Isolation and functional characterization of primary human proximal tubular epithelial cells from brain-dead organ...

Kidney proximal tubules (PT) utilize endocytosis to maintain body homeostasis. Effective PT repair mechanisms after acute kidney injury (AKI) are crucial to avoid the transition to chronic kidney disease (CKD). Cultured PT cells are often utilized to study these processes, but studies demonstrate loss of functional transporter expression in nearly all available cell lines. Here, we describe acquisition and culture of human PT cells isolated from the kidneys of brain-dead donors (bddPTC) not suitable for transplantation and designated to research. The kidney cortex was digested, and the cells filtered through strainers were cultured. After passage 1, the cells were labeled for CD10 and CD13, and bddPTC were isolated via fluorescence-activated sorting. bddPTC strongly expressed the brush border transport receptor megalin and aquaporin-1 in bddPTC. Functionally, uptake of the megalin ligand myoglobin was greater in bddPTC than HK2, a human PT cell line. Pretreatment with the megalin inhibitor cilastatin, megalin-targeted small interfering RNA, and endocytosis inhibitors reduced myoglobin uptake in bddPTC. Vascular cell adhesion protein 1, a marker for failed repair of PT after injury, was highly expressed in bddPTC. We provide reproducible means by which bddPTC may be used to study functional assessment and perhaps failed repair of PT.