Comparison of changes in urinary and blood levels of biomarkers associated with proximal tubular injury in rat models

Abstract

To investigate useful biomarkers associated with proximal tubular injury, we assessed changes in levels of a focused set of biomarkers in urine and blood. Male rats administered a single dose or four doses of gentamicin (GM, 240 mg/kg/day) or a single dose of cisplatin (CDDP, 5 mg/kg) were euthanized on days 2 (the day after initial dosing) 5, or 12. At each time point, histopathological examination of the kidney and immunohistochemistry for biomarkers, kidney injury molecule-1 (Kim-1), lipocalin (NGAL), clusterin (CLU), cystatin C (CysC) and β2-microglobulin (β2M) were performed. Biomarker levels were measured in urine and blood. In both treatment groups, degenerated/necrotic proximal tubules and regenerated tubules were mainly observed on days 5 and 12, respectively. At the same time as these tubular injuries, urinary Kim-1, CysC and β2M levels were increased. Moreover, urinary levels of CysC and β2M in GM-treated animals and Kim-1 in CDDP-treated animals increased (on day 2) prior to tubular injury on day 5. This was considered to reflect the characteristics of drug toxicity. Although almost all of the biomarkers in blood were not sufficiently sensitive to detect proximal tubular injury, urinary and plasma β2M levels simultaneously increased. Therefore, in addition to urinary Kim-1, CysC and β2M levels, plasma β2M levels were also considered useful for detecting proximal tubular injury.

Keywords: biomarker; cisplatin; gentamicin; immunohistochemistry; kidney; proximal tubular injury.

Fig. 1.

Histopathological findings in the kidneys of rats following GM or CDDP administration. The following findings were obtained in the GM group: eoshinophilic globules in and degeneration/necrosis of the proximal convoluted tubular epithelium in the cortex on day 5 (a), regeneration of the proximal convoluted tubular epithelium in the cortex on day 12 (b) and dilatation of the proximal tubule over the cortex and OSOM on day 12 (c). The following findings were observed in the CDDP group: nuclear chromatin margination of the proximal straight tubular epithelium (arrow) in the OSOM on day 2 (d) and degeneration/necrosis, regeneration, karyomegaly (arrow head) of the proximal straight tubular epithelium in the OSOM on day 5 (e) and day 12 (f). Hematoxylin and eosin staining. Bar = 50 μm (a, d, e, f), 100 μm (b, c).

Fig. 2.

Time course of the appearance of urinary and blood biomarkers in rats given a single administration or 4 administrations of gentamicin (GM, 240 mg/kg/day), rats given a single administration of cisplatin (CDDP, 5 mg/kg) or rats used as the vehicle control. The urinary and serum/plasma levels of Kim-1(a, b), NGAL (c, d), CLU (e, f), CysC (g, h) and β2M (i, j) were assayed at the indicated time points (the day after initial dosing shown as day 2). Individual data and mean values are expressed. The sample numbers were as follows: n = 12 for urinalysis on days 2 and 5 and blood analysis on day 2, except for the CDDP group (n = 10 for urinalysis on day 5), and n = 6 for urinalysis on day 12 and blood analysis on days 5 and 12, except for the CDDP group (n = 4 for urinalysis and blood analysis on day 12). *P<0.05 and **P<0.01 vs. control group (two-tailed Dunnett’s multiple comparison test).

Fig. 3.

Relationship between the urinary and serum/plasma levels of biomarkers. Pearson’s product-moment correlation coefficient was used to evaluate the linear association between the urinary and blood levels of each biomarker. Kim-1 (GM group, coefficient of correlation (R) = 0.96, P<0.01; CDDP group, R=0.71, P<0.01) (a), NGAL (GM group, R=0.89, P<0.01; CDDP group, R=0.95, P<0.01) (b), CLU (GM group, R=0.10, P=0.65; CDDP group, R=0.60, P<0.01) (c), CysC (GM group, R=0.25, P=0.26, CDDP group, R=0.40, P=0.07) (d), β2M (GM group, R=0.33, P=0.13, CDDP group, R=0.68, P<0.01) (e).

Fig. 4.

Kim-1 immunohistochemistry in the kidneys of rats following administration of GM or CDDP. Kim-1 immunoreactivity in a part of the proximal convoluted tubules in a nephron on day 5 (a), regenerated tubules on day 12 (b) and dilated tubules on day 12 (c) in the GM group. Kim-1 immunoreactivity in a few proximal straight tubules in the OSOM on day 2 (d) and the immunoreactivity in degenerate/necrotic and regenerated tubules on day 5 (e) and day 12 (f) in the CDDP group. Bar = 100 μm (a–f).

Fig. 5.

NGAL immunohistochemistry in the kidneys of rats following administration of GM or CDDP. NGAL immunoreactivity in a part of the proximal convoluted tubules sporadically on day 5 (a) and in a few regenerated tubules (b) and a part of dilated tubules on day 12 (c) in the GM group. NGAL immunoreactivity in a few proximal straight tubules in the OSOM on day 2 (d) and in degenerate/necrotic and regenerated tubules and their lumens on day 5 (e) and in a few degenerate/necrotic and regenerated tubules on day 12 (f) in the CDDP group. Bar = 100 μm (a–f).

Fig. 6.

CLU immunohistochemistry in the kidneys of rats following administration of GM or CDDP. CLU immunoreactivity in a few normal distal tubules in the cortex (a) and in a part of the dilated tubules on day 12 (b) in the GM group. CLU immunoreactivity in a part of degenerate/necrotic tubules and lumens of normal tubules in the medulla (c) and the distal tubules over the cortex to medulla on day 5 (d) and in a few degenerate/necrotic tubules in the medulla on day 12 (e) in the CDDP group. Bar = 100 μm (a, b), 200 μm (c–e).

Fig. 7.

CysC immunohistochemistry in the kidneys of rats following administration of GM, CDDP or a vehicle control. CysC immunoreactivity in the proximal convoluted tubules in control animals on day 5 (a, d). CysC immunoreactivity with a strong intensity in the proximal convoluted tubules of rats on day 5 (b) and immunoreactivity in a part of dilated tubules in addition to the proximal convoluted tubules on day 12 (c) in the GM group. CysC immunoreactivity in a part of the tubules including degenerated/necrotic and regenerated tubules and the tubular lumens of the medulla on day 5 (e). Bar = 200 μm (a, b, c), 400 μm (d, e).

Fig. 8.

β2M immunoreactivity in the proximal convoluted tubules of a control animal on day 5. Bar = 200 μm.