Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.
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.
Li
Min
M
Section of Nephrology and Hypertension, Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112-2632, USA.
Balamuthusamy
Saravanan
S
Simon
Eric E
EE
Batuman
Vecihi
V
D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis; D004705:Endocytosis / Q000187:drug effects*; D004847:Epithelial Cells / Q000473:pathology; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism* / Q000633:toxicity*; D015850:Interleukin-6 / Q000096:biosynthesis; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics*; D009101:Multiple Myeloma / Q000378:metabolism*; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000235:genetics*; D014162:Transfection
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D007145:Immunoglobulin kappa-Chains; D015850:Interleukin-6; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011956:Receptors, Cell Surface; C029486:intrinsic factor-cobalamin receptor