背景:肾结石病(KSD)通常伴有肾脏纤维化,以细胞外基质(ECM)的积累和重组为特征。在纤维化期间,固有的肾成纤维细胞被激活成为活跃产生ECM的肌成纤维细胞。然而,KSD中的成纤维细胞-肌成纤维细胞分化仍不清楚。因此,我们本研究检查了源自近端(HK-2)的分泌产物(分泌组)与暴露于草酸钙一水合物(COM)晶体的远端(MDCK)肾小管细胞激活肾成纤维细胞(BHK-21)。
方法:在无血清条件下用100µg/mlCOM晶体处理HK-2和MDCK细胞16小时。在没有COM处理的无血清培养基中维持的细胞作为对照。将来自每个样品的培养上清液的Secretome与新鲜的无血清培养基混合(1:1),然后再用于BHK-21培养24小时。
结果:分析表明,COM处理的HK-2分泌组显着诱导增殖,引起形态变化,主轴指数增加,和上调的成纤维细胞活化标志物(F-肌动蛋白,BHK-21细胞中的α-SMA和纤连蛋白)。然而,COM处理的MDCK分泌组对这些BHK-21参数没有显著影响。此外,转化生长因子-β1(TGF-β1)的水平,促纤维化因子,在COM处理的-HK-2分泌组中显著增加,但在COM处理的-MDCK分泌组中不显著增加。
结论:这些数据表明,第一次,暴露于COM晶体的近端和远端肾小管上皮细胞向常驻肾成纤维细胞发送不同的信息。只有来自近端肾小管细胞的分泌组,不是来自远端细胞,暴露于COM晶体后诱导肾成纤维细胞活化。这种差异效应部分是由于TGF-β1的分泌,仅在近端肾小管细胞中由COM晶体诱导。
BACKGROUND: Kidney stone disease (KSD) is commonly accompanied with renal fibrosis, characterized by accumulation and reorganization of extracellular matrix (ECM). During fibrogenesis, resident renal fibroblasts are activated to become myofibroblasts that actively produce ECM. However, such fibroblast-myofibroblast differentiation in KSD remained unclear. Our present study thus examined effects of secreted products (secretome) derived from proximal (HK-2) vs. distal (MDCK) renal tubular cells exposed to calcium oxalate monohydrate (COM) crystals on activation of renal fibroblasts (BHK-21).
METHODS: HK-2 and MDCK cells were treated with 100 µg/ml COM crystals under serum-free condition for 16 h. In parallel, the cells maintained in serum-free medium without COM treatment served as the control. Secretome derived from culture supernatant of each sample was mixed (1:1) with fresh serum-free medium and then used for BHK-21 culture for another 24 h.
RESULTS: Analyses revealed that COM-treated-HK-2 secretome significantly induced proliferation, caused morphological changes, increased spindle index, and upregulated fibroblast-activation markers (F-actin, α-SMA and fibronectin) in BHK-21 cells. However, COM-treated-MDCK secretome had no significant effects on these BHK-21 parameters. Moreover, level of transforming growth factor-β1 (TGF-β1), a profibrotic factor, significantly increased in the COM-treated-HK-2 secretome but not in the COM-treated-MDCK secretome.
CONCLUSIONS: These data indicate, for the first time, that proximal and distal tubular epithelial cells exposed to COM crystals send different messages to resident renal fibroblasts. Only the secretome derived from proximal tubular cells, not that from the distal cells, induces renal fibroblast activation after their exposure to COM crystals. Such differential effects are partly due to TGF-β1 secretion, which is induced by COM crystals only in proximal tubular cells.