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ADME PK

Chronic Exposure Nephrotoxicity Assay: 
A combined high content screening (HCS) approach using renal proximal tubule epithelial cells (RPTEC)

Detect therapeutically relevant pathophysiological nephrotoxicity of novel therapeutics using Cyprotex’s multi-parametric high content screening (HCS) human nephrotoxicity assay.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Background Information on Nephrotoxicity

  • Drug-induced nephrotoxicity (DIN) is a leading cause of renal failure in the clinic; creating a major concern within drug discovery programs.
  • Being a highly structured filtration network, with a rich blood flow, the kidney is often exposed to high concentrations of drugs and/or metabolites creating vulnerability to drug induced toxicity1.
  • Renal proximal tubule epithelial cells (RPTEC) are the predominant cell type in the kidney proximal tubule and one of the main sites for re-absorption and drug accumulation often resulting in tubular damage by interfering with mitochondrial function, impairing tubular transport, increasing oxidative stress or forming free radicals1,2,3.
  • A combined high content screening (HCS) approach allows a measure of multiple cell health markers including glutathione content (GSH), phospholipidosis (PLD), mitochondrial mass (mito mass) and mitochondrial membrane potential (MMP) alongside cellular ATP levels in a kidney relevant in vitro cell model in order to better predict drug induced nephrotoxicity (DIN).
Drugs cause approximately 20 percent of community- and hospital-acquired episodes of acute renal failure. Among older adults, the incidence of drug-induced nephrotoxicity may be as high as 66 percent.

2Naughton CA (2008) Drug-induced nephrotoxicity. Am Fam Physician 78(6); 743-750

Protocol

Protocol for Chronic Exposure Nephrotoxicity Assay

Cell Type Renal proximal tubule epithelial cells (RPTEC)
Analysis Platform Cellomics ArrayScan® (Thermo Scientific)
Analysis Method Combined High Content Screening (HCS)
Test Article Concentration* 8 point dose response curve with top concentration based on 100x Cmax or solubility limit
Number of Replicates* 3 replicates per concentration
Test Article Requirements 150 µL of a stock solution to achieve 100x Cmax (1000x top concentration to maintain 0.1% DMSO) or equivalent amount in solid compound.
Time Points* 9 days (216hrs)
Toxicity Markers* Cell loss

Nuclear size

DNA structure

Mitochondrial mass

Mitochondrial membrane potential

Phospholipidosis

Glutathione content

Cellular ATP
Quality Controls* Negative control: 0.1% DMSO (vehicle)

Positive controls: Sertraline and L-buthionine-sulfoximine
Data Delivery Minimum effective concentration (MEC) and AC50 values with dose response curves for each measured parameter.

*Other options available on request.

Data

Data from Cyprotex's Nephrotoxicity Assay

 
 
Figure 1
Representative high content screening (HCS) images of (a) (S)-(+)-camptothecin and (b) tobramycin in RPTECs labelled with Syto11 (blue) to detect DNA structure, monochlorobimane (mBCl) (green) to detect GSH content, LipidTOXTM Red (red) to detect phospholipidosis (PLD) and MitoTracker® Deep Red (yellow) to detect mitochondrial membrane potential (MMP).
 

(a).

(b).

Figure 2
Graphical representation of (a) cellular ATP content and GSH content response following 216 hours of cisplatin exposure and (b) cellular ATP content and phospholipidosis response following 216 hours of cyclosporin A exposure in RPTECs.

RPTECs were exposed to test compound for 216 hours, re-dosing occurred on 3 occasions over this period. At 216 hours the cell model was analysed using a Cellomics ArrayScan®  (Thermo Scientific) following incorporation of fluorescent dyes for cell health parameters including DNA structure (Syto11), GSH content (mBCl), phospholipidosis (HCS LipidTOX™ Red), mitochondrial dysfunction (MitoTracker® Deep Red). Subsequently cellular ATP content (CellTiter-Glo®, Promega) was determined.

CompoundHuman exposure Cmax (µM)*Known nephrotoxinMinimun effective concentration; MEC (µM)Most sensitive feature
(S)-(+) Camptothecin 0.083 Yes 0.003 Nuclear size
Acetaminophen 165.4 Yes 182 Glutathione content
Cisplatin 2 Yes 0.106 Glutathione content
Cyclosporin A 11 Yes 0.709 Phospholipidosis
Diclofenac 10.1 Yes 29 Cellular ATP level
Gentamycin 13 Yes 367 Mitochondrial membrane potential
Tobramycin 16 Yes 477 Mitochondrial mass
Phenacetin 12 Yes 397 Mitochondrial mass
Amikacin 34 Yes 344 -
Buspirone 0.009 No No response -
Piroxicam 12.79 No No response -
Flavoxate 1.788 No 117 Glutathione content
Flumazenil 1.21 No No response -
Levocarnitine 85.7 No No response -
Mecamylamine 0.142 No No response -
Propanthelien 0.44 No No response -

  < 1x Cmax   < 10x Cmax   < 30x Cmax   > 50x Cmax

 * Plasma Cmax values were taken from literature.

 
Table 1
Nephrotoxicity prediction of 16 reference compounds categorized according to literature data.

Utilising the RPTEC chronic exposure HCS assay all reference compound toxicities were correctly predicted with 100% accuracy, sensitivity and specificity within a 30x Cmax cut off (table 1). Multi-parametric high content screening allows detection of nephrotoxicity below therapeutic levels (Cmax) for cisplatin (MEC 0.106 µM; Cmax 2 µM) and cyclosporin A (MEC 0.709 µM; Cmax 11µM), highlighting the sensitivity of the assay.

The combination of an in vitro human relevant cell model with chronic compound exposures and multi-parametric endpoint assessment presents a viable screening strategy for the accurate in vivo relevant detection of novel therapeutics that cause nephrotoxicity early in drug development.

References

1Pazhayattil GS and Shirali AC (2014). Drug-induced impairment of renal function. Int J Nephrol Renovascular Dis 7; 457-468.
2 Naughton CA (2008). Drug-induced nephrotoxicity. Am Fam Physician 78(6): 743-750.
3 Ozer JS et al. (2010). A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function. Nat Biotechnol 25(5): 486-494.

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