Renal Nox4 contributes to systemic redox homeostasis by controlling glutathione, methionine, cysteine and folate metabolism

The NADPH oxidase Nox4 produces H2O2 and is highly expressed in the kidney. It has a protective role in diabetic renal disease as Nox4 knockout mice show an increased albuminuria. Interestingly, Nox4 expression is reduced in renal pathology, suggesting its role in normal renal function. To uncover the physiological role of Nox4 in the kidney, we first defined its localization by in situ hybridization (RNAscope) combined with immunofluorescence. Nox4 is selectively expressed in the proximal tubule. As this part of the kidney mediates mass reabsorption and secretion, WT (wild type) and Nox4*/* (tamoxifen-inducible Nox4 knockout mice) were put on a sugar/fat diet, with low sodium (130mg/kg chow), low micro nutrients and no protein. Urine samples (day 0, 3, 14), renal cortex and plasma (day 14) were analyzed by untargeted LC/MS for metabolites. In the urine, the knockout of Nox4 significantly increased the excretion of food components under normal chow (day 0). However, at day 3 of the diet, Nox4*/* had an increased excretion of xenobiotic metabolites. At day 14, the excretion of cysteine and cysteinylglycine disulfide was significantly reduced in Nox4*/*. Systematic differences in methionine, cysteine and glutathione metabolites which are controlled by Nrf2 were observed in plasma and renal cortex of Nox4*/* as compared to WT animals. In the renal cortex, Nox4*/* show reduced methionine, methionine sulfoxide, S-adenosylhomocysteine and S-methylcysteine. Plasma of Nox4*/* showed a reduction in cystathionine, cysteine s-sulfate and cysteine, indicating a reduced synthesis and therefore, attenuated reabsorption of these glutathione metabolites. Moreover, gamma-glutamyl amino acids (-glutamine, glycine, threonine and valine) were increased in plasma of Nox4*/* indicating a high activity of gamma glutamyl transferase during catalysis of glutathione in Nox4*/*. Metabolomics indicate that by Nrf2 activation, Nox4 controls the biosynthesis of glutathione as part of antioxidant response. By maintaining proper synthesis and reabsorption of metabolites, renal Nox4 maintains systemic redox homeostasis.