Urinary biomarkers for sensitive and specific detection of acute kidney injury in humans

Abstract

Acute kidney injury (AKI) is associated with high morbidity and mortality. The lack of sensitive and specific injury biomarkers has greatly impeded the development of therapeutic strategies to improve outcomes of AKI.The unique objective of this study was to evaluate the diagnostic performance of nine urinary biomarkers of AKI-kidney injury molecule-1 (KIM-1), neutrophil gelatinase associated lipocalin (NGAL), interleukin-18 (IL-18), hepatocyte growth factor (HGF), cystatin C (Cys), N-acetyl-beta-D-glucosaminidase (NAG), vascular endothelial growth factor (VEGF), chemokine interferon-inducible protein 10 (IP-10; CXCL10), and total protein-in a cross-sectional comparison of 204 patients with or without AKI.Median urinary concentrations of each biomarker were significantly higher in patients with AKI than in those without AKI (p < 0.001). The area under the receiver operating characteristics curve (AUC-ROC) for the combination of biomarkers using a logic regression model [risk score of 2.93*(NGAL > 5.72 and HGF > 0.17) + 2.93*(PROTEIN > 0.22) -2*(KIM < 0.58)] was greater (0.94) than individual biomarker AUC-ROCs. Age-adjusted levels of urinary KIM-1, NAG, HGF, VEGF, and total protein were significantly higher in patients who died or required renal replacement therapy (RRT) when compared to those who survived and did not require RRT.Our results demonstrate the comparative value of multiple biomarkers in the diagnosis and prognosis of AKI.

Keywords: acute kidney injury; biomarkers; hepatocyte growth factor; kidney injury molecule-1; neutrophil gelatinase associated lipocalin.

Figure 1

Evaluation of microbead‐based assay for quantitation of human urinary KIM‐1, NGAL, IL‐18, HGF, VEGF, and IP‐10. (A) Standard curve for human KIM‐1 was obtained using purified recombinant human KIM‐1 ectodomain fusion protein. It demonstrated linearity over five orders of magnitude from 40 pg/mL to 160,000 pg/mL with the lowest limit of detection (LLD) to be 4.4 pg/mL (B) Standard curve for human NGAL was obtained using a commercially available purified NGAL protein. The NGAL standard curve was also linear over five orders of magnitude from 0.49 to 1,000 ng/mL with the LLD of 534 pg/mL The standard curves for IL‐18 (C) and HGF (D) ranged from 0.12 to 2,000 pg/mL and 0.7 to 1,446 pg/mL with the LLD of 125 fg/mL and 709 fg/mL respectively. Similarly, the standard curves for VEGF (E) and IP‐10 (F) ranged from 7.8 to 31,982 pg/mL and 25 to 10,000 pg/mL with the LLD of 10 pg/mL and 32 pg/mL, respectively. The standard curves were plotted as five parameter logistic curves and repeated eight times on different sets of samples on different days using different sets of beads coupled with different batches of primary antibody. The inset in each panel documents the linearity of the maximum fluorescence intensity at lower concentrations.

Figure 2

Scatterplot of human urinary (A) KIM‐1, (B) protein, (C) NGAL, (D) HGF, (E) IP‐10, (F) Cystatin C, (G) IL‐18, (H) NAG, and (I) VEGF in patients with and without a clinical diagnosis of acute kidney injury. Urinary biomarker measurements were normalized to urine creatinine and were plotted on a logarithmic Y‐axis. The number of patients in each group is indicated below each category on the X‐axis.

Figure 2

Scatterplot of human urinary (A) KIM‐1, (B) protein, (C) NGAL, (D) HGF, (E) IP‐10, (F) Cystatin C, (G) IL‐18, (H) NAG, and (I) VEGF in patients with and without a clinical diagnosis of acute kidney injury. Urinary biomarker measurements were normalized to urine creatinine and were plotted on a logarithmic Y‐axis. The number of patients in each group is indicated below each category on the X‐axis.