ADME / Tox >> In Vitro Toxicity >> Nephrotoxicity

Determine your drug’s nephrotoxic potential early in discovery

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If not correctly assessed during lead optimization, drug candidates with undesired safety issues may slip through clinical development, resulting in high cost failures. Drug-induced nephrotoxicity is one such safety assessment that can and should be evaluated during lead optimization to determine a compound's potential to induce organ-specific cellular injury.

Eurofins' multiplexed nephrotoxicity assay provides an effective in vitro cell-based screening model to assess nephrotoxicity potential in cells derived directly from human kidney tissue. In vitro cell-based toxicity assays mimic in vivo tissue studies and in turn provide a reliable tool for safety evaluation in early stages of drug discovery. Eurofins offers two image-based High Content Analysis (HCA) assays using human renal proximal tubule epithelial cells (HRPTEpiC). These multi-endpoint analyses provide a highly encompassing assessment of nephrotoxicity, which also provides detailed mechanistic information underlying the observed toxicity.

Advantages of Eurofins' Nephrotoxicity Assays:

  • Pre-validated human renal proximal tubule epithelial cells (HRPTEpiC) harvested and purified from single donor source are used for reproducible assays
  • High Content Analysis (HCA) provides objective and more consistent data analysis compared to other methods
  • Low compound requirement - as little as 300 μl of a 20 mM solution is needed for testing at a final assay concentration of 100 μM
  • Relevant reference compound and vehicle controls are included on each plate

 

Cytotoxicity assessment undefined

A three parameter multiplexed study using HRPTEpiC cells is used to assessment compound induced nephrotoxicity in vitro.  A decrease in cell viability is a very sensitive marker to detect general toxicity. Activation of caspase 3 indicates an apoptotic mechanism. Phosphorylation of histone H3, a marker for mitotic block, reveals a defect in cell cycle regulation.

Features of Eurofins' cytotoxicity assessment

  • High Content Analysis, cytotoxicity parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Apoptosis - increased staining with an anti-active caspase antibody
    • Mitotic block - increased staining with an anti-phospho-histone-H3 antibody
  • 72 hr compound incubation
Table 1. Cytotoxicity in  HRPTEpiC induced by 72 hr treatment with staurosporine

Cell viability IC50                  (µM)              

Concentration (µM) to induce ≥ 5-fold of activated caspase-3 signal (apoptosis)         

Concentration (µM) to change ≥ 2-fold in phosphor-histone-H3 signal (mitosis)

0.026 ± 0.003

0.14 ± 0.05

0.0003 ± 0.00003

 

Cellular stress assessment

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A three parameter multiplexed study using HRPTEpiC cells is used to assessment compound induced cellular stress in vitro.  A decrease in cell viability is a very sensitive marker to detect general toxicity. Heat shock protein 27 (HSP 27) is expressed in response to cellular stress in an attempt to block the apoptosis pathway. Kidney injury molecule-1 (KIM-1) is a well-accepted marker of renal proximal tubule injury.

Features of Eurofins' cellular stress assessment

  • High Content Analysis, cellular stress parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Cellular stress - increased staining using an anti-HSP27 antibody
    • Kidney injury: increased staining using an anti-KIM-1 antibody
  • 48 hr compound incubation

 

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Figure 1. Cellular stress in HRPTEpiC induced by cantharidin treatment