Marunaka Rie, Taruno Akiyuki, Yamamoto Toshiro, Kanamura Narisato, Marunaka Yoshinori
Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan.
Department of Dental Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan.
Cell Physiol Biochem. 2018;50(1):363-377. doi: 10.1159/000494012. Epub 2018 Oct 11.
BACKGROUND/AIMS: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking.
Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs.
We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition.
The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.
背景/目的：上皮钠通道（ENaC）通过决定肾脏钠重吸收总量在血压调控中发挥关键作用，而肾脏钠重吸收总量受醛固酮、血管加压素、胰岛素和渗透压等多种因素调节。ENaC的细胞内转运过程主要通过决定上皮细胞顶端膜上ENaC的表达数量来调节肾脏集合管中ENaC介导的钠重吸收量。尽管我们之前报道蛋白酪氨酸激酶（PTK）有助于ENaC介导的上皮钠重吸收，但我们对PTK在ENaC细胞内转运中的作用尚无相关信息。
利用我们实验室最近建立的数学模型，我们研究了PTK抑制剂（PTKI）AG1296（10 μM：血小板衍生生长因子受体（PDGFR）抑制剂）和AG1478（10 μM：表皮生长因子受体（EGFR）抑制剂）对内源性表达ENaC的肾上皮A6细胞中ENaC细胞内转运速率的影响。
我们发现应用PTKI可使ENaC插入顶端膜的速率显著降低56%，ENaC的循环利用速率降低83%，单个ENaC停留在顶端膜的累积时间降低27%，ENaC首次插入后的整个寿命期降低47%，累积钠吸收降低61%，而应用PTKI后降解速率增加至3.8倍。这些观察结果表明，在低渗条件下的细胞内转运过程中，PTK有助于ENaC的插入、循环利用和降解过程。
本研究表明，在低渗条件下，应用可抑制EGFR和PDGFR的PTKI可降低肾A6上皮细胞中ENaC的插入速率（kI）和循环利用速率（kR），但增加降解速率（kD）。这些观察结果表明，低渗通过增加ENaC的插入速率（kI）和循环利用速率（kR）以及降低降解速率来增加ENaC的表面表达，而在EGFR和PDGFR相关PTK介导的途径中对内吞速率（kE）无显著影响。
