Limits...
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 hRPTEC 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, ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492764&req=5

fig03: Expression profile of biomarkers in hRPTEC 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, ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).

Mentions: Intriguingly, the results from 24 h showed different expression profiles for each biomarker. KIM-1 protein levels were increased in the culture medium by all six nephrotoxins (Fig. S6A), with some compounds also causing increases in NGAL (induced by AmB and doxorubicin, Fig. S6C). Similar expression patterns were also observed in cell lysates (Fig. S6B, D, and F). A more obvious dose-dependent overexpression of the biomarkers was observed at 48 h (Fig. S7). Much more significant dose-dependent responses of each biomarker were observed after 72 h of compound treatment (Fig.3). KIM-1, NGAL, and M-CSF protein release into culture medium was induced by medium and high concentrations of each compound and were dose dependent, although some increases in M-CSF were not statistically significant (Fig.3A, C, and E). KIM-1 protein levels in cell lysates showed similar increases as those in the culture medium (Fig.3B). Strikingly, NGAL protein expressing in cell lysates was increased up to 20-fold compared to the control cells and low-dose treatment (Fig.3D). The increase in biomarker expression over time is illustrated for one compound (Fig. S9).


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 hRPTEC 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, ****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

fig03: Expression profile of biomarkers in hRPTEC 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, ****P < 0.001, n ≥ 3 (three independent assays with two technical replicates in each assay).
Mentions: Intriguingly, the results from 24 h showed different expression profiles for each biomarker. KIM-1 protein levels were increased in the culture medium by all six nephrotoxins (Fig. S6A), with some compounds also causing increases in NGAL (induced by AmB and doxorubicin, Fig. S6C). Similar expression patterns were also observed in cell lysates (Fig. S6B, D, and F). A more obvious dose-dependent overexpression of the biomarkers was observed at 48 h (Fig. S7). Much more significant dose-dependent responses of each biomarker were observed after 72 h of compound treatment (Fig.3). KIM-1, NGAL, and M-CSF protein release into culture medium was induced by medium and high concentrations of each compound and were dose dependent, although some increases in M-CSF were not statistically significant (Fig.3A, C, and E). KIM-1 protein levels in cell lysates showed similar increases as those in the culture medium (Fig.3B). Strikingly, NGAL protein expressing in cell lysates was increased up to 20-fold compared to the control cells and low-dose treatment (Fig.3D). The increase in biomarker expression over time is illustrated for one compound (Fig. S9).

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