Renal ischemia-reperfusion (I/R) injury was investigated in inducible nitric oxide synthase (iNOS) knockout mice. After a 26-min bilateral renal pedicle clamp, serum creatinine concentrations (in mg/dl) in wild-type mice after a 24-h reperfusion were 0.25 ± 0.03 in sham-operated controls and 2.3 ± 0.38 in ischemic mice ( P < 0.01); after 48 h, concentrations (in mg/dl) were 0.25 ± 0.03 in controls and 2.0 ± 0.18 in ischemic mice ( P < 0.01). iNOS knockout mice demonstrated an attenuation of serum creatinine concentration after renal I/R injury. Serum creatinine concentrations (mg/dl) after a 24-h reperfusion were 2.3 ± 0.22 in wild-type ischemic and 1.21 ± 0.25 in iNOS knockout ischemic mice ( P < 0.05); after 48 h, concentrations were 2.0 ± 0.18 in wild-type ischemic and 0.96 ± 0.25 in iNOS knockout ischemic mice ( P< 0.01). Histological scoring of acute tubular necrosis in iNOS knockout mice was decreased compared with that in wild-type controls (0.88 ± 0.2 vs. 3.3 ± 0.3, P< 0.05). iNOS protein in the renal cortex of wild-type mice subjected to renal I/R injury was undetectable up to 48 h. However, a strong upregulation of heat shock protein 72 expression was observed in renal cortex of iNOS knockout mice under basal conditions. In conclusion, kidneys of iNOS knockout mice were protected against ischemic acute renal failure. This protective effect may be related to a compensatory upregulation of heat shock protein 72.

Background/Aims: Type 2 Diabetes Mellitus (T2DM) is characterized by insulin resistance (IR), but the underlying molecular mechanisms are incompletely understood. MicroRNAs (miRNAs) have been demonstrated to participate in the signalling pathways relevant to glucose metabolism in IR. The purpose of this study was to test whether the multiple-target anti-miRNA antisense oligonucleotides (MTg-AMO) technology, an innovative miRNA knockdown strategy, can be used to interfere with multiple miRNAs that play critical roles in regulating IR. Methods: An MTg-AMO carrying the antisense sequences targeting miR-106b, miR-27a and miR-30d was constructed (MTg-AMO_106b/27a/30d). Protein levels were determined by Western blot analysis, and transcript levels were detected by real-time RT-PCR (qRT-PCR). Insulin resistance was analysed with glucose consumption and glucose uptake assays. Results: We found that the protein level of glucose transporter 4 (GLUT4), Mitogen-activated protein kinase 14 (MAPK 14), Phosphatidylinositol 3-kinase regulatory subunit beta (PI3K regulatory subunit beta) and mRNA level of Slc2a4 (encode GLUT4), Mapk14 (encode MAPK 14) and Pik3r2 (encode PI3K regulatory subunit beta) were all significantly down-regulated in the skeletal muscle of diabetic rats and in insulin-resistant L6 cells. Overexpression of miR-106b, miR-27a and miR-30d in L6 cells decreased glucose consumption and glucose uptake, and reduced the expression of GLUT4, MAPK 14 and PI3K regulatory subunit beta. Conversely, silencing of endogenous miR-106b, miR-27a and miR-30d in insulin-resistant L6 cells enhanced glucose consumption and glucose uptake, and increased the expression of GLUT4, MAPK 14 and PI3K regulatory subunit beta. MTg-AMO_106b/27a/30d up-regulated the protein levels of GLUT4, MAPK 14 and PI3K regulatory subunit beta, enhanced glucose consumption and glucose uptake. Conclusion: Our data suggested that miR-106b, miR-27a and miR-30d play crucial roles in the regulation of glucose metabolism by targeting the GLUT4 signalling pathway in L6 cells. Moreover, MTg-AMO_106b/27a/30d offers more potent effects than regular singular AMOs.