CEDOC, Chronic Diseases Research Centre, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisbon, Portugal.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Adv Exp Med Biol. 2021;1306:109-120. doi: 10.1007/978-3-030-63908-2_8.
Our general goal was to non-invasively evaluate kidney tubular dysfunction. We developed a strategy based on cysteine (Cys) disulfide stress mechanism that underlies kidney dysfunction. There is scarce information regarding the fate of Cys-disulfides (CysSSX), but evidence shows they might be detoxified in proximal tubular cells by the action of N-acetyltransferase 8 (NAT8). This enzyme promotes the addition of an N-acetyl moiety to cysteine-S-conjugates, forming mercapturates that are eliminated in urine. Therefore, we developed a strategy to quantify mercapturates of CysSSX in urine as surrogate of disulfide stress and NAT8 activity in kidney tubular cells. We use a reduction agent for the selective reduction of disulfide bonds. The obtained N-acetylcysteine moiety of the mercapturates from cysteine disulfides was monitored by fluorescence detection. The method was applied to urine from mice and rat as well as individuals with healthy kidney and kidney disease.
我们的总体目标是非侵入性地评估肾小管功能障碍。我们开发了一种基于半胱氨酸（Cys）二硫键应激机制的策略，该机制是肾功能障碍的基础。关于 Cys-二硫化物（CysSSX）的命运信息很少，但有证据表明，它们可能在近端肾小管细胞中被 N-乙酰转移酶 8（NAT8）的作用解毒。该酶促进将 N-乙酰基部分添加到半胱氨酸-S-缀合物中，形成巯基尿酸盐，然后在尿液中排出。因此，我们开发了一种策略，以定量尿液中 CysSSX 的巯基尿酸盐作为二硫键应激和肾脏管状细胞中 NAT8 活性的替代物。我们使用还原剂选择性还原二硫键。通过荧光检测监测巯基尿酸盐中半胱氨酸二硫化物的 N-乙酰半胱氨酸部分。该方法应用于来自小鼠和大鼠的尿液以及具有健康肾脏和肾脏疾病的个体。
