Identification of the physiological function of the NADPH oxidase Nox4 in the kidney

已知的NADPH氧化酶的唯一功能是产生活性氧(ROS：H_2O_2或O_2·-)。NOx酶的调节、表达部位和产生的ROS类型不同。与所有其他NOx亚型不同的是，NOX4亚型在结构上是活跃的，在生理条件下几乎只在肾脏表达。尽管进行了大量研究，但NOX4的肾脏功能仍完全未知。本申请的目的是揭示这一功能，重点是近端肾小管的转运过程。来自野生型和NOX4-/-小鼠的血浆、尿液和肾组织的初步非靶向代谢组学表明，该酶控制硫氨基酸的代谢和/或复合体B维生素的重吸收。在这一应用中，这些方面将进一步探索，主要是在体内，使用全球和肾脏特异性诱导的NOX4基因敲除小鼠。低硫和维生素B缺乏饮食干预小鼠的血浆和尿液中的代谢物将通过有针对性的LC-MS/MS进行分析。此外，该酶的细胞内定位和相互作用伙伴将使用NOX4-FLAG敲入小鼠来鉴定，该小鼠是在为这一应用做准备时产生的。总而言之，该项目将使用活体模型来确定NOX4在肾脏中的生理功能。

The only known enzymatic function of NADPH oxidase enzymes of the Nox-family is the production of reactive oxygen species (ROS: H2O2 or O2•-). Nox enzymes differ in their regulation, site of expression and type of ROS produced. The isoform Nox4, differently to all other Nox isoforms, is constitutively active and under physiological conditions almost exclusively expressed in the kidney. Despite intense research, the renal function of Nox4 is, however, completely unknown. The aim of the present application is to uncover this function focusing on transport processes in the proximal tubule of the kidney. Preliminary untargeted metabolomics from plasma, urine and renal tissue of wild type and Nox4-/- mice suggested that the enzyme controls metabolism of sulphur amino acids and/or reabsorption of vitamins of the complex B. In this application, these aspects will be further explored, largely in vivo, using global and kidney-specific inducible Nox4 knockout mice. Metabolites will be analysed by targeted LC-MS/MS from plasma and urine of mice with low sulphur and vitamin B-deficient diet interventions. Moreover, the intracellular localization and interaction partners of the enzyme will be identified using a Nox4-FLAG knock-in mouse, generated in preparation for this application. In summary, the project will identify the physiological function of Nox4 in the kidney using in vivo models.