Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter

J Am Soc Nephrol. 2005 Aug;16(8):2338-45. doi: 10.1681/ASN.2004070599. Epub 2005 Jun 23.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Endocytosis
  • Endoplasmic Reticulum / metabolism
  • Histidine / chemistry
  • Infusions, Parenteral / methods
  • Kidney / metabolism*
  • Kidney Tubules / metabolism
  • LDL-Receptor Related Protein-Associated Protein / physiology*
  • Ligands
  • Low Density Lipoprotein Receptor-Related Protein-2 / metabolism
  • Low Density Lipoprotein Receptor-Related Protein-2 / physiology*
  • Male
  • Mice
  • Mice, Inbred ICR
  • Microscopy, Fluorescence
  • Microvilli / metabolism
  • Parathyroid Hormone / metabolism
  • Phosphates / chemistry
  • Phosphates / urine*
  • Protein Binding
  • Protein Sorting Signals
  • Protein Structure, Tertiary
  • Recombinant Proteins / administration & dosage*
  • Recombinant Proteins / chemistry
  • Sodium-Phosphate Cotransporter Proteins / metabolism*
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / physiology*
  • Symporters / metabolism*
  • Time Factors
  • Vitamin D-Binding Protein / chemistry

Substances

  • LDL-Receptor Related Protein-Associated Protein
  • Ligands
  • Low Density Lipoprotein Receptor-Related Protein-2
  • Parathyroid Hormone
  • Phosphates
  • Protein Sorting Signals
  • Recombinant Proteins
  • Slc34a1 protein, mouse
  • Sodium-Phosphate Cotransporter Proteins
  • Sodium-Phosphate Cotransporter Proteins, Type IIa
  • Symporters
  • Vitamin D-Binding Protein
  • Histidine