Metabolomics reveals a key role for fumarate in mediating the effects of NADPH oxidase 4 in diabetic kidney disease

TheNADPHoxidase (NOX) isoformNOX4 has been linked with diabetic kidney disease (DKD). However, a mechanistic understanDing of the downstream effects of NOX4 remains to be established. We report that podocyte-specific induction of NOX4 in vivo was sufficient to recapitulate the characteristic glomerular changes noted with DKD, incluDing glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, albuminuria, and podocyte dropout. Intervention with a NOX1/NOX4 inhibitor reduced albuminuria, glomerular hypertrophy, andmesangialmatrix accumulation in the F1 Akita model of DKD.Metabolomic analyses from thesemouse studies revealed that tricarboxylic acid (TCA) cycle-related urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by theNOX1/NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (inmouse and human tissue), and administration of theNOX1/NOX4 inhibitor increased glomerular FHlevels in diabeticmice. Induction ofNox4 in vitro and in the podocyte-specificNOX4 transgenicmouse led to reduced FHlevels. In vitro, fumarate stimulated endoplasmic reticulumstress, matrix gene expression, and expression of hypoxia-inducible factor-1a (HIF-1a) and TGF-b. Similar upregulation of renal HIF-1a and TGF-b expression was observed in NOX4 transgenicmice and diabetic mice and was attenuated by NOX1/NOX4 inhibition in diabetic mice. In conclusion, NOX4 is a major mediator of diabetesassociated glomerular dysfunction through targeting of renal FH, which increases fumarate levels. Fumarate is therefore a key link connectingmetabolic pathways to DKD pathogenesis, andmeasuring urinary fumarate levels may have application for monitoring renal NOX4 activity.