内源性哇巴因通过Na+,K+-ATPase α1/iRhom1相互作用保护草酸钙肾结石损伤生物学机制研究

内源性哇巴因（Endogenous ouabain, EO）是具有心血管活性的内分泌激素样物质，但我们发现EO在临床肾结石病人血液中异常高表达。为探知该现象，发现EO能显著保护草酸钙诱导的小鼠肾结石损伤，且该作用依赖肾小管上皮细胞amphiregulin (AREG)表达。作为EGFR配体，AREG基因转录后以前体蛋白(pro-AREG)存在，后经脱落酶ADAM17剪切成可溶性 AREG(sAREG )释放至胞外发挥作用。我们发现哇巴因能调控pro-AREG剪切成sAREG这一关键限速步骤，但机制不详。本研究将从细胞、分子与整体动物水平探索EO通过促进受体Na+,K+-ATPase α1亚基与iRhom1相互作用，进而稳定ADAM17细胞膜分布，促进sAREG生成，发挥保护作用的生物学机制。该机制阐明将有助于揭示EO在肾结石疾病中的新生理功能，为临床肾结石诊疗提供潜在生物标志物与理论依据。

Endogenous ouabain (EO) belongs to a novel endocrine substance that has cardiovascular activity. However, by taking advantage of highly-sensitive HPLC-MS-MS analytical method, we found for the first time that serum EO was upregulated in patients with nephrolithiasis and in mice with CaOx-induced renal injury.To understand the mechanism, ouabain was found to be able to protect CaOx-induced renal injury, and this effect was largely dependent on AREG expression in renal tubular cells. As EGFR ligand, AREG exists in a pro-protein form after transcription, that is, pro-AREG. After cleavage by membrane ADAM17, pro-AREG is converted into soluble AREG (sAREG) and released into extracellular milieu to bind with EGFR receptor on self-cell membrane or adjacent cells. In this study, we found ouabain was able to increase AREG gene transcription in renal tubular cells, however, overexpression of pro-AREG cDNA alone failed to mimic the stimulatory effect of ouabain on sAREG production and the protective effect of ouabain on CaOx-induced renal tubular cell injury, raising a possibility that ouabain may regulate the conversion of pro-AREG into sAREG, a critical rate-limiting step in AREG metabolism. To test this presumption, we used ouabain inhibitor or specific antibody to block the effect of EO, or supplemented with exogenous ouabain to mimic or amplify the effect of EO, then observed the biological effect of EO on Na+,K+-ATPase ɑ1/iRhom1 interaction, which increases membrane ADAM17 cleavage activity and sAREG production. The result of this study will highlight the importance of EO in the pathology of nephrolithiasis, and provide theoretical basis for the clinical diagnosis and therapy of nephrolithiasis.