Abstract: PUB455
Characterization of Myoglobin Endocytic Function in Primary Human Proximal Tubular Epithelial Cells
Session Information
Category: Pathology and Lab Medicine
- 1800 Pathology and Lab Medicine
Authors
- Funahashi, Yoshio, Oregon Health & Science University, Portland, United States
- Hutchens, Michael, Oregon Health & Science University, Portland, Oregon, United States
Background
The endocytic function of proximal tubular epithelial cells (PTEC) has important role in health and disease. Megalin is a multi-ligand endocytic receptor which mediates myoglobin uptake. We generated primary culture of PTEC from human kidneys (hPTEC), and characterized response to injury and myoglobin endocytic function.
Methods
Research-designated human donor kidneys were used to generate hPTEC. After dissection and digestion, hPTEC was isolated using cell strainers and fluorescence-activated cell sorting (FACS). After serum starvation or gentamicin treatment, cells were subjected to survival assay (MTT assay and FACS/DAPI), and injury assay (FACS/KIM-1). Megalin expression in hPTEC and HK2 were evaluated by western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence (IF). Cells were incubated with FITC conjugated myoglobin (FITC-Mgb) up to 120min, then FITC-Mgb uptake was evaluated by IF or intracellular FITC intensity using a plate reader. Megalin knockdown or inhibition was performed using siRNA or cilastatin (Cil). Dynamin inhibition was performed using dynasore (Dyn) and prochlorperazine (PCP).
Results
After FACS, hPTEC culture was 95% CD10/CD13 positive. hPTEC from 4 different kidneys with various cold ischemia time (16-26h) did not express KIM-1. hPTEC strongly expressed megalin by WB, qPCR and IF while HK2 did not. Serum starved cells demonstrated reduced viability and increased KIM-1 expression compared with control (MTT, DAPI, KIM-1, p<0.05 by One-way ANOVA). Compared with HK2, hPTEC demonstrated higher flux of FITC-Mgb uptake in first 20 min (p=0.004 by non-linear regression), which resulted in more FITC-Mgb uptake in hPTEC up to 120min. Megalin knockdown by siRNA caused reduced FITC-Mgb uptake in hPTEC (p<0.0001 by non-linear regression). Mgb uptake along a wide range of concentrations was reduced by Cil dose-dependently (Mgb:0 to 160ug/mL, Cil: 0 to 200mg/mL, p<0.0001 by non-linear regression). Dynamin inhibition by Dyn or PCP also reduced FITC-Mgb uptake.
Conclusion
hPTEC demonstrated healthy epithelial phenotype, high megalin expression, and megalin-mediated, dynamin-dependent endocytosis. They expressed KIM-1 in response to injury. hPTEC are a feasible platform for studies of endocytosis.