Novel assays for detection of urinary KIM-1 in mouse models of kidney injury

Abstract

Kidney injury molecule-1 (KIM-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a urinary biomarker to monitor preclinical nephrotoxicity in rats and on a case-by-case basis for the translation of potentially nephrotoxic drugs into first-in human studies. Although mouse models are widely employed in preclinical studies, few urinary biomarker studies have been performed in mice due to limited urine availability and lack of sensitive assays. Here, we report the development and validation of two different assays for quantitative assessment of mouse urinary KIM-1 (uKIM-1) and compare the sensitivity of KIM-1 relative to other standard markers in ischemia reperfusion and aristolochic acid (AA)-induced kidney injury in mice. A sensitive, reproducible, and quantitative microbead-based KIM-1 ELISA was established, which requires only 10 μl urine for triplicate determination with an assay range of 12.21 pg/ml to 50 ng/ml. The second assay is a laminar flow dipstick assay, which has an assay range of 195 pg/ml to 50 ng/ml and provides quantitative assessment of KIM-1 in 15 min. uKIM-1 levels increased with increasing time of ischemia or time after AA administration. After only 10-min ischemia followed by 24-h reperfusion, uKIM-1 was significantly elevated by 13-fold, whereas serum creatinine (sCr), blood urea nitrogen, N-acetyl-β-glucosaminidase (NAG), and proteinuria levels did not change. After AA administration, uKIM-1 levels were significantly upregulated by greater than threefold within 12 h, whereas sCr and NAG levels were unchanged. Mouse KIM-1 was stable for multiple freeze-thaw cycles, for up to 5 days at room temperature and up to at least an year when stored at -80°C.

FIG. 1.

Evaluation of mouse microbead-based KIM-1 assay. (A) Mouse recombinant KIM-1 protein was serially diluted and 13-point standard curve was generated by microbead-based ELISA assay. (B) Precision profile for KIM-1 assay. The intra- and interassay reproducibility of the assay was assessed by measuring 13 serially diluted samples (50,000 to 12.21 pg/ml of KIM-1). For the intra-assay variability, each sample was measured in six replicates within one plate. For the interassay variability, each sample was measured 5 times in five different plates. (C) Linearity of dilution of KIM-1 assay. Urine samples were diluted (1:2, 1:4, 1:8, 1:10, 1:16, 1:32, and 1:64) in sample diluents, and KIM-1 levels were evaluated in triplicate. The percent recovery is calculated.

FIG. 2.

Evaluation of KIM-1 stability in urine matrix. (A) Recovery of KIM-1 in urine samples stored at RT, 4°C, −20°C, and −80°C over different length of times. The plotted data are the means ± SD values of three samples. (B) Stability of uKIM-1 after multiple freeze-thaw cycles. KIM-1 levels were evaluated in freshly collected urine samples and after multiple freeze-thaw (FT; n = 5) cycles over 5 days.

FIG. 3.

Serum and urinary markers after administration of 5mg/kg AA. Male BALB/c mice were administered 5mg/kg AA IP in 0.9% saline. Blood, urine, and tissue were collected on days 0, 0.5, 1, 3, 5, 11, and 21. Urine was centrifuged at 3000rpm for 5min, and aliquots were stored at −80°C until further analysis. Serum markers, including sCr (A) and BUN (B), and urinary markers, NAG (C), Protein (D), and KIM-1 (E), were measured. For KIM-1 measurement, samples were diluted 1:10 in sample diluent. All urinary biomarkers were normalized to urinary creatinine levels. *p ≤ 0.05 compared with 0-h time point.

FIG. 4.

KIM-1 expression and histology in the mouse kidney after AA 5mg/kg administration. (A) Immunofluorescence staining of frozen kidney sections from AA-treated mice. Tissues were stained with KIM-1 antibody (R&D systems) as described in the Materials and Methods section. Tissue KIM-1 was clearly detected in 24h after AA (5mg/kg body weight) administration. (B) Western blot of the whole tissue kidney lysates collected from AA-treated (5mg/kg body weight) mouse. No KIM-1 was detected on day 0, but the protein levels became detectable by day 1 (24h) after AA administration. (C) Histology scoring of kidneys from AA-treated mice. Sections were evaluated for microvilli loss, necrosis, regeneration, inflammatory cell infiltration, and fibrosis on kidney tissues collected on day 0, 1, 2, 3, 5, 7, 11, and 21.

FIG. 5.

Correlation of sCr and uKIM-1 with histology score in AA-treated mice. Urine, serum, and kidneys were collected at different time periods after AA (5mg/kg) or vehicle treatment and analyzed for biomarkers and histopathology (PAS staining). uKIM-1 (A) and sCr (B) and plotted versus histopathology score. Animals with no renal histopathology (Histopathology score 0) were separated based on Vehicle treatment (Veh) or AA treatment (AA).

FIG. 6.

Comparison of uKIM-1 to conventional biomarkers 24h after different times of renal ischemia. Male BALB/c mice were subjected to 0 (sham), 10, 20, or 30min of bilateral ischemia by clamping the renal pedicles for the specific time. After reperfusion, urine, blood, and tissue were collected at 24h. sCr (A), BUN (B), urinary NAG (C), Protein (D), absolute uKIM-1 (E), and uKIM-1 normalized to urinary creatinine (F) were measured as described in the Materials and Methods section. *p ≤ 0.001 compared with 0-h time point and sham group, n = 10 for each group. (G) Immunofluorescence staining of frozen kidney sections from mice subjected to I/R injury. Tissues were stained with monoclonal KIM-1 antibody (R&D systems) as described in the Materials and Methods section. There is dose-dependent increase in KIM-1 expression seen with the increase in ischemia time.

FIG. 7.

Evaluation biomarker performance using ROC analysis. (A) ROC analysis of urinary biomarkers (KIM-1, NAG, and total protein) in mice subjected to ischemia/reperfusion over different degrees of histological injury (histopathology score 0–1, histopathology score 0–2, and histopathology score 0–3). (B) Performance of biomarkers, including sCr, uKIM-1 (absolute and normalized), NAG (normalized), and proteinuria (normalized) in detecting kidney injury over different histological grades.

FIG. 8.

Detection of KIM-1 using mouse Rena-Stick in renal I/R model. KIM-1 in standard solutions or urine samples is measured by mixing 60 μl of sample with 60 μl of TRIS-buffered solution and adding the mix to the port in Rena-Stick. Results were read in 15min. (A) A concentration-dependent decrease in the band intensity was observed with a decrease in recombinant mouse KIM-1 protein standard concentration. (B) A visual increase in KIM-1 band intensity with an increase in bilateral ischemia time in mice subjected to renal I/R, whereas no KIM-1 positive band was observed when sham-operated mouse urine was analyzed. (C) The band intensity was quantified using a hand-held lateral-flow reader, and a standard curve was drawn by plotting the recombinant mouse KIM-1 concentration on x-axis and band intensity units on y-axis. (D) A positive correlation between KIM-1 values obtained by quantification of band intensity using the lateral-flow reader and values obtained by microbead-based assay in urines collected from bilateral I/R mice.

FIG. 9.

Detection of KIM-1 using mouse Rena-Stick in AA model. KIM-1 in standard solution or urine samples is measured by mixing 60 μl of sample with 60 μl of TRIS-buffered solution and adding the mix to the sample port in Rena-Stick. Results were read in 15min. (A) A visual increase in KIM-1 band intensity at various time points after one dose of AA (5mg/kg). KIM-1 values obtained by quantification of band intensity using the lateral-flow reader were plotted versus values obtained by microbead-based assay in urines collected from AA-treated mice (B) and control mice (C).