MicroRNA-10 Negatively Regulates Inflammation In Diabetic Kidney Via Targeting NLRP3 Inflammasome

BackgroundChronic kidney disease is a public health problem all over the world.Diabetic kidney disease is the leading cause of chronic kidney disease and end-stage renal disease.It is predicted that by 2040,the number of diabetic patients aged 20-79 will increase to 642 million,of which 30-40%will become diabetic kidney disease.Therefore,all strategies that can delay or prevent diabetic kidney disease have extremely important social and economic significance.NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)is an intracellular sensor that detects a broad range of metabolic stress signals to result in the formation and activation of the NLRP3 inflammasome.Assembly of the NLRP3 inflammasome leads to.caspase-1-dependent release of cytokines IL-1β and IL-18,and triggers the pro-inflammatory cascade in diabetic kidney.Therefore,the mechanism of NLRP3 inflammasome activation maybe provide a new therapeutic target for renal inflammation in diabetic kidney disease.MicroRNAs are endogenously produced small non-coding single-stranded RNA that repress specific target gene expression by post-transcriptional regulation.Although many microRNAs have been studied in various kidney diseases,the role of kidney-specific microRNAs in diabetic kidney disease is still unclear.Methods1.Tissue-or organ-specific expression pattern and cellular location of microRNA-10 were evaluated by in situ hybridization,quantitative real-time PCR and cell fluorescence in situ hybridization2.In diabetic kidney disease models,including STZ-treated and db/db diabetic mice,the expression of microRNA-10,NLRP3 protein and inflammatory cells in the kidney was measured by in situ hybridization,immunohistochemistry and Western blot.And the relationship between microRNA-10 and NLRP3 protein was analyzed in kidney tissue section.3.To determine whether microRNA-10 interacts with NLRP3 mRNA 3’-untranslated region,we predicted the potential targets of microRNA-10 using a combination of several bioinformatics software programs.4.Luciferase assay was employed to validate whether microRNA-10 binds to human NLRP3 mRNA 3’-untranslated region.5.In vitro,we knocked out microRNA-10 by CRISPR-Cas 9 to observe the regulatory effect of microRNA-10 on NLRP3 inflammasome.6.In diabetic kidney disease mouse model,the lentiviral vector was used to knout out,overexpress or restore microRNA-10 in kidney to verify microRNA-10 regulating inflammation in diabetic kidney disease by NLRP3 inflammasome.7.In situ hybridization and immunohistochemistry were used to detect expression of microRNA-10,NLRP3 and macrophages infiltration in kidney biopsy from the patients diagnosed diabetic kidney disease.And the relationship between microRNA-10 and renal inflammation was analyzed.ResultsIn the present study,we identified a conserved and kidney-enriched microRNA,microRNA-10(including 2 members,miR-10a and miR-10b),expressed mainly in cytoplasma of tubular epithelial cells and glomerular podocytes.Downregulation of microRNA-10 expression in the kidney was observed in both mice and patients with diabetic kidney disease,which negatively correlated with the activation of NLRP3 inflammasome.We also found that microRNA-10 negatively regulated NLRP3 inflammasome signal by targeting the 3’-untranslated region of NLRP3 mRNA,inhibiting NLRP3 protein translation at post-transcriptional level,alleviating caspase-1 cleavage and IL-1β maturation,and attenuating inflammation in the diabetic kidney.ConclusionWe found that microRNA-10,which is highly expressed in the kidney,alleviated renal inflammation in diabetic kidney disease by inhibiting the activation of kidney NLRP3 inflammasome.These findings provide a new therapeutic target for renal inflammation in diabetic kidney disease.