Detection of Drug-Induced Acute Kidney Injury in Humans Using Urinary KIM-1, miR-21, -200c, and -423

Abstract

Drug-induced acute kidney injury (AKI) is often encountered in hospitalized patients. Although serum creatinine (SCr) is still routinely used for assessing AKI, it is known to be insensitive and nonspecific. Therefore, our objective was to evaluate kidney injury molecule 1 (KIM-1) in conjunction with microRNA (miR)-21, -200c, and -423 as urinary biomarkers for drug-induced AKI in humans. In a cross-sectional cohort of patients (n = 135) with acetaminophen (APAP) overdose, all 4 biomarkers were significantly (P < .004) higher not only in APAP-overdosed (OD) patients with AKI (based on SCr increase) but also in APAP-OD patients without clinical diagnosis of AKI compared with healthy volunteers. In a longitudinal cohort of patients with malignant mesothelioma receiving intraoperative cisplatin (Cp) therapy (n = 108) the 4 biomarkers increased significantly (P < .0014) over time after Cp administration, but could not be used to distinguish patients with or without AKI. Evidence for human proximal tubular epithelial cells (HPTECs) being the source of miRNAs in urine was obtained first, by in situ hybridization based confirmation of increase in miR-21 expression in the kidney sections of AKI patients and second, by increased levels of miR-21, -200c, and -423 in the medium of cultured HPTECs treated with Cp and 4-aminophenol (APAP degradation product). Target prediction analysis revealed 1102 mRNA targets of miR-21, -200c, and -423 that are associated with pathways perturbed in diverse pathological kidney conditions. In summary, we report noninvasive detection of AKI in humans by combining the sensitivity of KIM-1 along with mechanistic potentials of miR-21, -200c, and -423.

Keywords: KIM-1; acute kidney injury; biomarker; microRNAs; nephrotoxicity in patients.

FIG. 1

Urinary profiles of KIM-1, miR-21, -200c, and -423 in APAP-induced kidney injury. Levels of miR-21, -200c, and -423 and KIM-1 were measured in urine from healthy controls (n = 65) as well as in patients with an APAP-OD (n = 70). Within latter group approximately 62% of the patients developed clinically proven AKI (50% increase in SCr). MiRNA levels and KIM-1 concentrations were normalized to urinary creatinine. A, Both data sets were log2 transformed and presented as box plots (median with inter quartile range) with 5th and 95th percentile as whiskers. T-test was used for P-value calculation: *P < .004. B, Table with absolute levels as medians (interquartile range) of arbitrary miRNA levels (×10⁻²; 2^−ΔCt normalized by UCr) or absolute KIM-1 concentration (pg/mg urinary creatinine). UCr, urinary creatinine.

FIG. 2

KIM-1, miR-21, -200c, and -423 increase in the longitudinal Cp cohort in patients with and without clinical AKI. Levels of miR-21, -200c, and -423 as well as KIM-1 were measured in urine from patients (n = 108) before (Pre) and on 9 following time points after Cp treatment grouping patients based on their SCr dependent AKI status. Data sets were normalized to UCr, log2 transformed and presented as box plots (median with 25th and 75th percentiles) with 5th and 95th percentile as whiskers. T-test was used for P-value calculation to compare with the group baseline as well as within the two groups: *P < .0014. Broken lines represent the median level before treatment of No AKI developers.

FIG. 3

Localization of miR-21, -200c, and 423 in human kidney biopsies from patients with ATN. Pathological examination of the biopsy samples was performed with standard H&E stained sections. Using in situ hybridization, the expression patterns of miR-21, -200c, and -423 were assessed in kidney biopsies from normal controls and patients diagnosed with ATN. In situ hybridization settings were proven with U6 as positive control, having an abundant nuclear expression, and a scrabbled probe, not complementary to any known miRNA sequence, used as negative control. Star, tubular casts; arrow, positive miR-21 signal. For in situ: blue, positive probe staining; pink, counter staining; H&E pictures taken with 10× others with 20x objective; representative images of n = 3 controls and n = 3 ATN patients.

FIG. 4

HPTECs release miR-21, -200c, and -423 following toxicity. HPTECs were treated with Cp and 4-aminophenol (APAP degradation product) for 24 h followed by measurement of viability and miR-21, -200c, and -423. A, Dose-response curves after 24 h treatment with 0, 10, 31.6, 100, 316, and 1000 μM Cp and 4-aminophenol, respectively. Data are presented as means with standard deviation of percentage viability compared with the 0.5% DMSO treated control with fitted nonlinear dose–response curves (n = 3–6). Levels of miR-21, -200c, and -423 in the medium supernatant (B) as well as in the cells (C) after 24h treatment with 85 μM Cp and 100 μM 4-aminophenol, respectively. Data are presented as mean with SD (n = 3–4) of relative quantities (RQ, 2^−ΔΔCt). 1-way ANOVA with Dunnett’s test was used for P-value calculation: *P < .05, **P < .01, and ***P < .001.

FIG. 5

Target prediction analysis of miRNA biomarker candidates. IPA was used to identify mRNA targets of the 3 miRNAs (experimentally verified and highly predicted based on sequence complementarity). A, Within the pool of 1102 identified targets, IPA’s Core Analysis tool revealed associated pathways and pathological conditions. Bases on the ratio of genes found in the pool vs. genes related to the specific pathways or diseases, top 5 associated pathways and pathological conditions of the kidney were listed. B, All 49 targets associated with Renal Necrosis/Cell Death were plotted as network with the 3 miRNAs revealing overlapping targets as well as a complex interaction between the targets.