Role of osteopontin in early phase of renal crystal formation: immunohistochemical and microstructural comparisons with osteopontin knock-out mice
Role of osteopontin in early phase of renal crystal formation: immunohistochemical and microstructural comparisons with osteopontin knock-out mice
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DOI：
10.1007/s00240-011-0400-z
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发表时间：
2012-04-01
影响因子：
--
通讯作者：
Kohri, Kenjiro
中科院分区：
文献类型：
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作者：
Hirose, Masahito;Tozawa, Keiichi;Kohri, Kenjiro
骨桥蛋白（Osteopontin， OPN）是肾钙石的重要基质蛋白。然而，OPN在肾结晶形成早期的功能尚不明确。在这项研究中，我们通过超微结构观察和免疫透射电镜（tem）检测了对照和OPN敲除（OPN- ko）小鼠肾脏晶体形成早期OPN的表达。雄性野生型小鼠（C57BL/ 6,8周龄）和OPN-KO小鼠（C57BL/ 6,8周龄）腹腔注射乙醛酸酯（100 mg/kg）。分别于给药前、给药后6、12、24 h采集肾脏。我们采用透射电镜、免疫组化染色、western blotting和定量RT-PCR检测肾晶体形成与OPN超微结构定位的关系。乙醛酸盐给药后，肾皮质-髓质连接处的OPN蛋白表达逐渐升高，至12 h时，OPN mRNA表达升高，24 h时，OPN mRNA表达降低。超微结构观察，6 h时，OPN开始出现在肾远端小管细胞管腔侧，12 h时，OPN逐渐出现在小管腔内，OPN存在于小管腔内的晶核和塌陷的线粒体中。在OPN- ko小鼠中，线粒体出现塌陷，但24h未见晶核形成。本研究基于此结果提出，细胞损伤后管腔内线粒体、微绒毛等细胞器的出现可能是由于OPN的聚集能力导致晶核形成的起点。
Osteopontin (OPN) is an important matrix protein of renal calcium stone. However, the function of OPN in the early phase of renal crystal formation is not well defined. In this study, we examined OPN expression in the early phase of renal crystal formation with ultra-microstructural observations and immuno-TEM (transmission electron microscopy) in control and OPN knock-out (OPN-KO) mice. Glyoxylate (100 mg/kg) was intra-abdominally administered to male wild-type mice (C57BL/6, 8 weeks of age) and OPN-KO mice (C57BL/6, 8 weeks of age). Kidney was collected before and 6, 12, and 24 h after administration. We examined the relation between renal crystal formation and microstructural OPN location using TEM and immunohistochemical staining of OPN as well as western blotting and quantitative RT-PCR for OPN. OPN protein expression gradually increased in the renal cortex-medulla junction after glyoxylate administration, and OPN mRNA was increased until 12 h, but decreased at 24 h. In ultra-microstructural observation, OPN began to appear on the luminal side of renal distal tubular cells at 6 h and was gradually detected in the tubular lumen at 12 h. OPN was present in the crystal nuclei and collapsed mitochondria in the tubular lumen. In the OPN-KO mice, collapsed mitochondria were present, but no crystal nuclei formation were detected at 24 h. Based on the results this study proposed that the appearance of organelles, such as mitochondria and microvilli, in the tubular lumen after cell injury may be the starting point of crystal nucleus formation due to the aggregation ability of OPN.