Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds

Toxicol In Vitro. 2009 Sep;23(6):1170-8. doi: 10.1016/j.tiv.2009.06.003. Epub 2009 Jun 10.


Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.

Publication types

  • Validation Study

MeSH terms

  • Apoptosis / drug effects
  • Cells, Cultured
  • Cost-Benefit Analysis
  • Drug-Related Side Effects and Adverse Reactions*
  • Humans
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / drug effects*
  • Kidney Tubules, Proximal / metabolism
  • Necrosis / chemically induced
  • Perhexiline / toxicity
  • Staurosporine / toxicity
  • Time Factors
  • Toxicity Tests / economics
  • Toxicity Tests / methods*


  • Staurosporine
  • Perhexiline