Megalin-Dependent Internalization of Cadmium-Metallothionein and Cytotoxicity in Cultured Renal Proximal Tubule Cells
Megalin-Dependent Internalization of Cadmium-Metallothionein and Cytotoxicity in Cultured Renal Proximal Tubule Cells
复制标题
DOI：
10.1124/jpet.106.102574
复制
发表时间：
2006-08
影响因子：
3.5
通讯作者：
N. Wolff;Marouan Abouhamed;P. Verroust;F. Thévenod
中科院分区：
文献类型：
--
作者：
N. Wolff;Marouan Abouhamed;P. Verroust;F. Thévenod
慢性镉(Cd~(2+))暴露导致肾小管上皮细胞损伤。Cd~(2+)主要以金属硫蛋白-1(分子质量∼7 kDa)的络合物形式进入肾脏，在肾小球自由过滤。对于CD2+进入近端小管细胞，这些复合体被认为是通过与小蛋白配体(如megalin和cubilin)的受体内化，然后从内小体/溶酶体隔间的金属硫蛋白-1释放CD2+。为了探讨megalin在肾脏镉-金属硫蛋白-1重吸收中的作用，在肾近端小管细胞模型(WKPT-0293Cl.2细胞)上研究了megalin的表达及其对镉-金属硫蛋白-1内化和细胞毒性的依赖性。用逆转录聚合酶链式反应检测细胞表面(活体染色)和细胞内(通透性细胞)的megalin表达。Alexa Fluor488偶联金属硫蛋白-1的内化是浓度依赖的，在约15μM时饱和，在14.3μM时，受体相关蛋白(RAP)的1μM可显著抑制金属硫蛋白-1摄取30.9±6.6%(n=4)。加入1μM RAP、4 0 0μg/ml抗巨球蛋白抗体或5μM的Cubilin特异性配体apo-转铁蛋白后，7.14μM镉-金属硫蛋白-1作用24 h的细胞毒性分别降低41.0±7.6%、61.6±3.4%和26.2±1.8%(n=4~5)。近端肾小管上皮细胞中Cubilin的表达也在mRNA和蛋白水平得到证实。这些数据表明，肾近端小管镉-金属硫蛋白-1摄取和细胞死亡至少部分是由megalin介导的。
Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass ∼ 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 μM. At 14.3 μM, metallothionein-1 uptake could be significantly attenuated by 30.9 ± 6.6% (n = 4) by 1 μM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 μM cadmium-metallothionein-1 was significantly reduced by 41.0 ± 7.6%, 61.6 ± 3.4%, and 26.2 ± 1.8% (n = 4-5 each) by the presence of 1 μM RAP, 400 μg/ml anti-megalin antibody, or 5 μM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.