Yamate T, Kohri K, Umekawa T, Iguchi M, Kurita T
Department of Urology, Kinki University School of Medicine, Osaka, Japan.
J Urol. 1998 Oct;160(4):1506-12.
We previously suggested that osteopontin (OPN) plays an important role in the process of deposited calcium crystals adhesion to cells in the early stages of urolithiasis. To further confirm this theory, we tried to inhibit OPN expression at the translational level and examined its cellular biological consequence on the formation and adhesion process of crystals.
We synthesized antisense and sense oligonucleotide corresponding to an appropriate part of the coding sequence for OPN in Madin Darby canine kidney (MDCK) cells. With the aid of lipofection reagent DOTAP, antisense and sense oligonucleotide were introduced into MDCK cells grown in a confluent monolayer. After further incubation, inhibition of OPN expression in the cells was assessed by immunofluorescence photomicrography, and formation of calcium oxalate crystals was quantitated by incorporation of 45Ca into the stone and visualized by scanning electron microscopy (SEM).
Antisense oligonucleotide at concentrations higher than 20 microM inhibited synthesis of OPN. Incorporation of 45Ca into the calculus stone was inhibited by the addition of oligonucleotide in a concentration dependent manner in a range above 20 microM. More than 90% of incorporation was inhibited at 50 microM as compared to control. Inhibition of calcium crystal formation was confirmed by SEM.
OPN was shown as a major component in the extracellular matrix involving the formation and adhesion of calcium crystals in the distal renal tubular cells, suggesting that OPN plays an important role in stimulating deposition and adhesion of calculus crystals to cells in the early stages of urolithiasis.
我们之前曾提出，骨桥蛋白（OPN）在尿路结石形成早期钙晶体沉积至细胞的过程中发挥重要作用。为进一步证实该理论，我们试图在翻译水平抑制OPN表达，并研究其对晶体形成及黏附过程的细胞生物学影响。
我们合成了与Madin Darby犬肾（MDCK）细胞中OPN编码序列适当部分对应的反义寡核苷酸和正义寡核苷酸。借助脂质转染试剂DOTAP，将反义寡核苷酸和正义寡核苷酸导入长满单层的MDCK细胞。进一步孵育后，通过免疫荧光显微镜评估细胞中OPN表达的抑制情况，并通过将45Ca掺入结石中对草酸钙晶体的形成进行定量，通过扫描电子显微镜（SEM）观察其形态。
浓度高于20μM的反义寡核苷酸可抑制OPN的合成。在20μM以上的浓度范围内，添加寡核苷酸以浓度依赖的方式抑制了45Ca掺入结石。与对照组相比，在50μM时超过90%的掺入被抑制。SEM证实了钙晶体形成受到抑制。
OPN被证明是远端肾小管细胞外基质中参与钙晶体形成和黏附的主要成分，这表明OPN在尿路结石形成早期刺激结石晶体沉积和黏附至细胞的过程中发挥重要作用。
