Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters
Related Experiment Video
Updated: Jun 9, 2026
Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells
Published on: June 20, 2018
M J Palmoski1, B A Masters, O P Flint
1Department of Investigative Toxicology, Pharmaceutical Research and Development Division, Bristol-Myers Squibb, Syracuse, NY 13221-4755, USA.
Abstract:
With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12-14. The integrity of the monolayer was confirmed by exclusion of [(14)C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [(14)C]p-aminohippuric acid (PAH) and cation [(14)C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5-6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [(14)C]alpha-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.
Insights
This study developed a rabbit kidney proximal tubule cell culture system. The system effectively mimics in vivo renal function and polarity, proving useful for assessing nephrotoxicity.
Area of Science:
- Nephrology
- Cell Biology
- Toxicology
Background:
- Developing reliable in vitro models for renal toxicity assessment is crucial.
- Existing models may not fully replicate in vivo kidney cell function and polarity.
Purpose of the Study:
- To establish a kidney proximal tubule cell culture system for evaluating renal toxicity.
- To characterize the functional polarity of cultured rabbit proximal tubule cells.
Main Methods:
- Freshly isolated rabbit proximal tubules were cultured on Millipore cellulose filters (Millicell-HA).
- Cellular integrity and viability were assessed using [(14)C]inulin exclusion and protein synthesis assays.
- Transport of organic anions (p-aminohippuric acid) and cations (tetraethylammonium bromide) was measured across the cell monolayer.
- Uptake of alpha-methylglucopyranoside (glucose analog) was evaluated under varying conditions.
Main Results:
- The cell culture system demonstrated cell confluency by days 12-14 with peak DNA synthesis on day 6.
- Cultured cells exhibited functional polarity, with basolateral to apical transport rates 5-6 times higher than apical to basolateral for PAH and TEA.
- Specific inhibitors (probenecid, N-methylnicotinamide, quinine) modulated transporter activity.
- Apical glucose uptake was sodium-dependent and phloridzin-sensitive, mimicking in vivo characteristics.
Conclusions:
- Rabbit proximal tubule cells cultured on filter inserts maintain functional polarity.
- This in vitro system accurately reflects in vivo kidney cell transport mechanisms.
- The developed culture system is a valuable tool for investigating nephrotoxicity mechanisms.
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