Bidmon Bettina, Endemann Michaela, Müller Thomas, Arbeiter Klaus, Herkner Kurt, Aufricht Christoph
Kinderdialyse and Ludwig Boltzmann Institut, Department of Pediatrics, Universitätsklinik für Kinder und Jugendheilkunde, AKH Wien, Wien, Austria.
Kidney Int. 2002 Nov;62(5):1620-7. doi: 10.1046/j.1523-1755.2002.00617.x.
We recently designed an in vitro system based on differential Triton-extractability of Na,K-ATPase from the cytoskeletal protein fraction isolated from rat renal cortex after renal ischemia. In the present study, we hypothesized that heat shock protein (HSP)-70, HSP-25 and HSP-90 work synergistically to stabilize the cytoskeletal anchorage of Na,K-ATPase.
Cellular proteins were fractionated by differential centrifugation into cytoskeletal pellets (I-PEL) obtained early (exhibiting abnormally high Triton extractability of Na,K-ATPase) and non-cytoskeletal supernatants (R-SUP) obtained late (exhibiting high abundance of HSP) after renal ischemia. For assessment of the role of HSP-70, HSP-25 and HSP-90 upon in vitro re-compartmentalization, I-PEL was either incubated in R-SUP with/without HSP antibodies, or in buffer with/without HSPs at different titers and combinations. Effects were evaluated by changes of Triton extractability of Na,K-ATPase after co-incubation.
R-SUP was shown to contain significant amounts of HSP-70, HSP-25 and HSP-90. Incubation of I-PEL in R-SUP reduced Triton extractability of Na,K-ATPase. Addition of antibodies against each HSP significantly abolished these effects of R-SUP. Incubation of I-PEL with purified HSP-70, HSP-25 or HSP-90 each partly reproduced the effects of R-SUP, whereas the combination of all three HSP demonstrated a strong and more than additive effect on the cytoskeletal stabilization of Na,K-ATPase.
The molecular mechanisms responsible for postischemic re-compartmentalization of Na,K-ATPase in rat renal cortex likely involves interactions between HSP-70, HSP-25 and HSP-90, stress proteins known to be induced in the ischemic kidney.
我们最近设计了一种体外系统，该系统基于从肾缺血后大鼠肾皮质分离的细胞骨架蛋白组分中钠钾 - ATP酶的不同Triton可提取性。在本研究中，我们假设热休克蛋白（HSP）-70、HSP-25和HSP-90协同作用以稳定钠钾 - ATP酶的细胞骨架锚定。
通过差速离心将细胞蛋白分离为肾缺血后早期获得的细胞骨架沉淀（I-PEL，其钠钾 - ATP酶的Triton提取率异常高）和后期获得的非细胞骨架上清液（R-SUP，其HSP丰度高）。为了评估HSP-70、HSP-25和HSP-90在体外重新分隔中的作用，I-PEL要么在含有/不含有HSP抗体的R-SUP中孵育，要么在含有不同滴度和组合的HSP/不含HSP的缓冲液中孵育。通过共孵育后钠钾 - ATP酶的Triton提取率变化来评估效果。
R-SUP显示含有大量的HSP-70、HSP-25和HSP-90。I-PEL在R-SUP中孵育降低了钠钾 - ATP酶的Triton提取率。添加针对每种HSP的抗体显著消除了R-SUP的这些作用。I-PEL与纯化的HSP-70、HSP-25或HSP-90各自孵育部分重现了R-SUP的作用，而所有三种HSP的组合对钠钾 - ATP酶的细胞骨架稳定作用表现出强烈且大于相加的效果。
大鼠肾皮质中缺血后钠钾 - ATP酶重新分隔的分子机制可能涉及HSP-70、HSP-25和HSP-90之间的相互作用，这些应激蛋白在缺血性肾脏中被诱导产生。
