转化生长因子-β_1/Smad2/3介导肾小管上皮细胞转分化与幼鼠肾间质纤维化的相关性

Correlation between TGF-β_1/Smad2/3-induced epithelial to myofibroblast transformation and renal interstitial fibrosis in young rats

目的探讨转化生长因子β1(TGF-β1)与Smad2/3信号传导介导肾小管上皮细胞转分化(EMT)与幼鼠慢性肾间质纤维化的相关性,以及血管紧张素转换酶抑制剂(ACEI)的干预作用。方法本研究以80只幼年雌性SD大鼠为实验动物,制备7/8肾切除残肾慢性进展性肾功能衰竭模型。模型成功后随机将动物分为假手术组、7/8肾切除未治疗组和7/8肾切除干预治疗组。以模型制备后第2、4、8、12周为不同时间点。检测各时间点大鼠肾组织常规病理;采用免疫组织化学染色检测残肾组织中TGF-β1、Smad2/3:α-平滑肌肌动蛋白(α-SMA)、E-钙黏连蛋白及纤维连接蛋白(FN)在肾组织表达趋势。结果实验第2周时未治疗组肾小管间质大量炎性细胞浸润、肾小管上皮细胞肿胀、肾小管间隙加宽。至第12周未治疗组肾小管广泛扩张、基底膜增厚、皱缩或断裂,间质纤维化明显。而治疗组残肾组织病理变化较未治疗组有明显改善。免疫组织化学半定量分析显示未治疗组TGF-β1阳性细胞或阳性小管染色积分值呈逐渐升高趋势。对照组与未治疗组各时间点相比较差异有统计学意义(P<0.05)。Smad2/3表达趋势与TGF-β1相似。α-SMA蛋白表达趋势同步上调,E-钙黏连蛋白同步下调,未治疗组各时间点FN均呈现逐渐升高趋势(P<0.05)。未治疗组TGF-β1、Smad2/3、α-SMA、FN各指标不同时间点免疫组织化学蛋白染色积分值呈正相关,而与E-钙黏连蛋白表达趋势呈负相关(P<0.01)。干预组不同时间点TGF-β1、Smad2/3、α-SMA和FN蛋白表达趋势明显弱于未治疗组,但强于对照组,而E-钙黏连蛋白表达下调(P<0.01)。结论在幼年期肾损伤过程中,TGF-β1高表达与Smad2/3分子使其高表达,可能通过介导EMT导致肾小管间质区细胞外基质(ECM)异常合成与沉积,最终引起肾小管间质纤维化形成。早期给予ACEI干预可改善这种病理变化。

Objective To study the correlation between transforming growth faetor-β1（TGF-β1）, and Smad2/3 signaling pathway for regulation of renal epithelial to myofibroblast transformation （EMT） and chronic renal interstitial fibrosis in young rats, and to investigate the treated effects of angiotensin converting enzyme inhibitor （ACEI） on renal injury. Methods The 7/8 nephrectomy models were constructed using 80 female young SD rats and were randomly assigned into Sham group, 7/8 nephrectomy non-treatment group and 7/8 nephrectomy ACEI treatment group, respec- tively. All renal tissues were subjected to pathologic examination at various time points （weeks 2, 4, 8 and 12）. Immunohistochemieal assay was employed to detect the expression of TGF-β1 s-smooth muscle actin （α-SMA）, E-Cad- herin and fibroneetin （FN） in remnant renal tissues. The correlation between localized expression and time-dependent expression of various biomarkers at various time points was compared. The effect of ACEI treatment was also deter- mined. Results The renal interstitium was featured of marked inflammatory cell infiltration, renal epithelial-cell swelling, widened renal tubular space at week 2 in non-treatment group. In addition, extensive dilatation of renal tubes, basement membrane thickening, shrinkage or interruption as well as considerable interstitial fibrosis may be observed at week 12. These injuries were markedly ameliorated in treatment group. The cumulative score of cells and tubules in which TGF-β1 expression tested positive increased with time in non-treatment group, but not in treatment group, as shown by semi-quantitative immunohistochemical assay. The expression of Smad2/3 yielded similar trending with that of TGF-β1. The up-regulated expression of α-SMA, was in tune with the down-regulation of E-cadherin. Fur- thermore, non-treatment group was associated with increased scores of immunohistochemistry staining and quantifica- tion. The difference in immunohistochemistry staining and quantification score reached statistical significance between control group and non-treatment group at all time points （all P〈0.05）. The immunohistochemistry staining score of TGF-β1 was positively correlated with that of Smad2/3, α-SMA and FN and was negatively correlated with that of E- cadherin in non-treatment group at all time points （all P〈0.01）. Treatment group was characterized of significant down- regulated TGF-β1, Smad2/3, α-SMA and FN expression and unmarked down-regulation of E-cadherin compared with non-treatment group yet higher expression levels than control group （all P〈0.01）. ACEI treatment might ameliorate re- nal injury. Conclusion During the chronic progressive renal injury in young rats, TGF-β1 and Smad/2 overexpression may result in abnormal synthesis and precipitation of extracellular matrix in renal interstitium via meditation of EMT leading to renal tubule interstitial fibrosis. Treatment with ACEI in a timely fashion could be associated with remark- able amelioration or reverse all aforementioned injuries.