Yamagata Masayo, Ozono Keiichi, Hashimoto Yuta, Miyauchi Yoshiteru, Kondou Hiroki, Michigami Toshimi
Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
J Am Soc Nephrol. 2005 Aug;16(8):2338-45. doi: 10.1681/ASN.2004070599. Epub 2005 Jun 23.
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.
巨膜蛋白是一种多功能内吞受体，在肾近端小管中表达，在肾脏摄取各种蛋白质过程中发挥关键作用。据推测，巨膜蛋白依赖性内吞作用可能在肾脏磷酸盐重吸收中起作用。为了研究巨膜蛋白功能改变的短期影响，将39-kD受体相关蛋白（RAP）可溶性形式的重组蛋白腹腔注射给7周龄小鼠。通过细菌表达制备了缺乏氨基末端信号肽和羧基末端内质网滞留信号的组氨酸（His）标记的可溶性RAP（氨基酸39至356）（命名为His-sRAP）。在证实His-sRAP与巨膜蛋白之间的直接相互作用后，给小鼠单次腹腔注射His-sRAP（3.5mg/剂量）。免疫染色和蛋白质印迹分析表明，给药后1小时，近端小管细胞摄取了His-sRAP，巨膜蛋白的内化加速。此外，还观察到II型钠/磷酸盐共转运体（NaPi-II）的内化。然后检查了连续三次注射His-sRAP（3.5mg/剂量，每隔4小时注射三剂）的效果，发现包括维生素D结合蛋白在内的低分子量蛋白质的尿排泄增加，这与巨膜蛋白缺陷小鼠的报道结果一致。有趣的是，磷酸盐的尿排泄也增加了，刷状缘膜中NaPi-II的蛋白水平降低。25-羟基维生素D的血清浓度降低，而完整甲状旁腺激素的血浆水平在注射His-sRAP后未改变。结果表明，His-sRAP诱导的巨膜蛋白介导的内吞作用加速通过改变NaPi-II的亚细胞分布导致了磷酸盐尿。
