). In (megalin^lox/lox; apoE^Cre) mice, the bones were characterized by an increase in resorptive cavities, indicating a state of high bone turnover. On a vitamin D-deficient chow, vertebrates from (megalin^lox/lox; apoE^Cre) mice revealed a highly irregular and unmineralized bone surface that was largely covered by osteoids (Fig. 2D, F ). No such changes were seen in megalin^lox/lox animals in response to vitamin D depletion (Fig. 2B ). Using point counting, we quantified static and dynamic bone parameters in these mice. No differences in bone parameters between megalin^lox/lox and (megalin^lox/lox; apoE^Cre) on a normal diet were detected. On a vitamin D-deplete diet, in contrast, total bone mineral content in femur and tibia of (megalin^lox/lox; apoE^Cre) animals was drastically reduced (29.5 mg) compared with the controls (50.4 mg). The animals exhibited a significant increase in osteoid surface, osteoid width, and osteoid volume and a concomitant decrease in the mineralizing bone surfaces. Such features are indicative of osteomalacia as a consequence of dietary vitamin depletion.

View larger version (145K): [in this window] [in a new window]   Figure 2. Bone structure of megalinlox/lox and (megalinlox/lox; apoECre) mice. Undecalcified Goldner stained sections were produced from the lumbar vertebrates of megalinlox/lox (A, B) and (megalinlox/lox; apoECre) animals (C, D) on a normal or a vitamin D-deplete diet. E, F) Higher magnifications of sections from (megalinlox/lox; apoECre) mice on a normal (E) or a vitamin D-deplete chow (F). Arrows highlight osteoid surfaces on vertebral bodies of (megalinlox/lox; apoECre) mice (magnification: A—D) x100; E, F) x200).

CONCLUSION

Renal uptake and activation of 25-OH vitamin D₃ is a central regulatory step in vitamin D and bone metabolism, but the exact route that delivers the vitamin to PTC, the site of conversion, is controversial. We earlier demonstrated that the endocytic receptor megalin mediates cellular uptake of 25-OH vitamin D₃ bound to its carrier DBP, suggesting a crucial role of this receptor pathway in vitamin D conversion. Unfortunately, the perinatal lethality of the conventional megalin gene knockout precluded us from testing this hypothesis in detail. We have now generated a new mouse model with renal-specific megalin gene defect using Cre recombinase. Animals with kidney-specific megalin deficiency were viable and fertile. Yet a lack of receptor expression in the kidney resulted in severe plasma vitamin D deficiency, hypocalcemia, and osteomalacia as a consequence of hypovitaminosis D. Remarkably, the same phenotype is seen in mice lacking DBP, establishing the functional relationship of both proteins in vitamin D homeostasis. In conclusion, our data provide crucial in vivo evidence that delivery of 25-OH vitamin D₃ to the kidney involves glomerular filtration of 25-OH vitamin D₃/DBP complexes, followed by megalin-mediated retrieval from the lumen of the proximal tubules (Fig. 3 ). 25-OH Vitamin D₃ metabolites internalized via this pathway are converted into 1,25-(OH)₂ vitamin D₃ and resecreted into the circulation where they serve a crucial role in regulation of the systemic calcium and bone metabolism.

View larger version (17K): [in this window] [in a new window]   Figure 3. Model of megalin function in renal uptake and activation of 25-(OH) vitamin D3. Complexes of 25-(OH) vitamin D3 and DBP are filtered through the glomerulus and scavenged from the tubular lumen into PTC via megalin. The vitamin/carrier complexes are delivered to lysosomes, where DBP is degraded and the vitamin is released into the cytosol. 25-(OH) vitamin D3 molecules are hydroxylated to 1,25 (OH)2 vitamin D3 in mitochondria prior to resecretion into the interstitial fluid.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0578fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0578fje

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