Endocytosis of the somatostatin analogue, octreotide, by the proximal tubule-derived opossum kidney (OK) cell line

Background. Nephrotoxicity of cancer therapy using radiotabeled somatostatin analogues such as octreotide is due to ultrafiltration and reuptake by proximal tubular cells (PTCs). The mechanism of uptake is unknown. It could occur either by receptor-mediated endocytosis via a somatostatin receptor or, alternatively, the multiligand megalin/cubilin tandem receptor, or by fluid-phase endocytosis. To define the mechanisms of internalization and to identify potential receptors, we have studied the uptake and processing of octreotide by the PTC-derived opossum kidney (OK) cell line. Methods. We compared the kinetics of uptake and fate of In-111-diethylenetriamine pentaacetic acid (DTPA)-D-Phe(1)-octreotide and I-125-human serum albumin (I-125-HSA). To determine the contribution of receptor-mediated endocytosis, we tested competition for uptake by octreotide and somatostatin and by various megalin/cubilin ligands [receptor-associated protein (RAP), albumin, transferrin, insulin, polymixin B] or basic amino acids. Tie subcellular localization of fluorescein, isothiocyanate (FIT C)-D-Phe(1)-octreotide was studied by confocal microscopy. Results. Kinetics of uptake of In-111-DTPA-D-Phe(1)-octreotide and I-125-HSA by OK cells were comparable, but only the somatostatin analogue was significantly retained intact. All megalin/cubilin ligands and basic amino acids strongly inhibited 'I-HSA uptake, but these could not compete for >50% of In-111-DTPA-D-Phe(1)-octreotide uptake. The same was found for somatostatin and octreotide. The noncompetable uptake of In-111-DTPA- D-Phe(1)-octreotide was comparable to the clearance of Lucifer Yellow, a marker of fluid-phase endocytosis. By confocal microscopy, FITC-D-Phe(1)-octreotide colocalized with transferrin in endosomes, then accumulated in lysosomes. Conclusion. Receptor-mediated endocytosis via megalin/cubilin and fluid-phase endocytosis contribute about equally to the uptake of radiolabeled somatostatin analogues by OK cells.