Fujishiro Hitomi, Yamamoto Hazuki, Otera Nobuki, Oka Nanae, Jinno Mei, Himeno Seiichiro
Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
Toxics. 2020 Apr 1;8(2):24. doi: 10.3390/toxics8020024.
Cadmium (Cd) is an environmental pollutant known to cause dysfunctions of the tubular reabsorption of biomolecules in the kidney. Elevated levels of urinary excretion of low-molecular-weight proteins such as β-microglobulin (β-MG) have been used as an indicator of Cd-induced renal tubular dysfunctions. However, very few studies have examined the direct effects of Cd on the reabsorption efficiency of proteins using cultured renal cells. Here, we developed an in vitro assay system for quantifying the endocytic uptakes of fluorescent-labeled proteins by flow cytometry in S1 and S2 cells derived from mouse kidney proximal tubules. Endocytic uptakes of fluorescent-labeled albumin, transferrin, β-MG, and metallothionein into S1 cells were confirmed by fluorescence imaging and flow cytometry. The exposure of S1 and S2 cells to Cd at 1 and 3 µM for 3 days resulted in significant decreases in the uptakes of β-MG and metallothionein but not in those of albumin or transferrin. These results suggest that Cd affects the tubular reabsorption of low-molecular-weight proteins even at nonlethal concentrations. The in vitro assay system developed in this study to evaluate the endocytic uptakes of proteins may serve as a useful tool for detecting toxicants that cause renal tubular dysfunctions.
镉（Cd）是一种环境污染物，已知会导致肾脏中生物分子的肾小管重吸收功能障碍。尿中低分子量蛋白质如β-微球蛋白（β-MG）排泄水平升高已被用作镉诱导的肾小管功能障碍的指标。然而，很少有研究使用培养的肾细胞来研究镉对蛋白质重吸收效率的直接影响。在这里，我们开发了一种体外检测系统，用于通过流式细胞术定量来自小鼠肾近端小管的S1和S2细胞中荧光标记蛋白质的内吞摄取。通过荧光成像和流式细胞术证实了荧光标记的白蛋白、转铁蛋白、β-MG和金属硫蛋白在S1细胞中的内吞摄取。将S1和S2细胞暴露于1和3μM的镉中3天，导致β-MG和金属硫蛋白的摄取显著减少，但白蛋白或转铁蛋白的摄取没有减少。这些结果表明，即使在非致死浓度下，镉也会影响低分子量蛋白质的肾小管重吸收。本研究中开发的用于评估蛋白质内吞摄取的体外检测系统可能是检测导致肾小管功能障碍的有毒物质的有用工具。
