Bidmon B, Endemann M, Müller T, Arbeiter K, Herkner K, Aufricht C
Kinderdialyse, and Ludwig Boltzmann Institut, Department of Pediatrics, Universitatsklinik für Kinder- und Jugendheilkunde, AKH Wien, Wien, Austria.
Kidney Int. 2000 Dec;58(6):2400-7. doi: 10.1046/j.1523-1755.2000.00423.x.
Recent studies have suggested a role of heat shock protein (HSP)-70 in cytoskeletal repair during cellular recovery from renal ischemia. The aim of this study was to test the hypothesis that HSP-70 interacts in vitro with cytoskeletal elements obtained from rat renal cortex during early reflow after renal ischemia.
Cellular proteins were fractionated into cytoskeletal pellets and noncytoskeletal supernatants by Triton X-100 extraction of rat renal cortex obtained after 15 minutes or 18 hours of reflow after 45 minutes of renal ischemia, or from controls. Aliquots of isolated pellets were coincubated with aliquots of isolated supernatants in different combinations. A repeat Triton extraction was performed, and differential distribution of Na, K-ATPase or HSP-70 was assessed by Western blots and densitometric analysis.
Coincubation of cytoskeletal pellets obtained during early reflow after renal ischemia (exhibiting severe injury of the cytoskeletal anchorage of Na,K-ATPase) and noncytoskeletal supernatant obtained during later reflow (showing high HSP expression) resulted in specific translocation of HSP-70 from the supernatant into the pellet, functionally associated with dose-dependent stabilization of Na,K-ATPase within this cytoskeletal fraction. These effects could be reproduced by incubation with purified HSP-70 and were abolished by the addition of anti-HSP-70 antibodies.
These data support the hypothesis that HSP-70 interacts with cytoskeletal elements during the restoration of proximal tubule cell structure and polarity after renal ischemia. This experimental approach represents a new in vitro assay to study further the role of HSP in cellular repair.
最近的研究表明，热休克蛋白（HSP）-70在肾脏缺血后细胞恢复过程中的细胞骨架修复中发挥作用。本研究的目的是验证以下假设：在肾脏缺血后的早期再灌注过程中，HSP-70在体外与大鼠肾皮质获得的细胞骨架成分相互作用。
通过用 Triton X-100 提取肾脏缺血 45 分钟后再灌注 15 分钟或 18 小时后获得的大鼠肾皮质，或从对照组获得的肾皮质，将细胞蛋白分离为细胞骨架沉淀和非细胞骨架上清液。将分离的沉淀等分试样与分离的上清液等分试样以不同组合进行共孵育。进行重复的 Triton 提取，并通过 Western 印迹和光密度分析评估 Na、K-ATP 酶或 HSP-70 的差异分布。
肾脏缺血后早期再灌注期间获得的细胞骨架沉淀（显示 Na、K-ATP 酶的细胞骨架锚定严重受损）与后期再灌注期间获得的非细胞骨架上清液（显示高 HSP 表达）共孵育，导致 HSP-70 从 上清液特异性转运到沉淀中，在功能上与该细胞骨架部分内 Na、K-ATP 酶的剂量依赖性稳定相关。这些效应可以通过与纯化的 HSP-70 孵育来重现，并通过添加抗 HSP-70 抗体而消除。
这些数据支持以下假设：在肾脏缺血后近端小管细胞结构和极性恢复过程中，HSP-70 与细胞骨架成分相互作用。这种实验方法代表了一种新的体外测定方法，可进一步研究 HSP 在细胞修复中的作用。
