A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury

Abstract

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.

Fig 1. Serum microRNAs are diagnostic biomarkers of AKI in ICU patients.

(A) miRNAs were detected by RT-qPCR in serum samples from ICU patients and healthy controls. miRNA levels are expressed as ΔCp and data are presented as median and interquartile range. Asterisks indicate statistical significance (*P<0.05; ** P<0.01) (B) Spike-In raw Cp values as control. (C) ROC curve analysis.

Fig 2. Serum microRNAs indicate AKI severity.

Serum miRNAs were detected by qRT-PCR in samples from ICU patients at the moment of AKI diagnosis. (A) Serum levels of miR-210-3p, miR-126-3p, miR-29a-3p and miR-146a-5p correlate with AKI severity determined by AKIN classification and with increasing severity from 1 to 3 stage. miRNA expression levels are shown as ΔCp values and each patient is represented by an individual dot. Spearman correlation was performed for each miRNA and p-values are shown in the corresponding plots. Asterisks indicate statistical significance (*P<0.05 and ** P<0.01).

Fig 3. Serum miRNAs are AKI precocious biomarkers in cardiac surgery patients.

miR-26b-5p, miR-146a-5p, miR-93-3p and miR-127-3p serum levels are expressed as ΔCp values and data is presented as individual dots. AKI was diagnosed based on AKIN serum creatinine criteria. Spearman Rho correlation coefficient was calculated and p-values are indicated in each panel.

Fig 4. Serum miRNAs estimated before cardiac surgery identify patients in risk of AKI development.

(A) miRNAs were detected by RT-qPCR in serum samples obtained before surgery, expressed as ΔCp. Data are presented as median and interquartile range. Asterisks indicate statistical significance (*P<0.05; ** P<0.01). (B) Spike-In raw Cp values are shown as control.

Fig 5. ROC analysis of serum miRNAs as AKI risk biomarkers.

ROC curves of miR-26b-5p, miR-27a-3p, miR-93-3p and miR-127-3p expression levels in serum, determined by qRT-PCR, in samples obtained before surgery.