MiR-181d-5p Targets KLF6 To Improve Ischemia/Reperfusion-induced AKI Through Effects On Renal Function,Apoptosis And Inflammation

Objective: Renal tubular epithelial cell(RTEC)death and renal interstitial inflammation are the most crucial pathophysiological changes in acute kidney ischemia/reperfusion injury(IRI).The microRNA(mi R)-181 d family plays diverse roles in cell proliferation,apoptosis and inflammation,but its renal target and potential role in IRI are unknown.Methods:(1)Multiple bioinformatics softwares were applied to predict target gene for miR-181d-5p and Kruppel-like factor 6(KLF6)function.(2)In vivo experiment: C57BL/6J male mice,eight-week-old,were randomly divided into: normal group,sham group,and IRI group.The AAV-control,AAV-miR-181d-5p,AAV-shRNA were injected into the renal parenchyma respectively.Three weeks later,renal IRI models(bilateral kidney pedicle was clamped for 45 minutes)were built and the tissue and blood were taken for experiment.(1)Biochemical detection: Clinical blood biochemical analyzer to measure the level of Scr and BUN.(2)Histopathological examination: In situ hybridization and immunohistochemistry were used to observe the expression and distribution of mi R-181d-5p and KLF6;Hematoxylin-eosin(HE)staining was used to observe renal histopathology;TUNEL staining was used to observe apoptosis.(3)Molecular biology detection: qRT-PCR,Western Blot and ELISA were used to detect the expression of miR-181d-5p,KLF6,KIM-1,caspase-3,NF-?B,IL-6 and TNF-? in kidney tissue.(3)In vitro experiments: HK-2 cells were divided into normal group and H/R group(hypoxia 24 h reoxygenation 6h).miR-181d-5p mimics or miR-181d-5p inhibitors or their control sequences were transfected by liposome to overexpress or to inhibit miR-181d-5p expression.KLF6 overexpression plasmid(KLF6 plasmid)or KLF6 suppression plasmid(KLF6 RNAi)or empty plasmid were transfected by liposome to overexpression or to inhibition of KLF6 expression.(1)qRT-PCR,Western Blot and ELISA were used to detect the expression of miR-181d-5p,KLF6 and related factors of renal function,inflammation and apoptosis in cells and supernatants;(2)Annexin V-FITC/PI double-parameter method detection by flow cytometry were used to observe HK-2 cell apoptosis.(3)Double luciferase reporter gene were used to detect whether miR-181d-5p binded to KLF6 promoter.(4)ANOVA and Bonferroni test were used as statistical methods such as ANOVA and Bonferroni test to analyze the data.Results:(1)Here,we showed that the expression of miR-181d-5p decreased and KLF6 increased in a renal cell(HK-2)model of hypoxia/reoxygenation(H/R)injury and a mouse model of renal IRI.They were mainly distributed in in the renal tubules.(2)After renal I/R,overexpression of mi R-181d-5p significantly inhibited inflammatory mediators,reduced apoptosis and further improved renal function.(3)KLF6 exacerbated renal tubular epithelial cell damage and act as a NF-?B co-activator to aggravate the renal IRI inflammatory response.(4)Mechanistically,KLF6 was predicted as a new potential target gene of miR-181d-5p through bioinformatic analysis and luciferase reporter assay verification.(5)After overexpressing miR-181d-5p and inhibiting KLF6,the role of miR-181d-5p disappeared on the renal damage improvement.Conclusion: MiR-181d-5p upregulation produced protective anti-apoptotic and antiinflammatory effects against IRI in kidneys in vivo and H/R injury in HK-2 cells in vitro,and these effects were achieved by targeted inhibition of KLF6.Thus,our results provide novel insights into the molecular mechanisms associated with IRI and a potential novel therapeutic target.