Metabolomics Reveals a Key Role for Fumarate in Mediating the Effects of NADPH Oxidase 4 in Diabetic Kidney Disease

Abstract

The NADPH oxidase (NOX) isoform NOX4 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, and mesangial matrix accumulation in the F1 Akita model of DKD. Metabolomic analyses from these mouse studies revealed that tricarboxylic acid (TCA) cyclerelated urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by the NOX1/ NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (in mouse and human tissue), and administration of the NOX1/NOX4 inhibitor increased glomerular FH levels in diabetic mice. Induction of Nox4 in vitro and in the podocyte-specific NOX4 transgenic mouse led to reduced FH levels. In vitro, fumarate stimulated endoplasmic reticulum stress, 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 transgenic mice 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 connecting metabolic pathways to DKD pathogenesis, and measuring urinary fumarate levels may have application for monitoring renal NOX4 activity.

Tissue-specific metabolic reprogramming drives nutrient flux in diabetic complications

“…Succinate, a ligand of the G protein-coupled receptor GPR91, can activate the renin-angiotensin system (39)(40)(41), and the activation of angiotensin II may result in NADPH oxidase 4-generated (Nox4-generated) mitochondrial oxidative damage (42,43), leading to a feed-forward mechanism that promotes kidney injury in diabetes. Fumarate, which also accumulates in the diabetic kidney and urine of a type 1 model of DKD, is a potent inducer of HIF-1α and TGF-β, both of which could contribute to kidney disease pathology (44). Second, metabolic intermediates may alter metabolic enzyme activity through the generation of post-translational modifications.…”

Systems biology analysis reveals role of MDM2 in diabetic nephropathy

“…Tissue sections (5 μm) were stained with periodic acid-Schiff (PAS) and imaged by light microscopy. Immunofluorescent staining of WT1 in the cryosections was carried out as described (48). Briefly, sections were incubated overnight at 4°C with antibodies against WT1, washed with PBS, and incubated for 1 hour at room temperature with Alexa Fluor-conjugated secondary antibodies and visualized by a BIOREVO BZ-9000 fluorescence microscope (Keyence).…”

“…BUN was assessed with a colorimetric assay (DiaSys) and calculated as mg/dl. Urine metabolomics was performed as per our recent publications (3,48).…”

NADPH Oxidase 4 at the Nexus of Diabetes, Reactive Oxygen Species, and Renal Metabolism

“…In this issue of JASN, You et al 15 make a valuable contribution to the literature, expanding the role for NOX4 in diabetic kidney disease to include the regulation of intermediary metabolism. More specifically, You et al 15 show that NOX4 modulates levels of the tricarboxylic acid (TCA) cycle intermediate fumarate through its effects on fumarate hydratase (FH) expression (FH catalyzes the hydration of fumarate to malate).…”

“…More specifically, You et al 15 show that NOX4 modulates levels of the tricarboxylic acid (TCA) cycle intermediate fumarate through its effects on fumarate hydratase (FH) expression (FH catalyzes the hydration of fumarate to malate). Using a metabolomics screen, You et al 15 found dramatic increases in several TCA cycle intermediates in the urine of F1 Akita mice, a model of type 1 diabetic kidney disease, compared with F1 controls. Only fumarate levels, however, fell in a dose-dependent manner after NOX4 inhibition with GKT137831.…”