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Am J Physiol Renal Physiol 295: F82-F90, 2008. First published April 30, 2008; doi:10.1152/ajprenal.00091.2008
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Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells

Min Li, Saravanan Balamuthusamy, Eric E. Simon, and Vecihi Batuman

Section of Nephrology and Hypertension, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana

Submitted 21 February 2008 ; accepted in final form 26 April 2008

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.

myeloma kidney; RNA interference; nephrotoxicity; flow cytometry; receptor-mediated endocytosis


Address for reprint requests and other correspondence: V. Batuman, Section of Nephrology and Hypertension, Dept. of Medicine, Tulane Univ. School of Medicine, 1430 Tulane Ave., SL-45, New Orleans, LA 70112-2632






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