Fibrogenesis of interstitium in the progressive renal disease.

本研究旨在阐明肾小管上皮细胞在肾间质纤维化发病机制中的作用。首先，采用免疫组织化学方法研究了单侧梗阻(UUO)大鼠肾脏增殖细胞核抗原(PCNA)、骨桥蛋白(OPN)、α-平滑肌肌动蛋白(SMA)和巨噬细胞标记物(ED-1)的表达。结果表明，术后3d内增殖细胞核抗原阳性细胞明显增多，2周后逐渐减少。在该模型早期，增殖细胞核抗原阳性细胞主要为肾小管上皮细胞。术后4周间质细胞呈增殖细胞核抗原阳性反应。至术后5d，近端肾小管上皮细胞骨桥蛋白表达增加，ED-1阳性细胞增多。7d后α-SMA阳性细胞逐渐增多。UUO模型表观…肾间质体积的计点法测定2周后增加更明显。采用该动物模型，术后即刻给予免疫抑制剂咪唑宾5天，术后7天评价组织学指标。Mizoribin处理组大鼠肾间质中的浸润性细胞明显减少，而OPN在近端肾小管上皮细胞中的表达相似。与未治疗组相比，治疗组大鼠肾脏间质体积也显著减少。这些数据表明，在该模型中，单个核细胞向间质的渗透总是伴随着间质纤维化。然后我们决定研究物理力对肾小管上皮细胞行为的影响。将人近端肾小管上皮细胞培养在纤维粘连蛋白包被的硅橡胶上，然后施加20%的静态拉伸以产生物理作用力。采用双抗体夹心酶联免疫吸附试验(EL ISA)检测细胞在静态牵拉后24、48h培养上清液中转化生长因子-β(TGF-β)、白介素1(IL-1)和单核细胞趋化蛋白-1(MCP-1)的浓度，并与非牵张状态进行比较。24小时后，静态牵张组培养上清液中的转化生长因子-β浓度高于非牵张培养上清液。培养48h后，静态牵张组培养上清液中IL-1和MCP-1水平也明显高于非牵张培养上清液。我们的结果表明，物理力引起肾小管上皮细胞产生细胞因子和趋化因子，而这些分子是导致单个核细胞浸润的原因。从这些观察中，我们得出结论，在该模型中，肾小管上皮细胞在肾间质纤维化形成中起重要作用。较少

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