Urology Unit, Department of Surgery, Flinders Medical Centre and Flinders Clinical and Molecular Medicine, School of Medicine, Flinders University, Bedford Park, SA, Australia.
BJU Int. 2012 Apr;109(7):1100-9. doi: 10.1111/j.1464-410X.2011.10530.x. Epub 2011 Aug 24.
To determine the effects of intracrystalline (IC), surface-bound (SB) and combined IC + SB osteopontin (OPN) on the binding of urinary calcium oxalate dihydrate (COD) crystals to Madin-Darby canine kidney (MDCK-II) cells in ultrafiltered (UF) human urine.
(14)C-oxalic acid-labelled urinary COD crystals containing IC OPN were generated in pooled UF human urine containing human milk OPN at concentrations of 0, 1.0 and 5.0 mg/L. Additional labelled crystals were nucleated from a separate sample of the same pooled UF urine, to which were later added the same amounts of protein to produce crystals with SB OPN. COD crystals with IC+SB OPN were prepared using a combination of both techniques. Control crystals were prepared in the absence of OPN. Crystals were incubated with MDCK-II cells for up to 180 min in UF urine adjusted to 8 mm Ca(2+). Binding values for individual concentrations at specific time points and overall differences between binding curves were compared using the Mann-Whitney U-test. Crystal morphology and attachment to the cells were confirmed using field emission scanning electron microscopy (FESEM).
The sizes of crystals precipitated from UF urine in the presence of 0, 1 and 5 mg/L OPN were 21.9 µm, 19.3 µm and 16.5 µm, indicating that OPN had inhibited crystal growth in a dose-dependent fashion. Binding curves for control crystals were smooth, while those of the IC and IC+SB COD crystals associated with 1 and 5 mg/L OPN were bimodal, as were those of the 1 mg/L SB crystals. This suggests that OPN induces or potentiates a transient response that enables MDCK-II cells to release COD crystals after they have attached. Although OPN generally reduced the binding of urinary COD crystals to MDCK-II cells, at times it also appeared to mediate adhesion. It is possible therefore that OPN can reduce or increase crystal binding, and that our data represent the net effect of its opposing inhibitory or promotory properties.
In UF urine, OPN inhibits the growth of COD crystals and reduces the binding of urinary COD crystals to MDCK-II cells, regardless of whether it is IC, SB, or IC+SB. Future studies aimed at clarifying the effects of OPN, or indeed any urinary component, on crystal-cell interaction, should use crystals precipitated from urine and be performed under urinary conditions.
在超滤（UF）人尿中，确定晶内（IC）、表面结合（SB）和 IC+SB 骨桥蛋白（OPN）对尿液二水草酸钙（COD）晶体与 Madin-Darby 犬肾（MDCK-II）细胞结合的影响。
（14）C-草酸标记的尿液 COD 晶体，在含有人乳 OPN 的 UF 人尿中生成，浓度分别为 0、1.0 和 5.0mg/L。从同一 UF 尿样中分离出另一个结晶核，向其中添加相同量的蛋白，生成具有 SB OPN 的晶体。采用两种技术的组合制备 IC+SB OPN 的 COD 晶体。对照晶体在没有 OPN 的情况下制备。在 UF 尿液中，将晶体与 MDCK-II 细胞孵育 180 分钟，调整 Ca（2+）至 8mm。使用 Mann-Whitney U 检验比较特定时间点各浓度的结合值和结合曲线之间的总体差异。使用场发射扫描电子显微镜（FESEM）确认晶体形态和与细胞的附着。
在存在 0、1 和 5mg/L OPN 的 UF 尿液中沉淀的晶体尺寸分别为 21.9µm、19.3µm 和 16.5µm，表明 OPN 以剂量依赖性方式抑制了晶体生长。对照晶体的结合曲线较平滑，而与 1 和 5mg/L OPN 相关的 IC 和 IC+SB COD 晶体的结合曲线呈双峰状，1mg/L SB 晶体的结合曲线也是如此。这表明 OPN 诱导或增强了一种瞬态反应，使 MDCK-II 细胞在附着后能够释放 COD 晶体。尽管 OPN 通常会降低尿 COD 晶体与 MDCK-II 细胞的结合，但有时它似乎也介导了细胞的附着。因此，OPN 可以减少或增加晶体结合，并且我们的数据代表了其抑制或促进特性的相反作用的净效应。
在 UF 尿液中，OPN 抑制 COD 晶体的生长，并降低尿 COD 晶体与 MDCK-II 细胞的结合，无论其是 IC、SB 还是 IC+SB。未来旨在阐明 OPN 或任何尿液成分对晶体-细胞相互作用影响的研究，应使用尿液中沉淀的晶体，并在尿液条件下进行。
