Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter

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.

Retention of fetuin-A in renal tubular lumen protects the kidney from nephrocalcinosis in rats

“…Because the molecular weight of fetuin-A is smaller than that of albumin, it seems possible that fetuin-A passes through the slit diaphragm (11). Therefore, we examined whether fetuin-A was reabsorbed by the proximal tubular cells in a megalin-dependent manner (14,23). Here, we inhibited the function of megalin using HissRAP (23).…”

“…Therefore, we examined whether fetuin-A was reabsorbed by the proximal tubular cells in a megalin-dependent manner (14,23). Here, we inhibited the function of megalin using HissRAP (23). His-sRAP is a recombinant protein that encodes murine RAP corresponding to the amino acids 39 to 356, lacking the amino-terminal signal peptide and the carboxyterminal endoplasmic reticulum retention signal (23).…”

“…3-5 and 7) and intraperitoneal injection of leupeptin at a dose of 6 mg/100 g BW (Figs. 5 and 6), respectively (23). To assess the distribution of exogenous fetuin-A, purified rat fetuin-A (0.1 mg/ 10 g BW) was injected intravenously into mice (Fig.…”

“…A plasmid pET15b-sRAP encodes murine RAP corresponding to the amino acids 39 to 356 was kindly provided by Dr. T. Michigami, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan (23). His-sRAP protein was expressed in Escherichia coli BL21 (DE3) and was purified by Chelating Sepharose Fast Flow (GE Healthcare, Waukesha, WI).…”

“…The eluted His-sRAP was dialyzed against PBS and was concentrated by Amicon Ultra-15 (Millipore, Billerica, MA). HissRAP directly binds to megalin and induces acute impairment of megalin function (23).…”