PAX2和E-cadherin/α-SMA在肾间质纤维化大鼠肾小管上皮共表达的作用及意义

Role and significance of PAX2 and E-cadherin/α-SMA in renal tubular epithelium of renal interstitial fibrosis rats

目的阐明胚胎发育基因PAX2在肾间质纤维化大鼠中的再表达与肾小管上皮细胞-间充质转化细胞表型(epithelial-mesenchymal transition,EMT)特异性标志物E-cadherin和α-SMA的关系,探讨PAX2在肾间质纤维化中可能的作用机制。方法80只雄性Wistar大鼠随机分成假手术组(sham组)和模型组(UUO组),每组各40只。于术后3、5、7、14d(每组10只)处死取肾组织。光镜观察肾脏病理形态学改变,免疫组化双染检测PAX2和E-cadherin/α-SMA的关系、实时荧光定量PCR检测肾组织PAX2、E-cadherin和α-SMA mRNA的表达。结果①HE和Masson染色观察到UUO组出现明显的纤维化表现;②免疫组化双染结果发现sham组大鼠PAX2蛋白在肾小管上皮细胞无表达,E-cadherin蛋白大量表达于肾小管上皮细胞,α-SMA蛋白仅表达于小动脉的肌层,PAX2和E-cadherin/α-SMA之间无共表达;UUO组随着梗阻时间的延长PAX2和α-SMA表达更加明显,E-cadherin表达逐渐减少,PAX2和α-SMA蛋白共表达区域增加;③PCR结果显示sham组肾皮质中PAX2 mRNA少量表达,E-cadherin mRNA大量表达,α-SMA mRNA有微量表达;与sham组相比,3d UUO组PAX2 mRNA出现表达,E-cadherin mRNA表达减少,α-SMA mRNA表达增加,随着梗阻时间的延长,PAX2 mRNA和α-SMA mRNA表达量逐渐增加,E-cadherin mRNA表达量明显减少,以上三个指标UUO组与sham组及UUO组各时间点mRNA相对表达量比较均有统计学差异(P值均<0.05)。④相关分析表明:PAX2 mRNA水平与α-SMA mRNA水平呈正相关(r=0.993,P<0.05),与E-cadherin mRNA水平呈负相关(r=-0.983,P<0.05)。结论胚胎发育基因PAX2在肾间质纤维化大鼠肾小管上皮细胞再表达与EMT细胞表型特异性标志物E-cadherin/α-SMA蛋白和mRNA表达存在一定的联系,PAX2再表达参与了肾间质纤维化EMT过程。

Objective To elucidate the relationship between the reexpression of the embryonic developmental gene PAX2 in renal interstitial fibrosis rats and renal tubular epithelial-mesenchymal transition cell phenotype(EMT)specific markers E-cadherin andα-SMA,and to explore the role of PAX2 of action in renal interstitial fibrosis.Methods A total of 80 male Wistar rats were randomly divided into the sham group(n=40)and UUO model group(n=40).Renal tissues were sacrificed at 3,5,7 and 14 d(10 rats in each group)after operation.The pathological and morphological changes of the kidney were observed under light microscope.The relationship between PAX2 and E-cadherin/α-SMA was detected by immunohistochemical double staining.The expression of PAX2,E-cadherin andα-SMA mRNA in renal tissue was detected by real-time fluorescent quantitative PCR.Results①HE and Masson staining showed obvious fibrosis in the UUO group;②Double staining results showed that PAX2 protein was not expressed in renal tubular epithelial cells,E-cadherin protein was abundantly expressed in renal tubular epithelial cells,andα-SMA protein was expressed only in the muscular layer of small arteries.PAX2 There was no co-expression between E-cadherin andα-SMA;in the UUO group,the expression of PAX2 andα-SMA became more pronounced with the increase of the obstruction time,the expression of E-cadherin gradually decreased,and the area of co-expression of PAX2 andα-SMA protein increased;③PCR results:PAX2 mRNA was slightly expressed in the renal cortex of the sham group,E-cadherin mRNA was heavily expressed,andα-SMA mRNA was slightly expressed;compared with the sham group,PAX2 mRNA was expressed in the 3d UUO group,while the E-cadherin mRNA expression was reduced,andα-SMA mRNA expression increased.With the extension of obstruction time,PAX2 mRNA andα-SMA mRNA expression gradually increased,and E-cadherin mRNA expression decreased significantly.There were statistically significant differences in the relative expression of mRNA at each time point between the UUO group,the sham group and the UUO group(all P values<0.05).④Correlation analysis showed that PAX2 mRNA level was positively correlated withα-SMA mRNA level(r=0.993,P<0.05),and negatively correlated with E-cadherin mRNA level(r=-0.983,P<0.05).Conclusion The expression of embryonic development gene PAX2 in renal tubular epithelial cells of renal interstitial fibrosis rats has a certain relationship with the expression of E-cadherin/α-SMA protein and mRNA in phenotype-specific markers of EMT cells.The PAX2 re-expression is involved in the EMT process of renal interstitial fibrosis.