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Renal Toxicity Screening

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ciPTEC ASSAY

 

ciPTEC 

Understand the potential for nephrotoxicity and transporter function using the IONTOX ciPTEC Model

Glomerular filtration in the kidneys is the primary mode of drug excretion (1), making this organ highly vulnerable to drug-related injury.  The renal proximal tubes, in particular, play a major role in the elimination of drugs and their metabolites, and drug-induced damage to this region can subsequently result in acute kidney injury.Predicting the potential nephrotoxicity of a drug in preclinical stages is thus crucial however remains difficult; nephrotoxicity is typically detected only in late, clinical stages.

The ciPTEC renal toxicity screening assay overcomes these challenges by utilizing normal human-derived conditionally immortalized proximal tubular epithelial cells (ciPTEC), developed by Martijn Wilmer and colleagues (2).

These cells stably express a large host of functional enzymes and transporters used by the proximal tubes in vivo (2) for uptake, efflux and metabolism, which ultimately contribute to drug elimination.  Use of the ciPTEC cell model by IONTOX for these assays is under a license agreement.

ciPTEC Assay Overview

ciPTEC cells are cultured under optimal conditions to stimulate cellular differentiation and formation of an epithelial monolayer.  Achievement of this maturation can be validated through transporter function assays, as demonstrated by Nieskens et al (3), below.  The cells are then exposed to increasing concentrations of test compounds for 24, 48 and 72 hours  Toxicity is subsequently evaluated via markers of cell viability, including ATP concentrations, enzyme leakage, and increases in KIM-1, in treated versus untreated cultures. High throughput screening in 384-well plates available upon request.

 

Example ciPTEC Output 

 

Mature ciPTEC cells expressing  OAT1 or OAT3  were incubated with fluorescein to determine the functionality of each proximal tube transporter in culture.  Transporter mediated uptake of fluorescein in the ciPTEC-OAT1 and

ciPTEC-OAT3 lines is demonstrated in the panel to the left, following a 10-minute incubation with increasing concentrations of the fluorophore.  In a 60-minute time course, para-aminohippuric acid, a specific inhibitor of OAT-1 function, and estrone sulfate, an inhibitor of OAT-3, were co-incubated with fluorescein in ciPTEC-OAT1 and ciPTEC-OAT3 cell lines, respectively.  The results indicate that fluorescein uptake is indeed occurring via the OAT-1 and OAT-3 proximal tube transporters.

 

 

 

 

 

 

 

 

In figure A, B, and C Polymixin-B, an antibiotic known to induce nephrotoxicity, was administered to mature OAT-1 expressing ciPTEC cells (a, b) and non-OAT-1 expressing HK-2 cells (c) in increasing concentrations.  Following a 24 hour incubation, cell viability was assessed by ATP concentration relative to untreated controls.  In contrast to the ciPTEC lines, the HK-2 human proximal tubule line exhibits high sensitivity to the toxic properties of the polymixin antibiotic, demonstrating not only the importance of the OAT-1 transporter in the processes of drug elimination, but also the superiority of the ciPTEC model in assessing the potential for drug-based nephrotoxicity.

 

References

1. Le, J. (2016, April). Drug Excretion. Retrieved August 21, 2017, from Merck Manual: http://www.merckmanuals.com/professional/clinical-pharmacology/pharmacokinetics/drug-excretion
2. Wilmer, M. J., Saleem, M. A., Masereeuw, R., Ni, L., van der Velden, T. J., Russel, F. G., et al. (2010). Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters. Cell Tissue Res, 339, 449-457.
3. Nieskens, T. T., Peters, J. G., Schreurs, M. J., Smits, N., Woestenenk, R., Jansen, K., et al. (2016). A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity. The AAPS Journal, 18(2), 465-475.