Acute kidney injury (AKI) is characterized by a rapid decline of the kidney function. Drug-induced toxicity (i.e., nephrotoxicity) can explain 14-16 % of adult AKI cases and 16 % of pediatric AKI cases. In severe cases, AKI can progress to chronic kidney disease and eventually to end-stage kidney disease. The latter being treated with renal replacement therapy: dialysis or transplantation. This underscores the unmet need to better understand the mechanism of drug-induced nephrotoxicity and prevent drug-induced renal toxicity for compounds yet under development.

Clinically relevant drug-drug interactions (DDIs) belong to adverse effects associated with concomitant drug treatment modalities. DDIs may have significant harmful effects for a patient. Generally, metabolism of DDI’s in the liver may impact drug efficacy and renal elimination. Within this, DDIs can lead to decreased metabolism of either or both drugs resulting in elevated plasma drug levels or increased metabolism of either or both drugs causing reduced efficacy, formation of a toxic metabolite, or reduced drug transporter influx and efflux. For example, DDI at the site of the renal drug transporter may cause toxic events, i.e., when a compound is accumulating in the proximal tubule cells. Therefore, it is important to evaluate DDI of an investigational new drug. The FDA and the EMEA recommend assessment of potential DDIs in vitro as this highlights the impact of multiple clearance pathways as well as lead to better candidate compounds entering clinical studies.

Cell4Pharma is specialized in in vitro cell-based nephrotoxicity assays to identify potential renal toxic compounds at an early stage of drug discovery. conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) are being utilized for nephrotoxicity assays we provide and these cells show proximal tubule characteristics and express functional influx and efflux drug transporters (OAT1, OCT2, P-gp, MRP4, BCRP). The aforementioned reasons and more than 90 publications support the fact that our ciPTECs are highly stable and present an accurate model to assess nephrotoxicity in vitro.

Cell4Pharma has developed a unique conditionally immortalised human proximal tubule epitelial cell line (ciPTEC) expressing most relevant renal drug transporters endogenously. Our cells are protected under patent PCT/EP2010/066792. To improve the plethora of stable drug transporter functionality in ciPTEC, our product portfolio was extended with ciPTEC-OAT1 and ciPTEC-OAT3 with a market introduction in 2017. As such, Cell4Pharma’s ciPTEC platforms provide an excellent tool to predict pharmaco-kinetics, drug interactions and renal toxicity for potential pharmaceutical compounds.

Regulatory Guidelines for Drug Elimination Routes

The recent guidances for the pharmaceutical industry published by the Food and Drug Administration (FDA) in 2017 and the European Medicines Agency (EMA) in 2013 recommend that drug development should include identification of elimination routes via drug transport proteins and characterize drug-drug interactions (DDI). These include the investigation of renal drug transport studies at the sites of p-glycoprotein (Pgp), breast cancer resistance protein (BCRP), multidrug and toxin extrusion transport 1 and 2 (MATE1, MATE2-k), organic cation transporter 2 (OCT2) and organic anion transporter 1 and 3 (OAT1, OAT3). All transporters are present in our ciPTEC platforms.

LDH cytotoxicity assay, featuring conditionally immortalized proximal tubule epithelial cells (ciPTECs), provides an advanced platform for assessing the impact of test compounds on renal cells. In this process, ciPTECs are exposed to a carefully determined optimal concentration of the test compound, as identified in the viability assay, and the trials are conducted in duplicates. The subsequent quantification of lactate dehydrogenase (LDH) release serves as a reliable indicator of cytotoxic effects. This assay ensures a quantitative and comprehensive analysis of nephrotoxicity, allowing for precise measurement of cell damage.

For an oxidative stress assay in ciPTECs, we employ the CM-H2DCFDA probe, a cell-permeable indicator that reacts with intracellular reactive oxygen species (ROS). This reaction induces a fluorescent signal, allowing precise measurement of ROS levels in response to test compounds. Crucial for revealing oxidative stress-induced nephrotoxicity, a critical parameter in drug safety assessment, this assay provides a comprehensive analysis. By evaluating ROS levels, we gain insights into the potential impact of compounds on renal cells, ensuring a thorough understanding of their safety profile in the context of renal health.

N-Acetyl-β-D-Glucosaminidase (NAG) analysis in ciPTECs offers a focused approach to nephrotoxicity assessment. NAG, predominantly located in the lysosomes of proximal tubular cells, is a biomarker and elevated levels of NAG in response to a test compound serve as indicative signs of proximal tubular damage. Through this biomarker analysis, we provide valuable insights into renal health, aiding in the identification of potential nephrotoxic effects and contributing to a comprehensive evaluation of compound safety in the context of renal function.

Recognizing the substantial challenge of drug-induced nephrotoxicity in clinical settings, hindering both current treatments and new medication development, we present a high-content screening assay in ciPTEC. Utilizing fluorescent dyes, we assess cytoskeleton integrity (Phalloidin), mitochondrial function (Mitotracker), and nuclear abnormalities (DAPI). This model integrates multi-parametric data and drug exposure information, accurately identifying renal toxic drugs (sensitivity 75%, specificity 100%). With high throughput, a straightforward protocol, and cost-effectiveness in a 96-well format, our assay is ideal for early drug discovery. It facilitates the early identification, quantification, and mitigation of nephrotoxicity risks, contributing to a safer and more efficient drug development process.