Department of Nephrology, Faculty of Medicine, University of Thessaly, Larissa, Greece.
J Basic Clin Physiol Pharmacol. 2022 Aug 2;34(6):745-754. doi: 10.1515/jbcpp-2022-0128. eCollection 2023 Nov 1.
Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury (AKI). Experimental studies have shown that indoleamine 2,3-dioxygenase 1 (IDO) and the purinergic receptor P2X7 contribute to kidney I-R injury. We evaluated whether there is an interplay between IDO and P2X7 in the pathogenesis of I-R injury.
Primary renal proximal tubular epithelial cells (RPTECs) were subjected to anoxia or reoxygenation with or without specific inhibitors. Cell imaging, colorimetric assays, and Western blotting were used.
Cell imaging revealed that inhibition of IDO, or all the purinergic receptors with an ATPase, or specific inhibition of P2X7 rescued the cells from anoxia or reoxygenation-induced cell death. This was confirmed with LDH release assay, which also detected the ferroptotic nature of cell death due to reoxygenation. On the contrary, activated cleaved caspase 3 increased during anoxia, showing that apoptosis prevails. All the aforementioned treatments prevented caspase increase. Both anoxia and reoxygenation increased extracellular ATP, IDO, and P2X7 expression. IDO remained unaffected by the above-mentioned treatments. On the contrary, treatment with apyrase or inhibition of P2X7decreased extracellular ATP and P2X7 expression, which are also decreased by inhibition of IDO. The first indicates a positive feedback loop regarding P2X7 activation, expression and function, while the latter implies that IDO controls P2X7 expression.
In RPRECs subjected to anoxia or reoxygenation, IDO is upregulated, increasing P2X7 and contributing to anoxia or reoxygenation-induced cell death. Clarifying the molecular mechanisms implicated in kidney I-R injury is of particular interest since it may lead to new therapeutic strategies against AKI.
缺血再灌注（I-R）损伤是急性肾损伤（AKI）最常见的原因。实验研究表明，色氨酸 2,3-双加氧酶 1（IDO）和嘌呤能受体 P2X7 有助于肾 I-R 损伤。我们评估了 IDO 和 P2X7 之间是否存在相互作用在 I-R 损伤的发病机制中。
原代肾近端小管上皮细胞（RPTEC）在缺氧或再氧化条件下进行，或同时使用特异性抑制剂。细胞成像、比色测定和 Western blot 用于评估。
细胞成像显示，IDO 抑制剂、所有嘌呤能受体 ATPase 抑制剂或 P2X7 特异性抑制剂均可挽救细胞免受缺氧或再氧化诱导的细胞死亡。这一点通过 LDH 释放测定得到了证实，该测定还检测到由于再氧化引起的铁死亡性质的细胞死亡。相反，在缺氧期间，激活的 cleaved caspase 3 增加，表明细胞凋亡占主导地位。所有上述处理均阻止 caspase 增加。缺氧和再氧化均增加细胞外 ATP、IDO 和 P2X7 的表达。IDO 不受上述处理的影响。相反，用 apyrase 处理或抑制 P2X7 可降低细胞外 ATP 和 P2X7 的表达，而抑制 IDO 也可降低其表达。前者表明 P2X7 激活、表达和功能存在正反馈环，后者则表明 IDO 控制 P2X7 的表达。
在缺氧或再氧化的 RPRECs 中，IDO 上调，增加 P2X7，并有助于缺氧或再氧化诱导的细胞死亡。阐明与肾 I-R 损伤相关的分子机制尤其重要，因为它可能导致针对 AKI 的新治疗策略。
