Kidney Models Available
Introduction To Nephrotoxicity Testing In Vitro
Pharmaceutical, chemical, and cosmetic companies are responsible for knowing that their products are safe for human use and in the event of misuse that any adverse systemic effects are known.
An important challenge for these industries is the immediate need to reduce or replace animal testing. This means that in vitro alternatives must be found to test new drug candidates, chemical ingredients, or finished products.
In the past, companies could evaluate the safety of new drugs, chemicals, or finished products in animal studies. Although, some animal studies are still done on a routine basis there is real regulatory, consumer, and corporate pressure to reduce, refine, and even replace animal toxicity testing.
Immortalized HK-2 Cell Line To Rank Order Compounds
Current in vitro models for kidney testing focuses mainly on immortalized cell lines (e.g. HK-2). These models are useful for identifying intracellular mechanisms of toxicity and for toxicity screening studies that can be used to rank order a group of new molecular entities.
ciPTEC Model For Identifying Potential Nephrotoxicity Risk and Renal Drug- Drug Interactions
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.
Our 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
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. It is also possible to evaluate drug-drug interactions on uptake or efflux transporters in the ciPTEC cell lines used by IONTOX. High throughput screening in 384-well plates available upon request.
Example Output For Transporter Function Using The ciPTEC Assay
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.
Example Output for Toxicity
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.