Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis, and the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI specific siRNA in the absence and presence of an antioxidant tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in the PDI inhibited cells. Also, PDI inhibition caused Nrf2 (nuclear factor E2 related factor 2, a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase, and glutathione S-transferase activities, and increased oxidative stress. In PDI inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress, and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, while Keap1 silencing (Kelch-like ECH-associated protein 1, a Nrf2 regulatory protein) protected the cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. These studies suggest an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.

中文翻译：

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蛋白质二硫化物异构酶抑制损害 Keap1/Nrf2 信号传导和线粒体功能并诱导肾近端肾小管细胞凋亡。

肾近端肾小管细胞凋亡在肾脏健康和疾病中起着关键作用。然而，触发肾细胞凋亡的细胞分子仍然难以捉摸。在这里，我们评估了抑制蛋白质二硫化物异构酶 (PDI)（一种关键的硫氧还蛋白伴侣蛋白）对细胞凋亡的影响，以及人肾近端肾小管 (HK2) 细胞的潜在机制。在不存在和存在抗氧化剂 tempol 的情况下，用 PDI 特异性 siRNA 转染 HK2 细胞。PDI siRNA 转染导致 PDI 蛋白表达和酶活性降低约 70%。PDI 抑制增加 caspase-3 活性并诱导严重的细胞凋亡。通过线粒体细胞色素 c 水平、线粒体膜电位、耗氧量和 ATP 水平评估的线粒体功能在 PDI 抑制细胞中显着降低。还，PDI 抑制导致 Nrf2（核因子 E2 相关因子 2，一种氧化还原敏感转录因子）细胞质隔离，降低超氧化物歧化酶和谷胱甘肽 S 转移酶活性，并增加氧化应激。在 PDI 抑制细胞中，tempol 减少细胞凋亡、caspase-3 活性和氧化应激，并恢复 Nrf2 核易位和线粒体功能。在细胞中沉默 Nrf2 取消了 tempol 的有益作用，而 Keap1 沉默（Kelch 样 ECH 相关蛋白 1，一种 Nrf2 调节蛋白）保护细胞免受 PDI 抑制作用。总的来说，我们的数据表明 PDI 抑制减少了 Nrf2 核易位，导致氧化应激，进一步引发线粒体功能障碍和肾细胞凋亡。