Primary kidney cells

Abstract

Primary rabbit kidney epithelial cell cultures can be obtained that express renal proximal tubule functions. Toward these ends, renal proximal tubules are purified from the rabbit kidney by the method of Brendel and Meezan. To summarize, each kidney is perfused with iron oxide, which becomes associated with glomeruli. The renal cortex is sliced and homogenized to liberate nephron segments. Renal proximal tubules and glomeruli are purified by sieving. The glomeruli, covered with iron oxide, are removed using a magnet. After a brief collagenase treatment (to disrupt basement membrane), the tubules are plated in hormonally defined serum-free medium supplemented with 5 μg/mL bovine insulin, 5 μg/mL human transferrin, and 5 × 10⁻⁸ M hydrocortisone. After 5–6 d of incubation, confluent monolayers are obtained that possess multicellular domes, indicative of their capacity for transepithelial solute transport.

Fig. 1

Dome formation by rabbit kidney proximal tubule cell cultures. A photomicrograph was taken of a confluent monolayer under an inverted microscope at ×100 magnification. Panel A focuses on the cells in the monolayer, whereas panel B focuses on the cells in the dome.

Fig. 2

Transmission electron microscopy (TEM) of primary renal proximal tubule cells. Primary rabbit kidney proximal tubule cells were cultured on a plastic substratum in serum-free medium (as in Fig. 1), processed for TEM (9), and photographed at ×4000 magnification.

Fig. 3

Scanning electron microscopy (SEM) of primary renal proximal tubule cells. A monolayer of primary rabbit kidney proximal tubule cells was examined by SEM at ×6000 magnification (insert is ×30,000).