观察上调肾组织intermedin(IMD)表达对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响。

健康雄性Wistar大鼠随机分为假手术组、UUO组、IMD＋UUO组、空质粒＋UUO组。IMD＋UUO组和空质粒＋UUO组在输尿管结扎前分别将IMD-pcDNA3.1真核表达质粒和空质粒转入肾组织，real-time RT-PCR及免疫组化法检测转染效率。各组分别于术后7 d、14 d留取梗阻侧肾组织。HE、Masson染色观察肾组织病理变化；real-time RT-PCR检测肾组织中转化生长因子-β1(TGF-β1)、纤连蛋白(Fn1)的mRNA表达；Western印迹法检测Fn1的蛋白表达；免疫组化法检测TGF-β1的蛋白表达。

与假手术组相比，UUO组肾脏出现明显的病理改变，肾间质纤维化程度随梗阻时间延长加重(与假手术组比较，7 d, t＝11.927，P＝0.0003；14 d, t＝8.891，P＝0.0009)；IMD＋UUO组肾脏病理改变及肾间质纤维化程度较同时间点UUO组明显减轻(7 d, t＝3.892，P＝0.018；14 d, t＝4.047，P＝0.016)，而空质粒＋UUO组与UUO组无显著差别(7 d, t＝0.562，P＝0.604；14 d, t＝0.035，P＝0.974)。与同时间点假手术组相比，UUO组TGF-β1、Fn1的表达明显升高(TGF-β1 mRNA水平7 d, t＝4.432，P＝0.011；14 d, t＝4.873，P＝0.006；蛋白质水平7 d, t＝5.312，P＝0.006；14 d, t＝4.482，P＝0.011；Fn1 mRNA水平7 d, t＝6.053，P＝0.004；14 d, t＝7.345，P＝0.002；蛋白质水平7 d, t＝8.791，P＝0.009；14 d t＝8.027，P＝0.001)；转染IMD质粒后Fn1的表达较同时间点UUO组明显下降(mRNA水平7 d, t＝3.103，P＝0.036；14 d, t＝2.913，P＝0.044；蛋白质水平7 d, t＝2.955，P＝0.042；14 d, t＝2.991，P＝0.040)；而转染空质粒后Fn1的表达无明显变化(mRNA水平7 d, t＝0.095，P＝0.929；14 d, t＝0.158，P＝0.882；蛋白质水平7 d, t＝0.159，P＝0.881；14 d, t＝0.170，P＝0.874)。转染IMD和空质粒对TGF-β1的表达均无明显影响(转染IMD质粒mRNA水平7 d, t＝0.176，P＝0.869；14 d, t＝0.126，P＝0.906；蛋白质水平7 d, t＝0.198，P＝0.853；14 d, t＝0.196，P＝0.854；转染空质粒mRNA水平7 d, t＝0.100，P＝0.925；14 d, t＝0.097，P＝0.928；蛋白质水平7 d, t＝0.042，P＝0.968; 14 d, t＝0.060，P＝0.955)。

上调肾组织IMD的表达能明显减轻肾间质纤维化，但其作用不是通过直接抑制TGF-β1的表达实现的。

To investigate the effects of intermedin (IMD) overexpression on renal interstitial fibrosis in the obstructed kidney of rats with unilateral ureteral obstruction (UUO).

Male Wistar rats were randomly divided into sham-operated group, UUO group, IMD+ UUO group, and empty plasmid+ UUO group. For IMD+ UUO group or empty plasmid+ UUO group, pcDNA3.1-IMD plasmid or control empty vector was transfected into the left kidney via the renal artery by an ultrasound-microbubble-mediated system before the ureter was obstructed. The transfection rate was detected by real-time RT-PCR and immunohistochemistry. Groups of six animals were killed at 7 d and 14 d after operation. Kidneys were harvested for further analysis. Paraffin-embedded transverse kidney slices were stained with hematoxylin and eosin. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. mRNA expression levels of TGF-β1 and fibronectin (Fn1) were detected by real-time RT-PCR. Protein expression of TGF-β1 was detected by immunohistochemical staining. Protein expression of Fn1 was examined by Western blot analysis.

The ultrasound-microbubble-mediated delivery system yielded high expression of IMD in kidney cells. IMD overexpression remarkably attenuated UUO-induced tubular injury, and blunted fibrotic response as shown by decreased interstitial collagen deposition (7 d, t=3.892, P=0.018 vs UUO group; 14 d, t=4.047, P=0.016 vs UUO group) and downregulation of fibronectin (mRNA 7 d, t=3.103, P=0.036 vs UUO group; 14 d, t=2.913, P=0.044 vs UUO group; Protein 7 d, t=2.955, P=0.042 vs UUO group; 14 d, t=2.991, P=0.040 vs UUO group), whereas TGF-β1 upregulation was not affected (mRNA 7 d, t=0.176, P=0.869 vs UUO group; 14 d, t=0.126, P=0.906 vs UUO; Protein 7 d, t=0.198, P=0.853 vs UUO; 14 d, t=0.196, P=0.854 vs UUO group).

Our results indicated that kidney-specific IMD gene delivery inhibited renal fibrosis induced by UUO. The inhibitory effect of IMD on renal fibrosis was not achieved by directly inhibiting TGF-β1 expression.