Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter.
Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.
Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
Masayo
M
Yamagata
Keiichi
K
Ozono
Yuta
Y
Hashimoto
Yoshiteru
Y
Miyauchi
Hiroki
H
Kondou
Toshimi
T
Michigami
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007263:Infusions, Parenteral / Q000379:methods; D007668:Kidney / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism; D026563:LDL-Receptor Related Protein-Associated Protein / Q000502:physiology*; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism / Q000502:physiology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D008871:Microvilli / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000652:urine*; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000737:chemistry; D050608:Sodium-Phosphate Cotransporter Proteins / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000502:physiology*; D027981:Symporters / Q000378:metabolism*; D013997:Time Factors; D014809:Vitamin D-Binding Protein / Q000737:chemistry
D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D010281:Parathyroid Hormone; D010710:Phosphates; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C493555:Slc34a1 protein, mouse; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; D014809:Vitamin D-Binding Protein; D006639:Histidine