The kidney is the target for a wide variety of chemical agents, including heavy metals, haloalkenes, analgesics and antibiotics. The functional and metabolic characteristics of the proximal tubule (PT) predispose it as the primary site for xenobiotic damage. The aim of this study was to isolate and characterise human and rat PT cells in suspension and primary culture for use as defined models to investigate drug-induced PT cell damage in vitro . A second aim was to compare the response of human and rat systems to known nephrotoxins. Human and rat PT cells (90&'37 viable) were isolated from kidney cortex by collagenase digestion followed by isopycnic Percoll density centrifugation. This resulted in the formation of two distinct bands of cell at densities 1.040g/ml (A) and 1.060g/ml (B) for both preparations. Characterisation of human cells in terms of morphology, marker enzymes, retention of active transport systems and responsiveness to parathyroid hormone indicated that &'62 95&'37 of the cells in band B were proximal tubular. Each transport system demonstrated Michaelis-Menten kinetics; kinetic parameters suggested that a higher proportion of PT cells from the S1-S2 segment of the nephron were present in human isolates. Human isolated cells also contained levels of glutathione and cytochrome P450, in particular ethoxyresorufin-O-deethylase activity, a marker for the P4501A family, similar to the intact kidney. Both human and rat cells were successfully cultured in serum-supplemented medium (10&'37 v/v) with human cells reaching confluence by 3-4 days and rat by 5-6. Maximal attachment was seen when cells from both preparations were inoculated onto collagen coated plates with an additional layer of fibronectin. Only human cells were able to reach confluence on porous membranes and demonstated an enhanced morphology when compared to normal cultured cells. Cultured cells from both preparations retained an epithelial morphology and showed minimal secondary cell contamination as shown by light microscopy and in the case of human cultures additionally through immunohistochemical staining. Immunohistochemical staining also demonstrated that human cells in culture were depositing components of the extracellular matrix. The maintenance of PT cell function, throughout the time in culture, was shown following maintenance of active transport systems, in particular the glucose carrier system and on porous membranes the organic anion system. Only rat cells maintained the organic cation system in primary culture. In addition human cells maintained the preferential response to parathyroid hormone. Except for the transport of organic cations, the other carrier systems and responsiveness to hormones were evident at both sub-confluent and confluent stages of cell culture.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:306098 |
| Date | January 1992 |
| Creators | McLaren, John |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU545620 |
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