Abstract
There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and human macrophage colony stimulating factor (M-CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug-induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK-2) and primary (commercially available and freshly in-house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM-1, NGAL, and M-CSF assessed using ELISA and real-time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK-2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM-1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M-CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug-induced nephrotoxicity.
Highlights
Nephrotoxicity, or damage to the kidney, is a serious side effect of many marketed drugs, with 19–25% of acute renal failures caused, at least in part, by drug exposure (Bonventre et al 2010; McCullough et al 2013)
Renal cortex and outer stripe were cut into slices, washed with sterile phosphate buffered saline (PBS), minced, and the pieces were placed in a trypsinization flask filled with 300 mL of sterile, filtered Hanks’ buffer, containing 25 mmol/L NaHCO3, 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 0.5 mmol/L ethylene glycol tetraacetic acid (EGTA), 0.2% (w/v) bovine serum albumin, 50 lg/mL gentamicin, 1.3 mg/mL collagenase, and 0.59 mg/mL CaCl2, which was filtered prior to use
Traditional cytotoxicity assays are not reliable indicators for nephrotoxicity screening To investigate the reliability of traditional cytotoxicity assays in predicting drug-induced nephrotoxicity, MTT, and resazurin assays were performed using both human kidney 2 (HK-2) cells and primary human renal proximal tubule epithelial cells (hRPTECs)
Summary
Nephrotoxicity, or damage to the kidney, is a serious side effect of many marketed drugs, with 19–25% of acute renal failures caused, at least in part, by drug exposure (Bonventre et al 2010; McCullough et al 2013). There has been an intensive effort in recent years to identify in vivo biomarkers that can be used to selectively monitor kidney damage at an earlier stage (Dieterle et al 2010a; Hoffmann et al 2010; Ozer et al 2010; Sistare et al 2010; Huang et al 2014) Proteins such as kidney injury molecule-1 (KIM-1/TIM-1/HAVCR-1) (Hoffmann et al 2010; Vaidya et al 2010), neutrophil gelatinase-associated lipocalin (NGAL/LCN2) (Borkham-Kamphorst et al 2011; Paragas et al 2011), and human macrophage colony-stimulating factor 1 (M-CSF/CSF-1) (Wada et al 1997; Isbel et al 2001; Menke et al 2009) have been highlighted as proteins with clinical relevance to early detection of kidney injury. The U.S Food and Drug Administration (FDA) and European Medicines Agency (EMEA) have qualified seven renal safety biomarkers including KIM-1 for use in drug development (Dieterle et al 2010b)
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