长链非编码RNA C2dat1通过影响CaMK2D/NF-κB信号通路参与糖尿病肾间质纤维化

Long non-coding RNA C2dat1 involved in diabetic renal interstitial fibrosis by influencing CaMK2D/NF-κB signaling pathway

目的研究长链非编码RNA C2dat1在不同时期糖尿病肾病(diabetic kidney disease,DKD)大鼠肾脏中的表达变化以及其与肾间质纤维化的关系。方法48只雄性SD大鼠,随机分为两组:对照组和DKD组,每组24只。对照组给予普通饲料喂养,DKD组给予高脂饲料喂养,自由饮水。喂食8周后,干预前禁食不禁水12 h,DKD组予一次性腹腔注射链脲佐菌素,对照组给予同等剂量的柠檬酸钠缓冲液。72 h后,连续3 d测定随机末梢血糖浓度≥16.7 mmol/L,尿糖定性为阳性,糖尿病模型成功。在3、6、9和12周采集大鼠尿液、血液和肾脏样本。采用全自动生化仪检测尿蛋白24 h排泄率、尿肌酐和血清总胆固醇;HE染色观察肾脏组织病理变化;免疫组织化学染色检测钙/钙调蛋白依赖性蛋白激酶2D(calcium/calmodulin-dependent protein kinaseⅡdelta,CaMK2D)、p65、p50、α-SMA、E-cardherin相关蛋白质的表达;实时荧光定量PCR(quantitative real-time PCR,qPCR)检测lncRNA C2dat1和CaMK2D的表达;通过相关性分析,探究lncRNA C2dat1与α-SMA、E-cardherin和CaMK2D的关系。用高糖诱导肾小管上皮细胞HK-2,干预后24、48、72 h收集细胞,qPCR检测lncRNA C2dat1及CaMK2D的表达。结果与对照组大鼠比较,DKD组大鼠出现典型的多饮、多食,体重明显降低,血糖升高,尿蛋白24 h排泄率增加,尿肌酐降低,总胆固醇升高。HE染色结果显示,对照组大鼠肾小球结构完整,基底膜正常,未见系膜增生与炎症细胞浸润,而DKD组大鼠肾小球增大,基底膜均匀增厚。DKD组CaMK2D、p50、α-SMA表达高于对照组,E-cardherin表达低于对照组。DKD组lncRNA C2dat1和CaMK2D mRNA的表达高于对照组。在高糖诱导的HK-2细胞中,lncRNA C2dat1和CaMK2D的表达也高于对照组。相关性分析提示,lncRNA C2dat1与α-SMA和CaMK2D呈正相关,与E-cardherin呈负相关。结论在DKD进展过程中,高表达的lncRNA C2dat1可能通过调节CaMK2D表达激活NF-κB信号通路促进糖尿病肾间质纤维化。

Objective To study the changes in long non-coding RNA C2dat1 expression in kidney tissues of rats at different stages of diabetic kidney disease(DKD)and its relationship with renal interstitial fibrosis.Methods Forty-eight male SD rats were randomly divided into two groups with 24 rats in each group:control group and DKD group.The rats in the control group were fed with ordinary diet,while those in the DKD group were fed with high-fat diet and drank water freely.After eight weeks of feeding,the rats were fasted for 12 h with free access to water.Then,the DKD group was given a one-time intrabitoneal injection of streptozotocin and the control group was given an equal dose of sodium citrate buffer.After 72 h,the random peripheral blood glucose concentration(≥16.7 mmol/L for three consecutive days)and urine sugar(positive)were tested to assess the establishment of the diabetes model.Urine,blood and kidney samples were collected at 3,6,9 and 12 weeks.The urinary protein excretion rate within 24 h,urinary creatinine and serum total cholesterol were measured by automatic biochemical apparatus.Pathological changes in kidney tissues were observed by HE staining.The expression of calcium/calmodulin-dependent protein kinaseⅡdelta(CaMK2D),p65,p50,α-SMA and E-cardherin was detected by immunohistochemistry.Quantitative real-time PCR(qPCR)was used to detect the expression of lncRNA C2dat1 and CaMK2D.The relationship of lncRNA C2dat1 withα-SMA,E-cardherin and CaMK2D was analyzed by correlation analysis.In in vitro experiment,renal tubular epithelial cells HK-2 were induced by high glucose.The expression of lncRNA C2dat1 and CaMK2D in HK-2 cells was detected by qPCR after 24,48 and 72 h of intervention.Results The rats in the DKD group showed typical symptoms such as polydipsia,polyphagia,significant weight loss and increased blood glucose as compared with the rats in the control group.Results of the biochemical tests revealed that compared with the control group,the DKD group had increased 24 h excretion rate of urinary protein,decreased urinary creatinine and up-regulated total cholesterol.HE staining showed that the rats in the control group had intact glomeruli,normal basement membrane and no mesangial hyperplasia or inflammatory cell infiltration.However,enlarged glomeruli and evenly thickened basement membrane were observed in the DKD group.Immunohistochemistry indicated that the expression of CaMK2D,p50 andα-SMA was higher in the DKD group than in the control group,while the expression of E-cardherin was lower in the DKD group.qPCR results showed that the expression of lncRNA C2dat1 and CaMK2D at mRNA level was higher in the DKD group than in the control group.In in vitro experiment,the expression of lncRNA C2dat1 and CaMK2D at mRNA level was also higher in HK-2 cells induced by high glucose than in the control group.Correlation analysis indicated that lncRNA C2dat1 was positively correlated withα-SMA and CaMK2D,but negatively correlated with E-cardherin.Conclusions During the progression of DKD,the high expression of lncRNA C2dat1 might promote diabetic renal interstitial fibrosis by regulating the expression of CaMK2D to activate the NF-κB signaling pathway.