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Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells.

Huang JX, Kaeslin G, Ranall MV, Blaskovich MA, Becker B, Butler MS, Little MH, Lash LH, Cooper MA - Pharmacol Res Perspect (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland 306 Carmody Road, St Lucia, Queensland, 4072, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

mRNA levels of each biomarkers in hRPTEC cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 mRNA, (B) NGAL mRNA, and (C) M-CSF mRNA. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).
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fig04: mRNA levels of each biomarkers in hRPTEC cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 mRNA, (B) NGAL mRNA, and (C) M-CSF mRNA. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).

Mentions: The mRNA levels of each biomarker were again evaluated (Fig.4). KIM-1 mRNA did not show stimulation upon compound treatment, except for high-dose CsA and doxorubicin at 72 h (Fig.4A), while mRNA levels of NGAL showed a dose-dependent increase at 48 h treatment (Fig. S8F) and kept rising after 72 h exposure (Fig.4) to 100 μmol/L colistin (P < 0.01), 10 μmol/L cisplatin (P < 0.05), 20 μmol/L CsA (P < 0.05), and 1 μmol/L doxorubicin (P < 0.001) (Fig.4B). In addition, M-CSF mRNA levels displayed a similar pattern to NGAL mRNA, giving significant results in colistin, cisplatin, CsA, and doxorubicin treatment (Fig.4C). Other time points of mRNA expression levels are shown in Figure S8. These more widespread increases in mRNA expression were consistent with the greater increases in protein levels seen for multiple nephrotoxins in hRPTECs at 72 h, although the protein analysis provided more statistically significant increases.


Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells.

Huang JX, Kaeslin G, Ranall MV, Blaskovich MA, Becker B, Butler MS, Little MH, Lash LH, Cooper MA - Pharmacol Res Perspect (2015)

mRNA levels of each biomarkers in hRPTEC cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 mRNA, (B) NGAL mRNA, and (C) M-CSF mRNA. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492764&req=5

fig04: mRNA levels of each biomarkers in hRPTEC cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 mRNA, (B) NGAL mRNA, and (C) M-CSF mRNA. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).
Mentions: The mRNA levels of each biomarker were again evaluated (Fig.4). KIM-1 mRNA did not show stimulation upon compound treatment, except for high-dose CsA and doxorubicin at 72 h (Fig.4A), while mRNA levels of NGAL showed a dose-dependent increase at 48 h treatment (Fig. S8F) and kept rising after 72 h exposure (Fig.4) to 100 μmol/L colistin (P < 0.01), 10 μmol/L cisplatin (P < 0.05), 20 μmol/L CsA (P < 0.05), and 1 μmol/L doxorubicin (P < 0.001) (Fig.4B). In addition, M-CSF mRNA levels displayed a similar pattern to NGAL mRNA, giving significant results in colistin, cisplatin, CsA, and doxorubicin treatment (Fig.4C). Other time points of mRNA expression levels are shown in Figure S8. These more widespread increases in mRNA expression were consistent with the greater increases in protein levels seen for multiple nephrotoxins in hRPTECs at 72 h, although the protein analysis provided more statistically significant increases.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland 306 Carmody Road, St Lucia, Queensland, 4072, Australia.

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.

No MeSH data available.


Related in: MedlinePlus