活性维生素D_3对大鼠肾小管间质纤维化的影响及其机制研究

Effect of Active Vitamin D_3 on Rat Renal Tubulointerstitial Fibrosis and Its Possible Mechanism

目的:观察活性维生素D_3对大鼠肾小管间质纤维化的影响及其可能机制。方法:将40只雄性SD大鼠随机分为4组:假手术组(Sham组)、模型组(UUO组)、UUO+低剂量活性维生素D_3组(LV组)、UUO+大剂量活性维生素D_3组(HV组),每组10只。采用单侧(左)输尿管结扎法建立梗阻性肾小管间质纤维化大鼠模型。Sham组只游离左侧输尿管而不结扎。LV组与HV组分别予以0.03μg/(kg·d)、0.06μg/(kg·d)活性维生素D_3(溶于花生油)腹腔注射,Sham组和UUO组均予以等体积花生油腹腔注射。术后14 d处死大鼠,采集血、肾脏标本,并检测血清钙、磷、肌酐(SCr)、甲状旁腺激素(PTH)。制作肾脏病理组织切片,行H-E、Masson染色,在光镜下观察肾小管间质损伤及纤维化情况。采用免疫组织化学法检测肾小管间质中α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白(E-cadherin)与转化生长因子β(TGF-β)的表达。结果:与UUO组相比,LV组和HV组SCr水平[(39.0±1.83)μmol/L、(36.0±2.11)μmol/L比(43.1±5.55)μmol/L]、肾小管间质纤维化指数(2.00±0.12、1.70±0.10比2.80±0.11)、α-SMA表达(0.22±0.02、0.20±0.03比0.24±0.02)、TGF-β表达(0.26±0.03、0.25±0.03比0.32±0.04)均显著下降(P<0.05),E-cadherin表达(0.30±0.08、0.34±0.11比0.22±0.07)明显升高。与LV组相比,HV组SCr水平肾间质纤维化指数、肾间质损伤指数、α-SMA表达、TGF-β表达明显下降(P<0.05),E-cadherin表达上升(P<0.05)。相关性分析显示,TGF-β、α-SMA表达与肾间质纤维化指数正相关,E-cadherin表达与肾间质纤维化指数负相关;TGF-β表达与α-SMA表达正相关,E-cadherin与TGF-β负相关;α-SMA表达与E-cadherin表达负相关。结论:活性维生素D_3可改善UUO大鼠肾小管间质纤维化,大剂量活性维生素D_3改善作用优于小剂量,其机制可能是:通过抑制TGF-β的过度表达从而抑制UUO大鼠肾小管上皮细胞-肌成纤维细胞转分化进而改善肾小管间质纤维化。

Objective ：To observe the effect of active vitamin D3 on renal tubulointerstitial fibrosis and its possible mechanism . Methods ： Forty male SD rats were randomly divided into four groups ：sham operation group（Sham group ,n= 10） ,unilateral ureteral obstruction （UUO） model group （UUO group ,n= 10） ,UUO model treated with low dose of active vitamin D3 group （LV group ,n= 10） ,and UUO model treated with high dose of active vitamin D3 group （HV group ,n= 10） .UUO model was estabished by ligating the left ureter .The left ureter was only dissociated without ligation in Sham group .LV group and HV group were given active vitamin D3 （dissolved in peanut oil） at a dose of 0 .03 μg/（kg · d） and 0 .06 μg/（kg· d） ,respectively , by intraperitoneal injection .Sham group and UUO group were given the same volume of peanut oil by intraperitoneal injection . Rats were killed 14 days after surgery .Blood and kidney samples were collected .And serum calcium ,phosphorus ,creatinine （SCr） ,and parathyroid hormone （PT H ） were detected . The kidney pathological changes involving renal tubulointerstitial injury and fibrosis ,were observed after H-E and Masson staining with optical microscope .The expressions of α-smooth muscle actin （α-SMA ） 、E-cadherin , and transforming grow factor β （TGF-β） in renal tubulointerstitial tissues were detected by immunohistochemistry .Results ：Compared with those in UUO group ,the SCr level （[39 .0 ± 1 .83] μmol/L ,[36 .0 ± 2 .11]μmol/L vs .[43 .1 ± 5 .55] μmol/L） ,tubulointerstitial fibrosis index （2 .00 ± 0 .12 ,1 .70 ± 0 .10 vs .2 .80 ± 0 .11） ,expression of α-SMA （0 .22 ± 0 .02 ,0 .20 ± 0 .03 vs .0 .24 ± 0 .02） ,and expression of TGF-β （0 .26 ± 0 .03 ,0 .25 ± 0 .03 vs .0 .32 ± 0 .04）＆amp;nbsp;in LV group and HV group decreased significantly （P〈 0 .05） ,while the expression of E-cadherin （0 .30 ± 0 .08 ,0 .34 ± 0 .11 vs .0 .22 ± 0 .07 ） increased . Compared with those in LV group , the SCr level , tubulointerstitial fibrosis index , tubulointerstitial injury index ,expression of α-SMA ,and expression of TGF-β ,in HV group decreased significantly （ P 〈0 .05） ,while the expression of E-cadherin increased （P〈 0 .05） .Correlation analysis showed that the expressions of TGF-β andα-SMA were positively correlated with tubulointerstitial fibrosis index . E-cadherin was negatively correlated with tubulointerstitial fibrosis index .TGF-β was positively correlated with α-SMA .E-cadherin was negatively correlated with TGF-β .α-SMA was negatively correlated with E-cadherin .Conclusions ： Active vitamin D3 can relieve renal tubulointerstitial fibrosis in UUO rats .Its possible mechanism is that the tubular epithelial-myofibroblast transdifferentiation （EM T ） is suppressed by inhibiting the over expression of TGF-β so as to relieve the renal tubulointerstitial fibrosis .The effect of the high dose of active vitamin D3 for relieving renal tubulointerstitial fibrosis is superior to that of low dose of active vitamin D3 .