Brown Adipose Tissue Ameliorates Diabetic Nephropathy Through The MiR-30b Pathway By Targeting Runx1

BackgroundDiabetic kidney disease(DKD)is one of the most frequent diabetic microangiopathy.With the increasing global prevalence of diabetes in recent decades,DKD has become the main cause of end-stage renal disease,and it is urgent to find more effective therapeutic targets and methods.Brown adipose tissue(BAT),as an active endocrine and thermal energy-depleting organ,is regarded as a potential target for ameliorating obesity,diabetes and related metabolic diseases.In recent years,growing evidence has shown that lots of microRNAs(miRNAs)can be secreted to the bloodstream from adipose tissue,and these miRNAs act as signalosomes in the crosstalk among various organs,such as the liver.Therefore,we speculate that BAT can alleviate or reverse the progression of DKD by secreting miRNAs to target kidneyrelated genes,there is no direct evidence for this relationship between BAT and the kidney.In the previous study,we found that BAT transplantation or activation could significantly alleviate the progression of renal injury in diabetic mice.Furthermore,we found that the miR-30b level in the bloodstream of diabetic mice was significantly increased,and its renal target gene,Runx1,was down-regulated.Therefore,we speculated that BAT activation might influence the kidney by secreting miR-30b and then alleviate DKD.Therefore,this study mainly explores the possible mechanisms of BAT-derived miR-30b targeting two key fibrotic regulators Runx1 and Snail1,and their role in the development of DKD.Materials and Methods1.Subjects:C57BL/6J male mice(4～5 weeks old);Human proximal tubule cell line(HK-2)2.Experimental grouping and intervention:Cell experiment:HK-2 cells treat with TGFβ1.Animal experiment:high-fat diet combined with streptozotocin to establish a mouse model of diabetes,diabetic mice were randomly divided into subgroups:miR-30b overexpression:(1)healthy control group(Con);(2)AgomiR-30b intervention group(DM-30b);(3)AgomiR-NC intervention group(DM-NC).Runx1 knockdown or overexpression:(1)healthy control group(Con);(2)Runx1 knockdown group(DM+AAV-shRunx1);(3)Runx1 knockdown control group(DM+AAV-Scr);(4)Runxl overexpression group(DM+AAV-Runx1);(5)Runx1 overexpression control group(DM+AAV-NC).3.Quantitative Real-Time PCR4.Western Blot5.ELISA6.Luciferase activity assay7.Renal pathology8.Statistical AnalysisResults1.BAT-derived miR-30b alleviates the development of DKD In vivo experiments,administration of miR-30b in diabetic mice significantly reduced blood glucose and 24h urinary albumin,increased renal miR-30b levels,improved renal pathological changes,and down-regulated renal fibrosis indicators of FN,COL1 and SNAIL 1 and RUNX1 expression;In vitro experiments,overexpression of miR-30b in HK-2 cells significantly improved the expression of FN,COL1,α-SMA and CDH1 fibrosis-related genes;conversely,suppression of miR-30b revealed the opposite function.2.Possible molecular mechanism of miR-30b in DKDIn vitro experiments,miR-30b directly inhibited the key fibrotic genes of RUNX1 and SNAIL1 expression,and RUNX1 significantly regulated the downstream PI3K3D,thereby reducing the expression of fibrosis-related genes FN and COL1 in HK-2 cells.3.The effect of miR-30b renal target gene RUNX1 on DKDIn vivo experiments,suppression of RUNX1 significantly reduced urinary albumin in diabetic mice,relieved renal pathological changes,and improved renal fibrotic indicators such as TGFβ1,FN,COL1,α-SMA and SNAIL1 expression;otherwise,overexpression of RUNX1 showed the opposite change.ConclusionsBAT provides renoprotective effects against diabetes by increasing circulating miR30b levels,subsequently targets renal RUNX1 and SNAIL1,and regulates downstream PI3K pathways,inhibiting the process of EMT in renal tubular epithelial cells and improving renal fibrosis.