Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutical Sciences

First Advisor

Mary Kennedy

Second Advisor

Phillip Gerk

Abstract

Background: Intra-amniotic infections (IAIs) are common complications of labor and delivery. If inadequately treated, these infections can lead to significant morbidity and mortality in the mother and the fetus. Intrapartum aminoglycoside (AG) administration is recommended for the management of IAIs. AGs are known to cross the placenta and achieve bactericidal concentrations in fetal serum. However, the highest and most persistent fetal levels are achieved in renal tissue. So, the fetus may be vulnerable to the nephrotoxic effects of AGs. Megalin, a 600 kDaendocytic receptor, is responsible for the uptake of AGs into renal proximal tubular epithelial cells. This receptor is also expressed in human term placenta and it is reasonable to speculate that it is similarly involved in the placental transport of AGs. However, the mechanisms responsible for placental AG uptake and transport have not yet been characterized. Objective: To evaluate the role of megalin in the transport of AGs across human placenta. Specific aims: (1) To assess and compare megalin expression in term and preterm placental villous tissue, and (2) assess the functional activity of megalin in in vitro placental models. Methods: (1) Following IRB approval, placental tissue samples were collected from pregnant women undergoing term or preterm deliveries. Placental villous tissueswere used to quantify megalin expression by western blotting and q-PCR (2) The human choriocarcinoma cell line (BeWo cells) were grown on Transwell plates, and then megalin expression and function were assessed. Results: Megalin protein and mRNA expression were confirmed in samples of human placental villous tissues. Megalin mRNA expression declined steeply with gestational age till week 31 of gestation then it plateaued thereafter. Also, the expression in the early preterm (n=2) was six fold higher than that of both late preterm (n=3) and term placenta (n=10) (p<0.05). The uptake of 3H-gentamicin by the BeWo cells was time-dependent, saturable (Vmax=42.9 ± 4.9 nmol/mg protein/min; Km=2.93±0.68mM) and partially inhibited by megalin inhibitors. Conclusion: Megalin is expressed in human placental villous tissues as well as the BeWo cells. When grown on Transwell® plates, the BeWo cells appear to be the most appropriate model to study the in vitro transport of AGs across the apical membrane. Time, temperature and concentration dependence of gentamicin uptake in the BeWo cells indicate protein-mediated transport. The inhibition data are consistent with megalin-mediated endocytosis of AGs.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2012

Available for download on Thursday, December 14, 2017

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