Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter
Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter
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DOI：
10.1681/asn.2004070599
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发表时间：
2005-08-01
影响因子：
13.6
通讯作者：
Michigami, T
中科院分区：
文献类型：
--
作者：
Yamagata, M;Ozono, K;Michigami, T
巨蛋白是一种多功能内吞受体，在肾近曲小管中表达，在肾脏摄取各种蛋白质中发挥关键作用。据推测，巨蛋白依赖性内吞作用可能在肾磷酸盐重吸收中发挥作用。为了解决巨蛋白功能改变的短期影响，将可溶形式的 39-kD 受体相关蛋白 (RAP) 的重组蛋白腹腔内给予 7 周龄的小鼠。通过细菌表达制备缺乏氨基末端信号肽和羧基末端内质网保留信号的组氨酸（His）标记的可溶性RAP（氨基酸39至356）（称为His-sRAP）。在确认His-sRAP和巨蛋白之间的直接相互作用后，对小鼠进行单次腹膜内施用His-sRAP（3.5毫克/剂）。免疫染色和蛋白质印迹分析表明，给药后 1 小时，近端肾小管细胞中 His-sRAP 的摄取和巨蛋白的加速内化。此外，还观察到 II 型钠/磷酸盐协同转运蛋白 (NaPi-II) 的内化。然后检查了三次连续施用 His-sRAP（3.5 毫克/剂，三剂间隔 4 小时）的效果，发现低分子量蛋白质（包括维生素 D 结合蛋白）的尿排泄增加，这与巨蛋白缺陷小鼠的研究结果一致。有趣的是，尿液中磷酸盐的排泄也增加了，刷状缘膜中NaPi-II的蛋白水平降低了。给予His-sRAP后，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没有改变。结果表明，His-sRAP 诱导的巨蛋白介导的内吞作用加速，通过改变 NaPi-II 的亚细胞分布而引起磷酸尿。
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 intraperitorteally 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 intraperitorteal administration of His-sRAP (3.5 mg/dose). Immuno-staining 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.