Kohda Yuka, Matsunaga Yoshiko, Shiota Ryugo, Satoh Tomohiko, Kishi Yuko, Kawai Yoshiko, Gemba Munekazu
Division of Pharmacology, Osaka University of Pharmaceutical Sciences, 4-21-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
J Toxicol Sci. 2006 Aug;31(3):207-17. doi: 10.2131/jts.31.207.
Zinc is an essential nutrient that can also be toxic. We have previously reported that zinc-related renal toxicity is due, in part, to free radical generation in the renal epithelial cell line, LLC-PK(1) cells. We have also shown that an MEK1/2 inhibitor, U0126, markedly inhibits zinc-induced renal cell injury. In this study, we investigated the role of an upstream MEK/ERK pathway, Raf-1 kinase pathway, and the transcription factor and ERK substrate Elk-1, in rat renal cortical slices exposed to zinc. Immediately after preparing slices from rat renal cortex, the slices were incubated in medium containing Raf-1 and MEK inhibitors. ERK1/2 and Elk-1 activation were determined by Western blot analysis for phosphorylated ERK (pERK) 1/2 and phosphorylated Elk-1 (pElk-1) in nuclear fractions prepared from slices exposed to zinc. Zinc caused not only increases in 4-hydroxynonenal (4-HNE) modified protein and lipid peroxidation, as an index of oxidant stress, and decreases in PAH accumulation, as that of renal cell injury in the slices. Zinc also induced a rapid increase in ERK/Elk-1 activity accompanied by increased expressions of pERK and pElk-1 in the nuclear fraction. A Raf-1 kinase inhibitor and an MEK1/2 inhibitor U0126 significantly attenuated zinc-induced decreases PAH accumulation in the slices. The Raf-1 kinase inhibitor and U0126 also suppressed ERK1/2 activation in nuclear fractions prepared from slices treated with zinc. The present results suggest that a Raf-1/MEK/ERK1/2 pathway and the ERK substrate Elk-1 are involved in free radical-induced injury in rat renal cortical slices exposed to zinc.
锌是一种必需营养素，但也可能具有毒性。我们先前曾报道，锌相关的肾毒性部分归因于肾上皮细胞系LLC-PK(1)细胞中自由基的产生。我们还表明，MEK1/2抑制剂U0126可显著抑制锌诱导的肾细胞损伤。在本研究中，我们研究了上游MEK/ERK途径、Raf-1激酶途径以及转录因子和ERK底物Elk-1在暴露于锌的大鼠肾皮质切片中的作用。从大鼠肾皮质制备切片后，立即将切片在含有Raf-1和MEK抑制剂的培养基中孵育。通过蛋白质印迹分析测定暴露于锌的切片制备的核级分中磷酸化ERK (pERK) 1/2和磷酸化Elk-1 (pElk-1)，以确定ERK1/2和Elk-1的激活情况。锌不仅导致作为氧化应激指标的4-羟基壬烯醛(4-HNE)修饰蛋白增加和脂质过氧化增加，以及作为切片中肾细胞损伤指标的PAH积累减少。锌还诱导ERK/Elk-1活性迅速增加，同时核级分中pERK和pElk-1的表达增加。Raf-1激酶抑制剂和MEK1/2抑制剂U0126显著减轻了锌诱导的切片中PAH积累的减少。Raf-1激酶抑制剂和U0126还抑制了用锌处理的切片制备的核级分中ERK1/2的激活。目前的结果表明，Raf-1/MEK/ERK1/2途径和ERK底物Elk-1参与了暴露于锌的大鼠肾皮质切片中自由基诱导的损伤。
