Overexpression of miRNA-5100 with PEI-NPs led to change in expression of many mRNAs

The aim of this study was to identify miRNAs that regulate AKI and develop their applications as diagnostic biomarkers and therapeutic agents. First, kidney tissues from two different AKI mouse models, namely, AKI induced by the administration of lipopolysaccharide (LPS) causing sepsis (LPS-AKI mice) and AKI induced by renal ischemia–reperfusion injury (IRI-AKI mice), were exhaustively screened for their changes of miRNA expression compared with that of control mice by microarray analysis followed by quantitative RT-PCR. The initial profiling newly identified miRNA-5100, whose expression levels significantly decreased in kidneys in both LPS-AKI mice and IRI-AKI mice. Next, the administration of miRNA-5100-mimic conjugated with a nonviral vector, polyethylenimine nanoparticles (PEI-NPs), via the tail vein significantly induced miRNA-5100 overexpression in the kidney and prevented the development of IRI-AKI mice by inhibiting apoptosis and inflammation in vivo. Furthermore, serum levels of miRNA-5100 in patients with AKI were identified as significantly lower than those of healthy subjects. ROC analysis showed that the serum expression level of miRNA-5100 can identify AKI (cut-off value 0.14, AUC 0.96, sensitivity 1.00, specificity 0.833, p<0.05). These results suggest that miRNA-5100 regulates AKI and may be useful as a novel diagnostic biomarker and therapeutic target for AKI. The effect of miRNA-5100-mimic treatment was investigated by an in vivo, nonviral vector gene transfection method 38. Polyethyleneimine is a crude nonlipid polycationic polymer with a positive charge on its surface. miRNAs are mixed with a negative charge to create a complex by mixing them. miRNAs with PEI-NPs have high biocompatibility, stability, and transfection ability. The sequence of the miRNA-5100 mimic used was 5-UCGAAUCCCAGCGGUGCCUCU-3. Complexes of miRNA-5100 mimics and PEI-NPs were formed at an N/P ratio of 6 with miRNA-5100 at a concentration of 5 nmol in a volume of 200 μl (GeneDesign). Complexes of control miRNA and PEI-NPs were similarly formed with control miRNA and an N/P ratio of 6 at a concentration of 5 nmol in a volume of 200 μl. miRNA with PEI-NPs was administered by tail vein injection in all mouse models. The sequence of control miRNA was 5-GGUUCGUACGUACACUGUUCA-3(GeneDesign). Four different protocols for preventing AKI were used in this study for comparison (sham model, IRI-AKI model, IRI-AKI+miRNA-5100-mimic-PEI-NPs model, IRI-AKI+control-miRNA-PEI-NPs model). In the sham model, a midline incision was made in the abdominal skin, muscle, and peritoneal membrane. In the IRI-AKI mouse model, an operation involving right nephrectomy and left renal clamp and reperfusion (for 45 min) was performed. In the IRI-AKI+miRNA-5100-mimic-PEI-NPs model, miRNA-5100-mimic with PEI-NPs was injected via the tail vein 2 days before the IRI procedure. The mixture was adjusted to be 5 nmol miRNA-5100-mimic with PEI-NPs in a volume of 200 μl. In the IRI-AKI+control-miRNA-PEI-NPs model, control miRNA with PEI-NPs was injected via the tail vein 2 days before the IRI procedure. The mixture was adjusted to be 5 nmol control miRNA with PEI-NPs in a volume of 200 μl. All mouse models underwent blood sampling and removal of the left kidney 24 h after the operation. A blood sample was extracted from the vena cava from each animal.