Patel Amit B, Robertson William G, Choong Simon, Hothersall John S
Centre for Treatment and Prevention of Kidney Stone disease, Institute of Urology, University College London, UK.
BJU Int. 2006 Nov;98(5):1094-9. doi: 10.1111/j.1464-410X.2006.06478.x.
To investigate whether the antioxidant protection attributed to small heat-shock proteins (sHSPs) affects calcium oxalate stone formation, a pro-oxidant disease.
Canine distal tubular epithelial cells (Madin-Darby canine kidney, MDCK cells) were grown as confluent monolayers. Treatment regimens included control and HS-treated cells (37 degrees C and 42 degrees C for 1 h) with or without calcium oxalate monohydrate (COM) or free oxalate treatment (28 microg/cm2) 16 h later. In digitonin-permeabilized cells, O2- was measured by lucigenin-enhanced chemiluminescence over a 5-min period, to measure mitochondrial O2- production. Protein expression was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis Western blot analysis using specific antibodies.
COM significantly increased O2- production in MDCK cells. HS treatment, which up-regulated HSP25 expression, significantly decreased this O2- production (P < 0.05) but had no effect in control cells. In COM-treated cells (20 h) there was a marked and significant down-regulation of both HSP 25, HSP 70 and heme oxygenase-1 expression compared to cells treated with HS alone (P < 0.05). Free oxalate had no effect on HSP 25 expression.
The results suggest that the COM-induced increase in mitochondrial O2- production in MDCK cells is ameliorated by HSP 25 up-regulation via HS. Specific COM inhibition of HSP 25, HSP 70 and heme oxygenase-1 up-regulation suggests that COM-induced reactive oxygen species damage is unable to benefit from HSP-associated physiological resistance.
探讨小热休克蛋白（sHSPs）的抗氧化保护作用是否会影响草酸钙结石形成，这是一种促氧化疾病。
犬远端肾小管上皮细胞（Madin-Darby犬肾细胞，MDCK细胞）生长为汇合单层。处理方案包括对照细胞和热休克处理的细胞（37℃和42℃处理1小时），16小时后分别给予或不给予一水合草酸钙（COM）或游离草酸处理（28μg/cm²）。在用洋地黄皂苷通透处理的细胞中，通过荧光素增强化学发光法在5分钟内测量超氧阴离子（O₂⁻），以测定线粒体O₂⁻的产生。使用特异性抗体通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳蛋白质印迹分析评估蛋白质表达。
COM显著增加了MDCK细胞中O₂⁻的产生。热休克处理上调了HSP25的表达，显著降低了这种O₂⁻的产生（P<0.05），但对对照细胞没有影响。与单独用热休克处理的细胞相比，在COM处理的细胞（20小时）中，HSP 25、HSP 70和血红素加氧酶-1的表达均有明显且显著的下调（P<0.05）。游离草酸对HSP 25的表达没有影响。
结果表明，通过热休克上调HSP 25可改善COM诱导的MDCK细胞线粒体O₂⁻产生增加。COM对HSP 25、HSP 70和血红素加氧酶-1上调的特异性抑制表明，COM诱导的活性氧损伤无法从与HSP相关的生理抗性中获益。
