尿外泌体miR-155与2型糖尿病肾病发病及严重程度的相关性

Correlation between urinary exosomal miR-155 and the onset and severity of type 2 diabetic kidney disease

目的探讨2型糖尿病(type 2 diabetes mellitus,T2DM)患者尿外泌体微小RNA(exosomal microRNA,exo-miR)-155与糖尿病肾疾病(diabetic kidney disease,DKD)发病及严重程度的关系。方法于2019年1—5月从上海健康医学院崇明医院内分泌与代谢科招募5例T2DM正常白蛋白尿患者和5例2型DKD患者作为微小RNA筛选队列,采集患者尿液样本提取尿外泌体,并利用miRCURY LNA阵列检测尿exo-miR特征谱。随后于2019年6月至2022年10月通过该院内分泌与代谢科纳入符合入组标准且年龄、性别相匹配的351例T2DM患者作为验证队列。根据尿白蛋白/肌酐比值(urinary albumin/creatinine ratio,UACR)分为3组:正常白蛋白尿组(UACR<30 mg/g,n=143)、微量白蛋白尿组(30 mg/g≤UACR≤300 mg/g,n=171)和大量白蛋白尿组(UACR>300 mg/g,n=37)。根据DKD诊断指南,将微量白蛋白尿和大量白蛋白尿患者归为DKD组。采用实时荧光定量PCR法检测尿exo-miR-155表达水平。结果透射电镜、纳米颗粒跟踪分析和Western印迹观察分析结果均显示尿外泌体提取成功。在筛选队列中,按照P<0.05且变化倍数(FC)>1.5的筛选标准,与T2DM患者尿液样本相比,DKD组尿外泌体样本中筛出226个差异表达miRNA,其中miR-155上调倍数最高(FC=32.75,P<0.001)。在验证队列中,与正常白蛋白尿组[0.76(0.55,0.95)]相比,大量白蛋白尿组[1.84(1.18,2.42)]尿exo-miR-155水平升高最明显(Z=-7.411,P<0.001),微量白蛋白尿组[0.86(0.69,1.25)]次之(Z=-4.092,P<0.001),而且大量白蛋白尿组尿exo-miR-155水平也明显高于微量白蛋白尿组(Z=-5.841,P<0.001)。相关分析显示,尿exo-miR-155水平与UACR呈正相关(rs=0.329,P<0.001),与估算肾小球滤过率呈负相关(rs=-0.249,P=0.015)。受试者工作特征曲线分析显示,尿exo-miR-155水平预测T2DM患者进展为DKD的曲线下面积为0.892(95%CI 0.859~0.925),对应截断值为0.982,灵敏度和特异度分别为71.9%和87.7%。多因素Logistic回归分析结果显示尿exo-miR-155≥0.982是T2DM患者进展为DKD的独立危险因素(OR=3.310,95%CI 1.981~5.530,P<0.001)。结论尿exo-miR-155在微量白蛋白尿和大量白蛋白尿T2DM患者中表达水平较高,其与白蛋白尿程度相关,可作为识别有潜在DKD的预测标志物。

Objective To explore the relationship between urinary exosomal microRNA(exo-miR)-155 in patients with type 2 diabetes mellitus(T2DM)and the onset and severity of diabetic kidney disease(DKD).Methods From January to May 2019,5 patients with T2DM normoalbuminuria and 5 patients with type 2 DKD were recruited from the Department of Endocrinology and Metabolism of Chongming Hospital of Shanghai Health Medical College as a microRNA screening cohort.Urine samples were collected to extract urinary exosomes,and the urine exo-miR spectrum was detected and analyzed using the miRCURY LNA array.From June 2019 to October 2022,351 patients with T2DM who met the enrollment criteria and were matched by age and sex were included in the validation cohort in the Department of Endocrinology and Metabolism of the hospital.Patients were divided into 3 groups according to urinary albumin/creatinine ratio(UACR):normoalbuminuria group(UACR<30 mg/g,n=143),microalbuminuria group(30 mg/g≤UACR≤300 mg/g,n=171)and macroalbuminuria group(UACR>300 mg/g,n=37).According to DKD diagnostic guidelines,microalbuminuria group and macroalbuminuria group were classified into DKD group.Real-time fluorescence quantitative PCR was used to detect the expression level of exo-miR-155 in urine.Results The results of transmission electron microscopy,nanoparticle tracking analysis,and Western blotting showed that the extraction of exosome vesicles was successful.In the screening cohort,according to the screening criteria of P<0.05 and fold changes(FC)>1.5,226 differentially expressed miRNAs were identified from the urinary exosomes of the DKD group compared to the T2DM group.Among them,miR-155 ranged highest(FC=32.75,P<0.001).In the validation cohort,compared with the normoalbuminuria group[0.76(0.55,0.95)],the macroalbuminuria group[1.84(1.18,2.42)]had the most significant increase in urinary exo-miR-155 level(Z=-7.411,P<0.001),followed by the microalbuminuria group[0.86(0.69,1.25)](Z=-4.092,P<0.001),and the urinary exo-miR-155 level in the macroalbuminuria group was significantly higher than that in the microalbuminuria group(Z=-5.841,P<0.001).The correlation analysis showed that urinary exo-miR-155 level was positively correlated with UACR(rs=0.329,P<0.001)and negatively correlated with estimated glomerular filtration rate(rs=-0.249,P=0.015).The results of receiver operating characteristic curve analysis showed that the area under the curve of urinary exo-miR-155 level predicted DKD progression in T2DM patients was 0.892(95%CI 0.859-0.925),corresponding cutoff value was 0.982,and the sensitivity and specificity were 71.9%and 87.7%,respectively.Multivariate logistic regression analysis showed that urinary exo-miR-155≥0.982 was an independent risk factor for progression to DKD in T2DM patients(OR=3.310,95%CI 1.981-5.530,P<0.001).Conclusion The expression level of urinary exo-miR-155 is increased in T2DM patients with microalbuminuria and macroalbuminuria,which is related to the degree of albuminuria,and can be used as a predictive marker to identify potential DKD.