The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta

Abstract

Abstract. Aminoglycosides (AG) are known to readily cross the placenta, although the mechanisms responsible for placental transport have not been characterized. Megalin is expressed in human placenta, and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in placental transport. However, the role of megalin in placental AG uptake has not been established. An in vitro model to study megalin-mediated placental transport has also not been previously described. The objectives of this study, therefore, were to evaluate the human choriocarcinoma (BeWo) cell line as a model to study megalin-mediated placental transport and to assess the uptake kinetics of gentamicin, an AG antibiotic, using this in vitro model. BeWo cells were grown on Transwell® plates, and megalin expression and functional activity were assessed. Uptake of 3 H-gentamicin was also evaluated in the presence and absence of megalin inhibitors. Expression of megalin protein and mRNA in BeWo cells were confirmed via immunoblot and qPCR analysis. Uptake of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) (a megalin substrate) was time-, concentration-, and temperature-dependent consistent with a transporter-mediated process. FITC-BSA uptake was also significantly reduced in the presence of unlabeled gentamicin (a megalin substrate) and sodium maleate (to induce megalin shedding) suggesting that megalin is functionally active in BeWo cells. Gentamicin uptake exhibited time and temperature dependence, saturability and Michaelis-Menten kinetics, all of which suggest a transporter-mediated process. Gentamicin uptake was also significantly reduced in the presence of the megalin inhibitors RAP and EDTA suggesting that megalin is likely involved in gentamicin uptake.

Uteroplacental insufficiency reduces rat plasma leptin concentrations and alters placental leptin transporters: ameliorated with enhanced milk intake and nutrition

“…2C; P = 0.039, Student's t test). The molecular weight of megalin we observed is similar to those reported in several studies (Lebeau et al 2001;Zou et al 2004;Briffa et al 2015a), including in placental BeWo cells (Akour et al 2015).…”

Cytotoxicity of Endocytosis and Efflux Inhibitors in the BeWo Cell Line

“…To elucidate the absorption mechanism, different endocytosis inhibitors were used. For our experiments, we have used amiloride as an inhibitor of micropinocytosis [ 7 ], chlorpromazine as an inhibitor of clathrin-mediated endocytosis [ 8 ], colchicine as an inhibitor of micropinocytosis [ 4 ], filipin as a lipid raft inhibitor [ 9 ], gentamicin as an inhibitor of megalin [ 10 ], methyl-β-cyclodextrin as a lipid raft inhibitor [ 9 ], as well as GF120918 [ 11 ] and verapamil [ 12 ] as efflux inhibitors.…”

Scavenger Receptor Class B Member 1 Independent Uptake of Transthyretin by Cultured Hepatocytes Is Regulated by High Density Lipoprotein

“…Megalin is a membrane receptor that has been shown to be involved in TTR uptake by other tissues such as kidney [4] and astrocytes [9]. Megalin is not expressed by liver and specifically not in HepG2 [21] cells. Aside from SR-B1 and Megalin, no other TTR receptors have been identified to date.…”