Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium

Abstract

Sensitive biomarkers are needed to detect kidney injury at the earliest stages. The objective of this study was to determine whether the appearance of kidney injury molecule-1 (Kim-1) protein ectodomain in urine and kidney injury molecule-1/hepatitis A viral cellular receptor-1 (Kim-1/Havcr1) gene expression in kidney tissue may be more predictive of renal injury after exposure to nephrotoxicants when compared to traditionally used biomarkers. Male Sprague-Dawley rats were injected with a range of doses of gentamicin, mercury (Hg; HgCl2), or chromium (Cr; K2Cr2O7). The results showed that increases in urinary Kim-1 and kidney Kim-1/Havcr1 gene expression paralleled the degree of severity of renal histopathology and were detected at lower doses of nephrotoxicants when compared to blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-beta-D-glucosaminidase (NAG). In a time course study, urinary Kim-1 was elevated within 24 h after exposure to gentamicin (100 mg/kg), Hg (0.25 mg/kg), or Cr (5 mg/kg) and remained elevated through 72 h. NAG responses were nephrotoxicant dependent with elevations occurring early (gentamicin), late (Cr), or no change (Hg). At 72 h, after treatment with any of the three nephrotoxicants, there was increased Kim-1 immunoreactivity and necrosis involving approximately 50% of the proximal tubules; however, only urinary Kim-1 was significantly increased, while BUN, serum creatinine, and NAG were not different from controls. In rats treated with the hepatotoxicant galactosamine (1.1 mg/kg), serum alanine aminotransferase was increased, but no increase in urinary Kim-1 was observed. Urinary Kim-1 and kidney Kim-1/Havcr1 expression appear to be sensitive and tissue-specific biomarkers that will improve detection of early acute kidney injury following exposure to nephrotoxic chemicals and drugs.

FIG. 1

Representative photomicrographs of renal lesions and Kim-1 immunoreactivity in rats at 72 h following treatment with gentamicin, Hg, or Cr. Panels A, C, E, and G (X200) and I (X630), H&E stain; B, D, F, and H (X200) and J (X400), immunoperoxidase staining for Kim-1. Treatments represented in panels are as follows: panels A and B—saline: no renal lesion and no immunoperoxidase staining for Kim-1; panels C and D—gentamicin, 100 mg/kg, sc: necrotic tubular epithelial cells in proximal tubules showing positive Kim-1 immunoreactivity; Panels E and F—Hg, 0.25 mg/kg, iv: necrotic and vacuolated proximal tubules showing a strong positive reaction for Kim-1; panels G and H—Cr, 5 mg/kg, sc: severe necrotic cell in proximal tubules associated with Kim-1 immunoreactivity; panels I and J—Cr, 5 mg/kg, sc: cancer cells of renal cell carcinoma showing a strong positive reaction for Kim-1.

FIG. 2

Comparison of urinary Kim-1 and kidney Kim-1/Havcr1 gene expression with routinely used biomarkers BUN, serum creatinine, and urinary NAG and nephropathy score after various doses of gentamicin. Following the third daily injection of gentamicin as described in the Materials and Methods, rats were placed individually in plastic metabolism cages and urine collected for 24 h, after which rats were euthanized and blood and kidneys were collected. Urinary NAG and Kim-1 are normalized by urinary creatinine. Relative fold changes of BUN, creatinine, NAG, urinary Kim-1, and kidney Kim-1/Havcr1 expression for each animal were calculated by dividing the value for each individual animal level by the level averaged from all corresponding vehicle (saline or distilled water)-treated animals and are displayed in box and whisker plots. BUN (control) = 16.6 ± 3.3 mg/dL; serum creatinine (control) = 0.5 ± 0.0 mg/dL; urinary NAG (control) = 20.3 ± 10.6 (U/g creatinine); urinary Kim-1 (control) = 165.9 ± 141 (pg/mg creatinine); kidney Kim-1/Havcr1 (control) = 1.2 ± 0.7. Asterisks indicate statistically significant difference ( p < 0.05) compared to control group (n = 6 for each group).

FIG. 3

Comparison of urinary Kim-1 and kidney Kim-1/Havcr1 expression with routinely used biomarkers BUN, creatinine, and NAG and nephropathy score after treatment with various doses of Hg (HgCl₂). Immediately following the single injection of Hg as described in the Materials and Methods, rats were placed individually in plastic metabolism cages and urine collected for 24 h, after which rats were euthanized and blood and kidneys were collected. Urinary NAG and Kim-1 are normalized by urinary creatinine. Relative fold changes of BUN, serum creatinine, urinary NAG, urinary Kim-1, and kidney Kim-1/Havcr1 expression for each animal were calculated by dividing the value for each individual animal level by the level averaged from all corresponding vehicle (saline or distilled water)-treated animals and are displayed in box and whisker plots. BUN (control) = 16.6 ± 3.3 mg/dL; creatinine (control) = 0.5 ± 0.0 mg/dL; NAG (control) = 20.3 ± 10.6 (U/g creatinine); urinary Kim-1 (control) = 165.9 ± 141 (pg/mg creatinine); kidney Kim-1/Havcr1 = 1.2 ± 0.7. Asterisks indicate statistically significant difference ( p < 0.05) compared to control group (n = 6 for each group).

FIG. 4

Comparison of urine Kim-1 and kidney Kim-1/Havcr1 expression with routinely used biomarkers BUN, creatinine, and NAG and nephropathy score after treatment with various doses of Cr (K₂Cr₂O₇). Immediately following the single injection of Cr as described in the Materials and Methods, rats were placed individually in plastic metabolism cages and urine collected for 24 h, after which rats were euthanized and blood and kidneys were collected. Urinary NAG and Kim-1 are normalized by urinary creatinine. Relative fold changes of BUN, creatinine, NAG, urinary Kim-1, and kidney Kim-1 expression for each animal were calculated by dividing the value for each individual animal level by the level averaged from all corresponding vehicle (saline or distilled water)-treated animals and are displayed in box and whisker plots. BUN (control) = 16.6 ± 3.3 mg/dL; serum creatinine (control) = 0.5 ± 0.0 mg/dL; NAG (control) =20.3 ± 10.6 (U/g creatinine); urinary Kim-1 (control) = 165.9 ± 141 (pg/mg creatinine); kidney Kim-1/Havcr1 = 1.2 ± 0.7. Asterisks indicate statistically significant difference (p < 0.05) compared to control group (n = 6 for each group).

FIG. 5

NAG and Kim-1 levels in urine collected for various intervals after treatment with gentamicin (100 mg/kg, sc). A 24-h preinjection urine sample was collected prior to the injections of gentamicin as described in Materials and Methods, and urine was collected from the same rats in metabolism cages at intervals of 0–4, 4–8, 8–24, 24–48, and 48–72 h commencing immediately after the third sc injection. Urinary NAG and Kim-1 are normalized to urinary creatinine. Relative fold changes of NAG and urinary Kim-1 for each animal at each time interval were calculated by dividing the amount by the average amount from pretreatment (-24–0 h) samples of all animals including control and chemical-treated groups. NAG (pretreatment) = 12.7 ± 3.9 U/g creatinine; urinary Kim-1 (pretreatment) = 59.1 ± 17.6 pg/mg creatinine. Asterisks indicate statistically significant difference (p < 0.05) compared to control group (n = 6 for each group).

FIG. 6

NAG and Kim-1 levels in urine for various intervals after treatment with Hg (0.25 mg/kg, iv). A 24-h preinjection urine sample was collected, and urine was collected from the same rats in metabolism cages at intervals of 0–4, 4–8, 8–24, 24–48, and 48–72 h commencing immediately after the single injection. Urinary NAG and Kim-1 are normalized to urinary creatinine. Relative fold changes of NAG and urinary Kim-1 for each animal at each time interval were calculated by dividing the amount by the average amount from pretreatment samples of all animals including control and chemical-treated groups. NAG (pretreatment) = 12.7 ± 3.9 U/g creatinine; urinary Kim-1 (pretreatment) = 59.1 ± 17.6 pg/mg creatinine. Asterisks indicate statistically significant difference ( p < 0.05) compared to control group (n = 6 for each group).

FIG. 7

NAG and Kim-1 in urine for various intervals following treatment with Cr (5 mg/kg). A 24-h preinjection urine sample was collected, and urine was collected from the same rats in metabolism cages at intervals of 0–4, 4–8, 8–24, 24–48, and 48–72 h commencing immediately after the single injection. Urinary NAG and Kim-1 are normalized by urinary creatinine. Relative fold changes of NAG and urinary Kim-1 for each animal at each time interval were calculated by dividing the amount by the average amount from pretreatment samples of all animals including control and chemical-treated groups. NAG (pretreatment) = 12.7 ± 3.9 U/g creatinine; urinary Kim-1 (pretreatment) = 59.1 ± 17.6 pg/mg creatinine. Asterisks indicate statistically significant difference ( p < 0.05) compared to control group (n = 6 for each group).

FIG. 8

Comparison of urinary Kim-1 with routinely used biomarkers BUN, creatinine, and NAG and nephropathy score for tubule injury 72 h after gentamicin (100 mg/kg, sc), Hg (0.25 mg/kg, iv), Cr (5 mg/kg, sc) administration. Urinary NAG and Kim-1 are normalized to urinary creatinine. Asterisks indicate statistically significant difference ( p < 0.05) compared to control group (n = 6 for each group).