支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及其机制探讨

Role of scaffolding protein JLP on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction and its underlying mechanism

目的观察支架蛋白JLP基因缺失对单侧输尿管梗阻（UUO）小鼠肾间质纤维化的影响，探讨JLP在梗阻性肾病肾纤维化形成中的作用和可能机制。方法将jlp＋/＋生型（jlp＋/＋）小鼠和jlp敲除（jlp＋/＋）小鼠分为4组：jlp“假手术组（jlp＋/＋-Sham）；jlp＋/＋假手术组（jlp＋/＋-Sham）；jlp＋/＋模型组（jlp＋/＋-UUO）和jl旷模型组（jlp-／-UUO）。分别于术后7d和14d处死小鼠。Masson染色观察肾间质纤维化改变；免疫荧光、免疫组化及Western印迹法观察JLP在各组小鼠肾组织的表达与分布；免疫组化法检测肾组织α-平滑肌肌动蛋白（α-SMA）、I型胶原纤维（COL-I）、Ⅲ型胶原纤维（COL-Ⅲ）、转化生长因子β1（TGF-B1）表达；Western印迹检测肾组织α-SMA、COL-I、COL-Ⅲ、TGF-β1、p-Smad2及p-Smad3蛋白表达。结果jlp＋/＋小鼠肾组织表达JLP，主要分布于肾小管。Masson染色结果显示，与jlp＋/＋．UUO组比较，jlp-／-UUO组肾间质胶原纤维增加，纤维化程度加重（均P〈0．05）。免疫组化结果显示，与jlp＋/＋-UUO组比较，jlp＋/＋．UUO组小鼠肾皮质α-SMA、COL-I、COL-Ⅲ和TGF-β1表达明显升高（均P〈0．05）。Western印迹结果显示，与jlp＋/＋．UUO组比较，jlp＋/＋-UUO组α-SMA、COL-I、COL-Ⅲ和TGF-β1蛋白表达明显升高（均P〈0．05），p-Smad2和p-Smad3蛋白表达也明显升高（均P〈0．05）。结论支架蛋白JLP在预防。肾纤维化的形成中起重要作用，这一作用可能是通过抑制TGF-β1的表达和肌成纤维细胞生成来实现的。

[Abstract] Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction （UUO）, and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy. Methods jlp Wild type （jlp＋/＋） and jlp deficient （jlp-/-） mice were divided into four groups： jlp＋/＋- and jlp-/--sham-operated groups（jlp-/--sham group and jlp＋/＋-sham group）, jlp＋/＋- and jlp-/--unilateral ureteral obstruction （UUO）-operated groups （jlp-/--UUO group and jlp＋/＋-UUO group）. Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining. The expression of JLP in jlp ＋/＋ renal tissue was assayed by immunohistochemistry staining, immunofiuorescence and Western blotting. Immunohistochemical staining was used to detect the expression of a-smooth muscle actin （α-SMA）, collagen I （COL- I ）, collagen Ⅲ （COL-Ⅲ） and transforming growth factor-β1 （TGF-β1） in sham and UUO groups. Besides, the α-SMA, COL- I, COL-Ⅲ, TGF-β1, p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups. Results The expression of JLP was mainly demonstrated in the renal tubules of mice. A large amount of collagen deposition was observed in the renal interstitial area in jlp-/-- UUO group compared to jlp＋/- UUO group. Similarly, the expression of a-SMA, COL- I, COL-IU and TGF-β1 was significantly increased in the kidney cortices in jlp-/-- UUO-operated groups. Meanwhile, Western blotting showed that the expression of α-SMA, COL- I, COL-Ⅲ, and TGF-β1 protein was obviously higher in jlp-/-- UUO group. Moreover, the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/-- UUO group. Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production. [