Gadolinium modulates gentamicin uptake via an endocytosis-independent pathway in HK-2 human renal proximal tubular cell line

Abstract

The aim of this study was to characterize the uptake mechanism of gentamicin, an aminoglycoside antibiotic, in human renal proximal tubular cell line HK-2. Sodium-dependent uptake of d-[3H]glucose and l-[3H]alanine was observed in HK-2 cells, indicating that the cells employed in this study retain functional characteristics of the renal proximal tubular cells. On the other hand, mRNA and protein expression of megalin, an endocytic receptor which is responsible for the internalization of gentamicin into the renal proximal tubular cells, was very faint in HK-2 cells. Various aminoglycoside antibiotics including amikacin and kanamycin inhibited the uptake of [3H]gentamicin. Colchicine and cytochalasin D, general endocytosis inhibitors, had no significant effect on [3H]gentamicin uptake in HK-2 cells, which was in contrast to the results observed in OK cells, a renal proximal tubular cell line expressing megalin. Furthermore, unlike OK cells, [3H]gentamicin uptake in HK-2 cells was not inhibited by N-WASP181-200, a cationic 20-amino acid peptide. Ruthenium red, a nonspecific cation channel blocker, decreased the uptake of [3H]gentamicin in HK-2 cells. In contrast, the trivalent cation gadolinium biphasically modulated [3H]gentamicin uptake with a maximum increase at 0.3mM gadolinium. The enhanced effect of gadolinium on [3H]gentamicin uptake was independent of gadolinium-induced increase in intracellular calcium concentration. These findings indicate that gentamicin is primarily taken up via an endocytosis-independent pathway in HK-2 cells with very low expression of megalin, and that the uptake of gentamicin is modulated by gadolinium.