Küng Catharina J, Haykir Betül, Schnitzbauer Udo, Egli-Spichtig Daniela, Hernando Nati, Wagner Carsten A
Institute of Physiology, University of Zurich and National Center of Competence in Research Kidney.CH, Zurich, Switzerland.
Am J Physiol Renal Physiol. 2021 Dec 1;321(6):F785-F798. doi: 10.1152/ajprenal.00250.2021. Epub 2021 Nov 1.
Na-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc, located at the brush-border membrane of renal proximal tubules, are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of ERK1/2 (phospho-ERK1/2). FGF23 also downregulates expression of renal 1-α-hydroxylase () and upregulates 24-hydroxylase (), thus reducing plasma calcitriol levels. Here, we examined the time course of FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway toward degradation in vivo. Mice were injected intraperitoneally with recombinant human (rh)FGF23 in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 min after rhFGF23 administration, and increased phosphorylation was still detected 480 min after injection. Colocalization of phospho-ERK1/2 with NaPi-IIa was seen at 60 and 120 min and partly at 480 min. The abundance of both cotransporters was reduced 240 min after rhFGF23 administration, with a further reduction at 480 min. NaPi-IIa and NaPi-IIc were found to colocalize with clathrin and early endosomal antigen 1 as early as 120 min after rhFGF23 injection. Both cotransporters partially colocalized with cathepsin B and lysosomal-associated membrane protein-1, markers of lysosomes, 120 min after rhFGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 h upon rhFGF23 injection. Both cotransporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking toward the lysosome as early as 120 min after rhFGF23 administration. The hormone fibroblast growth factor 23 (FGF23) controls phosphate homeostasis by regulating renal phosphate excretion. FGF23 acts on several phosphate transporters in the kidney. Here, we define the time course of this action and demonstrate how phosphate transporters NaPi-IIa and NaPi-IIc are internalized.
位于肾近端小管刷状缘膜的钠依赖性磷酸盐共转运体NaPi-IIa和NaPi-IIc受多种因素调节，包括成纤维细胞生长因子23（FGF23）。FGF23在激活涉及ERK1/2磷酸化（磷酸化ERK1/2）的信号通路后，下调NaPi-IIa和NaPi-IIc的丰度。FGF23还下调肾1-α-羟化酶（）的表达并上调24-羟化酶（），从而降低血浆骨化三醇水平。在此，我们研究了FGF23诱导的NaPi-IIa和NaPi-IIc内化的时间进程及其在体内的细胞内降解途径。在不存在（生化分析）或存在（免疫组织化学）溶酶体蛋白酶抑制剂亮抑酶肽的情况下，给小鼠腹腔注射重组人（rh）FGF23。rhFGF23给药后60分钟，ERK1/2的磷酸化增强，注射后480分钟仍检测到磷酸化增加。在60分钟和120分钟时可见磷酸化ERK1/2与NaPi-IIa共定位，在480分钟时部分共定位。rhFGF23给药后240分钟，两种共转运体的丰度降低，480分钟时进一步降低。发现rhFGF23注射后120分钟，NaPi-IIa和NaPi-IIc最早与网格蛋白和早期内体抗原1共定位。rhFGF23注射后120分钟，两种共转运体均与溶酶体标记物组织蛋白酶B和溶酶体相关膜蛋白-1部分共定位。因此，rhFGF23注射后2小时内，NaPi-IIa和NaPi-IIc被内化。两种共转运体共享网格蛋白介导的内吞途径，该途径首先导致早期内体，最终在rhFGF23给药后120分钟最早导致向溶酶体的运输。激素成纤维细胞生长因子23（FGF23）通过调节肾磷酸盐排泄来控制磷酸盐稳态。FGF23作用于肾脏中的几种磷酸盐转运体。在此，我们确定了这种作用的时间进程，并证明了磷酸盐转运体NaPi-IIa和NaPi-IIc是如何被内化的。
