di Mari J F, Davis R, Safirstein R L
University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0562, USA.
Am J Physiol. 1999 Aug;277(2):F195-203. doi: 10.1152/ajprenal.1999.277.2.F195.
Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.
缺血/再灌注（I/R）损伤会引发肾脏的功能和形态学改变。坏死，主要发生在近端小管（PT），是这种肾损伤模型的标志，而远端肾单位的细胞存活下来，表面看起来完好无损。我们研究了肾单位各个区域细胞结局的差异是否可能归因于丝裂原活化蛋白激酶（MAPKs）对I/R损伤反应的节段性差异激活。虽然c-Jun氨基末端激酶（JNK）在外髓质的皮质和内带均被激活，但细胞外调节激酶（ERK）途径仅在内带被激活，内带中厚壁升支（TAL）细胞占主导。这些研究与ERK激活对存活至关重要的观点一致。为了直接验证这一假设，我们研究了一个体外系统，在该系统中可以检测这些途径的调控及其对细胞存活的影响。通过黄嘌呤氧化酶对次黄嘌呤的分解代谢，在培养的小鼠PT和TAL细胞中诱导氧化损伤。通过细胞计数、光学显微镜、碘化丙啶摄取和荧光激活细胞分选（FACS）分析评估，发现PT细胞比TAL细胞对氧化应激更敏感。免疫沉淀/激酶分析显示，两种细胞类型中均发生JNK激活，而ERK激活仅发生在TAL细胞中。然后我们研究了ERK途径的丝裂原活化蛋白激酶激酶（MEK）-1抑制剂PD-098059对PT和TAL存活的影响。在TAL细胞中，氧化应激暴露后ERK抑制使细胞存活率降低近四倍（P < 0.001）。在PT细胞中，胰岛素样生长因子I（IGF-I）激活ERK途径使存活率提高了三倍（P < 0.001），而这种IGF-I增强的细胞存活被PD-098059抑制。这些结果表明，缺血后肾脏中的细胞存活可能依赖于ERK激活，提示该途径可能是I/R损伤治疗的靶点。
