Predictive value of miR-582-5p for onset of sepsis-induced acute kidney injury and its functional role during disease development

Abstract

Introduction: Acute kidney injury (AKI) is a common complication of sepsis, characterized by sharply declining renal function. As a global concern, understanding its pathogenesis and improving diagnosis and therapy face significant challenges. MicroRNAs are involved in the progression of a variety of diseases.This research was focused on differences in expression and clinical predictive value of miR-582-5p in sepsis-induced AKI.

Material and methods: Blood and urine samples were collected from 180 patients. Sepsis-induced AKI was imitated in vitro by human kidney 2 (HK2) cells treated with 10 µg/ml lipopolysaccharide (LPS). The relative expression of miR-582-5p and HMGB2 in different conditions was quantified by qRT-PCR. Regulation of gene expression was performed by cell transfection. Cell viability and apoptosis were detected subsequently. Kidney injury and inflammatory assessment were analyzed by means of ELISA. Estimation of oxidative stress was performed using the corresponding kit. The dual luciferase reporter system verified the targeting relationship between miR-582-5p and HMGB2.

Results: Relative expression of miR-582-5p was lower in sepsis patients who suffered AKI later, along with LPS-induced HK2 cells. Both weak viability and elevated apoptosis were reversed by up-regulated miR-582-5p in HK2 cells exposed to LPS. In addition, the concentration of inflammatory factors and oxidation levels showed a significant decrease, based on up-regulated miR-582-5p. The clinical predictive value of miR-582-5p was visualized by a ROC curve with high sensitivity and specificity.

Conclusions: Up-regulated miR-582-5p reduced sepsis-induced AKI, and HMGB2 was a potential downstream target of miR-582-5p.

Keywords: acute kidney injury; miR-582-5p; sepsis.

Fig. 1

Expression of miR-582-5p in sepsis patients and its diagnostic performance. A) Relative expression of miR-582-5p in sepsis-induced acute kidney injury (AKI) patients (n = 90), compared to the non-AKI groups (n = 90). B) Evaluation of the diagnostic ability of miR-582-5p. Each sample was bio-replicated 3 times. A) Student’s t test. ***p < 0.001 compared to non-AKI group

Fig. 2

Function of miR-582-5p in LPS-induced HK2 cells. A) Cell viability decreased with time. B) Relative expression of miR-582-5p decreased with time, assessed by CCK-8 kit. C) Successful cell transfection verified by qRT-PCR of miR-582-5p. D) Poor cell viability was improved by increased miR-582-5p. E) The proportion of cell apoptosis decreased with increased miR-582-5p. F) Up-regulated miR-582-5p diminished the inflammatory response. Three repetitions were required for each experiment and each sample was bio-replicated 3 times. A-G) Student’s t test. *p < 0.05 compared to control; ***p < 0.001 vs. control group; ##p < 0.01 vs. LPS + mimic-NC; ###p < 0.001 vs. LPS + mimic-NC. G) Highly expressed miR-582-5p enhanced the ability to resist oxidative stress. Three repetitions were required for each experiment and each sample was bio-replicated 3 times. G) Student’s t test. *p < 0.05 compared to control; ***p < 0.001 vs. control group; ##p < 0.01 vs. LPS + mimic-NC; ###p < 0.001 vs. LPS + mimic-NC

Fig. 3

MiR-582-5p regulated HMGB2 in a targeted manner. A) Complementary binding sites between miR-582-5p and HMGB2. B) Dual-luciferase reporter assay to analyze the interaction of miR-582-5p and HMGB2 in HK-2 cells. C) Expression level of HMGB2 measured by qRT-PCR. D) Correlation between miR-582-5p and HMGB2 by Pearson correlation analysis. Three repetitions were required for each experiment and each sample was bio-replicated 3 times. B, C) One-way analysis of variance. Student’s t test. ***p < 0.001 compared to control group, ###p < 0.001 compared to LPS + mimic-NC