Fibrogenesis of interstitium in the progressive renal disease.

本研究的目的是阐明小管上皮细胞在肾间质纤维化发病机制中的作用。首先，采用免疫组化技术检测单侧梗阻（UUO）大鼠肾脏中增殖细胞核抗原（PCNA）、骨桥蛋白（OPN）、α -平滑肌肌动蛋白（SMA）和巨噬细胞标志物（ED-1）的表达。结果显示，PCNA阳性细胞在术后3天内显著增加，2周后逐渐减少。模型早期PCNA阳性细胞以小管上皮细胞为主。术后4周间质细胞PCNA阳性。近端小管上皮细胞中OPN的表达和ED-1阳性细胞的浸润也增加至术后5 d。7 d后α - sma阳性细胞逐渐增多。用点计数法测定UUO模型肾组织间质体积，结果显示，2周后肾组织间质体积明显增大。采用该动物模型，术后立即给予免疫抑制剂米佐利宾5 d，术后7 d评价组织学参数。米佐瑞滨处理的动物显示肾间质浸润细胞显著减少，这与近端肾小管上皮细胞中OPN的表达相似。与未治疗的动物相比，治疗动物的肾间质体积也显著减少。这些数据表明，在该模型中，单个核细胞浸润到间质中总是伴随着间质纤维化。然后我们决定研究物理力对小管上皮细胞行为的影响。将人近端小管上皮细胞培养在包被纤维连接蛋白的硅橡胶上，然后用20%的静拉伸力诱导物理力。在静态拉伸开始后24和48小时，用ELISA法测定培养上清中tgf - β、IL-1和MCP-1的浓度，并与非拉伸条件进行比较。24h后，静态拉伸组上清中tgf - β的浓度高于非拉伸培养上清。48h后，静态拉伸组培养上清中IL-1和MCP-1水平也较非拉伸组升高。我们的研究结果表明，物理力引起小管上皮细胞产生细胞因子和趋化因子，这些分子负责单核细胞浸润。通过这些观察，我们得出结论，小管上皮细胞在肾间质纤维化中起重要作用。少

The aim of this study is to clarify the role of tubular epithelial cells in the pathogenesis of renal interstitial fibrosis. First of all, we investigated the expression of proliferating cell nuclear antigen (PCNA), osteopontin (OPN), alpha-smooth muscle actin (SMA), and macrophage cell marker (ED-1) in unilateral obstruction (UUO)-treated rat kidney using immunohistochemical technique. The results showed that PCNA positive cells dramatically increased within 3 days after operation and gradually decreased by 2 weeks. PCNA positive cells were mainly tubular epithelial cells in early stage of this model. However interstitial cells became positive for PCNA after 4 weeks from operation. Expression of OPN in proximal tubular epithelial cells and infiltration of ED-1 positive cells were also increased until 5 days after operation. alpha-SMA positive cells were gradually increased after 7 days. Interstitial volume that was evaluated by point counting method in the kidney of UUO model apparent … More ly increased after 2 weeks. Using this animal model, we administrated mizoribin that is a kind of immunosuppresive agents for 5 days immediately after operation, and then evaluated histological parameters after 7 days from operation. Animals treated with mizoribin showed significant reduction of infiltrating cells in the renal interstitium, in spit of the similar expression of OPN in the proximal tubular epithelial cells. The interstitial volume of the kidney in the treated animal also dramatically reduced compared with that in non-treated animal. These data indicate that infiltration of mononuclear cells into the interstitium was always followed by interstitial fibrosis in this model. Then we decided to investigate the effects of physical force on cellular behavior of tubular epithelial cells. Human proximal tubular epithelial cells were cultured on fibronectine-coated silicon rubber, and then 20 % of static-stretch was employed to induce physical force. Concentration of TGF-beta, IL-1, and MCP-1 in culture supernatants were measured using ELISA at 24 and 48 hours after initiation of static stretch and compared with non-stretch condition. After 24hrs, the concentration of TGF-beta in the supernatant of the static-stretch group showed higher level than that of non-stretch culture supernatant. After 48hrs, IL-1 and MCP-1 level of culture supernatant were also increased in the static-stretch group compared with non-stretch culture supernatant. Our results indicate that the physical force evokes the production of cytokines and chemokines from tubular epithelial cells, and that these molecules are responsible for mononuclear cell infiltration. From these observations, we concluded that tubular epithelial cells have important roles for fibrogenesis of renal interstitium in this model. Less