蛋白磷酸酶2A 在肾间质纤维化中的作用

目的：探讨蛋白磷酸酶2A(protein phosphatase 2A，PP2A)在大鼠单侧输尿管梗阻(unilateral ureteral obstruction，UUO)及TGF-β1刺激的人近端肾小管上皮细胞-2(human kidney proximal tubular epithelial-2，HK-2)的肾纤维 化模型中的作用。方法：1)15只雄性SD大鼠随机分成假手术组( sham组)、模型组(UUO组)和UUO+冈田酸(okadaic acid，OA)干预组(OA组)，每组各5只。术后OA组每日给予1.8%酒精稀释的OA 30 μg/kg，胃管饲喂72 h，对照组和模型 组给予相等体积的1.8%酒精胃管饲喂，72 h后处死大鼠，收集血和肾组织，检测肾功能并采用免疫组织化学、Western 印迹和RT-PCR法检测肾组织PP2A的c亚基(PP2Ac)、纤维连接蛋白(fi bronectin，FN)、胶原-I(collagen-I，Col-I)、E-钙黏 蛋白(E-cadherin，E-cad)和α平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)的蛋白及mRNA的表达。2)采用台盼蓝排斥 实验及MTT 法找出适宜的OA浓度。常规培养HK-2细胞，随机分为对照组、TGF-β1组(TGF-β1 5 ng/mL干预24 h)、TGF- β1+OA组(TGF-β1 5 ng/mL+OA 40 nmol/L，同时干预24 h)，Western印迹检测肾小管上皮细胞PP2Ac，FN，Col-I，E-cad 和α-SMA 蛋白的表达。结果：1)肾功能表明UUO组尿素氮和肌酐较sham组升高，OA组尿素氮、肌酐均比UUO组下 降(均P<0.05)。免疫组织化学、Western印迹和RT-PCR均显示：与sham组比较，UUO组PP2Ac，FN，Col-I和α-SMA表 达升高，而E-cad表达下降(均P<0.05)；与UUO组比较，OA组PP2Ac，FN，Col-I和α-SMA表达下降，E-cad表达升高(均 P<0.05)；2) OA 40 nmol/L为最适宜的实验质量浓度；Western印迹显示：与对照组比较，TGF-β1组PP2Ac，FN，Col-I 和α-SMA表达升高，E-cad表达下降(均P<0.05)；与TGF-β1组比较，TGF-β1+OA组PP2Ac，FN，Col-I和α-SMA表达下 降，E-cad表达升高(均P<0.05)。结论：PP2A能促进肾间质纤维化。

Role of protein phosphatase 2A in renal interstitial fibrosis

Objective: To explore the role of protein phosphatase 2A (PP2A) in renal interstitial fibrosis by using rat model of unilateral ureteral obstructive (UUO) or cell model of human kidney proximal tubular epithelial (HK)-2 cells treated with transforming growth factor-β1 (TGF-β1). Methods: 1) A total of 15 Sprague-Dawley rats were randomly divided into a sham group, a UUO group and an okadaic acid (OA) treated group (OA group) (n=5 in each group). The OA 30 μg/(kg·d)], diluted with 1.8% alcohol, was given to the rats in the OA group through gastric tube after at 72 h after the surgery, while the equal volume of 1.8% alcohol was given to the rats in the sham group and the UUO group. After sacrificing rats, the blood and kidney were collected to detect the renal function and the expression of PP2Ac, fibronectin (FN), collagen-I (Col-I), E-cadherin (E-cad) and α-smooth muscle actin (α-SMA) by immunohistochemistry, Western blot and RT-PCR, respectively; 2) The likely concentration of OA was determined by Trypan blue dye exclusive assay and methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The HK-2 cells were incubated with serum-free Dulbecco’s modified eagle medium (DMEM) for 24 h; then they were divided into a control group, a TGF-β1 group (treated with 5 ng/mL TGF-β1 for 24 h) and a TGF-β1+OA group (treated with 5 ng/mL TGF-β1 and 40 nmol/L OA for 24 h). The HK-2 cells were collected and the expression of PP2Ac, FN, Col-I, E-cad and α-SMA were detected by Western blot. Results: 1) Compared with the sham group, the BUN and Scr in the UUO group increased (both P<0.05); compared with the UUO group, the BUN and Scr in the OA group decreased (both P<0.05); the expression of PP2Ac, FN, Col-I and α-SMA was up-regulated while the expression of E-cad was down-regulated in the UUO group compared with those in the sham group (all P<0.05). The expression of PP2Ac, FN, Col-I and α-SMA was down-regulated while the expressions of E-cad was up-regulated in the OA group compared with those in the UUO group (all P<0.05); 2) The likely concentration of OA was 40 nmol/L. Western blot showed that the expression of PP2Ac, FN, Col-I and α-SMA was up-regulated while the expressions of E-cad was down-regulated in the TGF-β1 group compared with those in the control group (all P<0.05); the expression of PP2Ac, FN, Col-I and α-SMA were down-regulated while the expression of E-cad was up-regulated in the TGF-β1+OA group compared with those in the TGF-β1 group (all P<0.05). Conclusion: PP2A might be able to promote the renal interstitial fibrosis.