实验性慢性肾缺血模型肾小管-间质细胞a-平滑肌肌动蛋白的表达和意义

目的:观察单侧肾动脉狭窄(RAS)大鼠模型慢性肾缺血组织中肾小管-间质a-平滑肌肌动蛋白(a-SMA)的表达和意义。 方法:建立Goldblatt单侧RAS大鼠模型,观察大鼠慢性缺血肾脏在90天内不同阶段的病理改变;应用免疫组化法检测肾小管-间质a-SMA及波形蛋白(vimentin)在不同时间点的表达;应用免疫荧光双标记激光共聚焦显微镜观察细胞角蛋白(cytokeratin,CK)与a-SMA在肾小管上皮细胞中的共定位情况。 结果:RAS术后第5天首先出现肾小管vimentin阳性,术后第7天肾间质可见少量的a-SMA阳性细胞并随时间递增。通过连续切片观察到,vimentin阳性的肾小管周围肾间质细胞a-SMA表达增强,并与肾小管间质纤维化程度基本一致。当慢性缺血状态持续存在时,后期(术后第45天至90天)少数损伤的肾小管上皮细胞表达a-SMA。应用免疫荧光双标记激光共聚焦显微镜观察发现,部分a-SMA阳性的肾小管上皮细胞同时表达CK,并且个别细胞还有向间质游走的趋势。 结论:在慢性肾缺血早期Vimentin阳性的肾小管上皮细胞本身可能诱导了肾间质固有细胞发生肌成纤维细胞转分化,参与肾间质纤维化的发生和发展;在慢性肾缺血后期,肾小管上皮细胞转分化可能是导致肾纤维化进行性发展的关键环节。

Expression of a-SMA in renal tubulointerstitium of the rat with chronic renal ischemia

Objective: Chronic ischaemic renal disease is increasingly recognized as a potentially treatable cause of chronic renal failure (CRF). The pathophysiology of chronic renal ischemia, resulting in tubular cell injury and leading to tubulointerstitial fibrosis, remains unclear. Myofibroblast formation, identified by the alpha-smooth muscle actin-positive (a-SMA) cells, is a key event in renal interstitial fibrosis. Presently there is some evidence to support the hypothesis that transdifferentiation from tubular epithelial cells into myofibroblast-like cells plays a role in interstitial fibrosis of some renal diseases. But the precise mechanism of the cells phenotypic transformations in tubulointerstitium, with chronic renal ischemia, remains largely unknown. The present study was designed to observe the morphologic change alternation by establishing rat model with unilateral renal artery stenosis(RAS), and to investigate the expression and significance of a-SMA in renal tubulointerstitium of the rat with chronic renal ischemia. Methodology: Male Sprague-Dawley rats were subjected to unilateral stenosis of the left main renal artery for 90 days, in order to observe the renal pathologic change in different stages. In renal tubulointerstitium, the expression of a-SMA and vimentin was detected by immunohistochemistry at the points of different time. Using immunofluorescent double-labeling, the colocalization between cytokeratin and a-SMA in renal epithelial cell was observed with cofocal laser-scanning microscopy. Results: In the early period of chronic renal ischemia, vimentin in tubule first appeared to be positive at day 5 after RAS. The a-SMA expression in interstitium increased progressively starting at day 7. By using consecutive renal tissue slicing, the enhanced expression of a-SMA in renal interstitium can be observed in the circumference of renal tubular cells with vimentin-positive. When chronic renal ischemia lasting existence, the expression of a-SMA was observed in a few renal tubular cells from day 45 to day 90 after RAS. The expression of a-SMA in renal tubulointerstitium was significantly related with the degree of tubulointerstitial fibrosis(r=0.823, P<0.01). Using immunofluorescent double-labelling, the colocalization between cytokeratin and a-SMA in renal epithelial cell was observed with cofocal laser-scanning microscopy, and some of them seemed to be migrating to interstitium. It provided the immunomorphological evidence that tubular epithelial cells can undergo phenotypic transdifferentiation toward a myofibroblast-like cell during progressive chronic ischaemic tubulointerstitial fibrosis. Conclusion: In the early stages of chronic renal ischemia, the renal tubular epithelia cells with vimentin-positive, may induce the transdifferentiation from tubulointerstial cells to myofibroblasts, and participate in the occurence and development of renal interstitial fibrosis in the rat. In the later stages of chronic renal ischemia, the transformation of renal tubular cells to myofibroblasts may be the key event that leads to progressive tubulointerstitial fibrosis.