Performance of novel kidney biomarkers in preclinical toxicity studies

Abstract

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

FIG. 1.

Immunohistochemical analysis showing expression and immunolocalization of (a) clusterin, (b) Kim-1, (c) Lcn2, (d) vimentin, and (e) Timp-1 in kidneys of male Wistar rats treated with BI-2, BI-3, or gentamicin. Representative images from cortical regions of controls (insets) and high-dose animals after 14 days of treatment (3 days for BI-3 and Lcn2) are shown. Images were acquired at ×200 magnifications using an Olympus CH-2 microscope fitted with an Olympus E330 digital camera.

FIG. 2.

Analysis of (a) BUN, (b) serum creatinine, (c) urinary clusterin, (d) urinary Kim-1, (e) urinary Lcn2, and (f) urinary Timp-1 in male Wistar rats treated with BI-2 for 1, 3, or 12/14 days. Data are presented as individual animals and color coded according to histopathology scores for PTD. Mean values of five individual animals per dose group are indicated by a black line. Statistical analysis was performed by ANOVA and Dunnett’s test. Statistical changes are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. Note: for urinary markers after 1 and 3 days of treatment, histopathology scores shown are from groups of rats treated in parallel, that is, animals no. 1–15 and 16–30, respectively.

FIG. 3.

Determination of (a) BUN, (b) serum creatinine, (c) urinary clusterin, (d) urinary Kim-1, (e) urinary Lcn2, and (f) urinary Timp-1 in male Wistar rats treated with BI-3 for 1, 3, or 12/14 days. Data are presented as individual animals and color coded according to histopathology scores for PTD. Mean values of five individual animals per dose group are indicated by a black line. Statistical analysis was performed by ANOVA and Dunnett’s test. Statistical changes are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. Note: for urinary markers on days 1 and 3, histopathology scores shown are from groups of rats treated in parallel, that is, animals no. 1–15 and 16–30, respectively.

FIG. 4.

Analysis of (a) BUN, (b) serum creatinine, (c) urinary clusterin, (d) urinary Kim-1, (e) urinary Lcn2, and (f) urinary Timp-1 in male Wistar rats treated with gentamicin for 1, 3, or 12/14 days. Data are presented as individual animals and color coded according to histopathology scores for PTD. Mean values of five individual animals per dose group are indicated by a black line. Statistical analysis was performed by ANOVA and Dunnett’s test. Statistical changes are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. Note: for urinary markers after 1 and 3 days of treatment, histopathology scores shown are from groups of rats treated in parallel, that is, animals no. 1–15 and 16–30, respectively.

FIG. 5.

Determination of (a) BUN, (b) serum creatinine, (c) urinary clusterin, (d) urinary Kim-1, (e) urinary Lcn2, and (f) urinary Timp-1 in male Wistar rats treated with IMM125 for 1, 3, or 12/14 days. Data are presented as individual animals and color coded according to histopathology scores for PTD. Mean values of five individual animals per dose group are indicated by a black line. Statistical analysis was performed by ANOVA and Dunnett’s test. Statistical changes are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. Note: for urinary markers after 1 and 3 days of treatment, histopathology scores shown are from groups of rats treated in parallel, that is, animals no. 1–15 and 16–30, respectively.

FIG. 6.

ROC curves for (a) gene expression of candidate biomarkers and (b) urinary biomarkers compared to traditional clinical chemistry parameters. The area under the ROC curve, which serves as measure for the overall ability of a biomarker to discriminate healthy versus injured animals, is shown in the legend.