Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells
Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells
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DOI：
10.1152/ajprenal.00091.2008
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发表时间：
2008-07-01
影响因子：
4.2
通讯作者：
Batuman, Vecihi
中科院分区：
文献类型：
--
作者：
Li, Min;Balamuthusamy, Saravanan;Batuman, Vecihi
使用靶向特异性短干扰（si）RNA，我们沉默了串联内吞受体megalin和cubilin基因在培养的人肾近端小管上皮细胞。通过siRNA转染导致高达90%的megalin和cubilin蛋白和mRNA表达的抑制。在HK-2细胞暴露于κ-轻链长达24小时，轻链内吞减少无论是巨蛋白或cubilin沉默细胞显着，但不完全。然而，cubilin和megalin基因的同时沉默导致轻链内吞作用的几乎完全抑制，如通过测量细胞质中的κ-轻链蛋白浓度和通过使用FITC标记的κ-轻链的流式细胞术所确定的。在这些细胞中，轻链诱导的细胞因子反应（白细胞介素-6和单核细胞趋化蛋白-1）和上皮间质转化以及相关的细胞和形态学改变也显着抑制。结果表明，轻链内吞作用主要由巨蛋白-cubilin串联内吞受体介导，并将内吞作用确定为轻链细胞毒性的关键步骤。阻断轻链内吞作用可防止其对人肾近曲小管细胞的肾毒性作用。
Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin-or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.