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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

Expression profile of biomarkers in HK-2 cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 protein concentration in culture medium; (B) KIM-1 protein concentration in cell lysates; (C) NGAL protein concentration in culture medium; (D) NGAL protein concentration in cell lysates; (E) M-CSF protein concentration in culture medium; and (F) M-CSF protein concentration in cell lysates. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005, n ≥ 3 (three independent assays with two technical replicates in each assay).
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fig01: Expression profile of biomarkers in HK-2 cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 protein concentration in culture medium; (B) KIM-1 protein concentration in cell lysates; (C) NGAL protein concentration in culture medium; (D) NGAL protein concentration in cell lysates; (E) M-CSF protein concentration in culture medium; and (F) M-CSF protein concentration in cell lysates. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005, n ≥ 3 (three independent assays with two technical replicates in each assay).

Mentions: We first investigated the effects of the six nephrotoxic compounds on the protein levels of KIM-1, NGAL and M-CSF using a quantitative ELISA kit. HK-2 cells were cultured in 96-well plates and incubated with three concentrations (high, medium, and low) of each compound. The high concentrations used for inducing biomarker expression were varied for each compound so that they were lower than the respective CC50, in order to induce cell injury but reduce the possibility of nonspecific toxic events, while the low concentration was 10-fold less. Both culture medium and cell lysates were assessed after 4, 24, 48 (Figs. S1–S3), and 72 h (Fig.1) exposure to the compounds. Limited biomarker overexpression was observed compared to the control groups, and there was no significant dose-dependent increase at the 4 h time point (Fig. S1). An AmB–induced M-CSF increase in HK-2 cell lysate was observed at 24 h (Fig. S2F); however, the result may not be reliable as it was reaching the detection sensitivity limitation of the ELISA kits (around 10 pg/mL). After 48 h, CsA induced an increase in NGAL, while gentamicin and AmB induced increases in M-CSF in culture media (Fig. S3C and E). Nevertheless, dose-dependent overexpression became less obvious at 72 h in culture medium (Fig.1A, C, and E). Biomarker overexpression was observed in cell lysates for some compounds at 72 h, but was not consistent for all compounds or biomarkers (Fig.1B, D, and F).


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)

Expression profile of biomarkers in HK-2 cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 protein concentration in culture medium; (B) KIM-1 protein concentration in cell lysates; (C) NGAL protein concentration in culture medium; (D) NGAL protein concentration in cell lysates; (E) M-CSF protein concentration in culture medium; and (F) M-CSF protein concentration in cell lysates. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005, 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

fig01: Expression profile of biomarkers in HK-2 cells after nephrotoxic compound treatment for 72 h. (A) KIM-1 protein concentration in culture medium; (B) KIM-1 protein concentration in cell lysates; (C) NGAL protein concentration in culture medium; (D) NGAL protein concentration in cell lysates; (E) M-CSF protein concentration in culture medium; and (F) M-CSF protein concentration in cell lysates. Data are presented as Mean ± Standard deviation. Significantly different *P < 0.05; **P < 0.01; ***P < 0.005, n ≥ 3 (three independent assays with two technical replicates in each assay).
Mentions: We first investigated the effects of the six nephrotoxic compounds on the protein levels of KIM-1, NGAL and M-CSF using a quantitative ELISA kit. HK-2 cells were cultured in 96-well plates and incubated with three concentrations (high, medium, and low) of each compound. The high concentrations used for inducing biomarker expression were varied for each compound so that they were lower than the respective CC50, in order to induce cell injury but reduce the possibility of nonspecific toxic events, while the low concentration was 10-fold less. Both culture medium and cell lysates were assessed after 4, 24, 48 (Figs. S1–S3), and 72 h (Fig.1) exposure to the compounds. Limited biomarker overexpression was observed compared to the control groups, and there was no significant dose-dependent increase at the 4 h time point (Fig. S1). An AmB–induced M-CSF increase in HK-2 cell lysate was observed at 24 h (Fig. S2F); however, the result may not be reliable as it was reaching the detection sensitivity limitation of the ELISA kits (around 10 pg/mL). After 48 h, CsA induced an increase in NGAL, while gentamicin and AmB induced increases in M-CSF in culture media (Fig. S3C and E). Nevertheless, dose-dependent overexpression became less obvious at 72 h in culture medium (Fig.1A, C, and E). Biomarker overexpression was observed in cell lysates for some compounds at 72 h, but was not consistent for all compounds or biomarkers (Fig.1B, D, and F).

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