Functional analysis of megalin, a key molecule of reabsorption in renal tubular cells ; making and using RAP-transgenic mice
| Project/Area Number |
13671134
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | Osaka University (2002)
Research Institute, Osaka Medical Center for Maternal and Child Health (2001) |
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Principal Investigator |
OZONO Keiichi Osaka University Graduate School of Medicine, Department of Pediatrics, Professor, 医学系研究科, 教授 (20270770)
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| Co-Investigator(Kenkyū-buntansha) |
MICHIGAMI Toshimi Osaka Medical Center and Research Institute, Department of Environmental Medicine, Section Chief, 環境影響部門, 主任研究員 (00301804)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | megalin / receptor-associated protein / vitamin D / GST-fusion protein / transgenic mouse / receptor-associated protein(RAP) |
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| Research Abstract |
Megalin is a multi-ligand endocytic receptor present in renal proximal tubules and involved in the uptake of proteins. Although it has been reported that megalin-knockout mice exhibit impaired renal uptake of 25-hydroxyvitamin D, the function of megalin is not fully understood, partially because most of the knockout mice die perinatally from holoprosencephaly. In this project, to elucidate the function of megalin in kidney, we administered soluble form of recombinant receptor associated protein (RAP) to mice after making a soluble recombinant RAP protein. RAP is a 39-kD protein binding to members of the LDL receptor family including megalin, and inhibits the binding of all other ligands to these receptors. Ligand blot analysis revealed the interaction between soluble RAP (sRAP) expressed in E. Coli and megalin. Male ICR mice were given I.p. administration of sRAP, and urine samples were collected. Urinary excretion of low-molecular-weight proteins was increased by administration of sRAP. Western blot analysis using antibody against vitamin D binding protein (DBP) confirmed the increased urinary loss of DBP by sRAP administration. Immunostaining using anti-His antibody demonstrated the apical localization of sRAP in the proximal tubules, suggesting that systemically administered sRAP targeted the proximal tubules and was internalized after binding to megalin. These results indicate that administration of recombinant sRAP might provide a useful in vivo model to investigate the roles of renal uptake of proteins in mineral metabolism. However, our vector for the expression of RAP was integrated into germ line but was not expressed in mice in vivo. Thus, we are changing the construct of RAP expression vector and will make transgenic mice for RAP using the new vector.
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Report
(3 results)
Research Products
(4 results)
