Background and aims: Diabetes-induced overproduction of reactive oxygen species (ROS) in the kidney is a key factor in the development and progression of renal complications associated with diabetes. Chronic hyperglycaemia disrupts the renal environment, promoting inflammation, progressive albuminuria, and fibrosis in diabetic kidney disease (DKD). Unlike rodents, humans possess the NADPH oxidase isoform NOX5, which plays a significant role in renal ROS generation. This study investigates the specific impact of NOX5 on renal pathology using a NOX5 transgenic mouse model and a human kidney organoid model of DKD. Furthermore, this research compares NOX5 with the well-characterized NADPH oxidase, NOX4, to delineate their respective contributions to DKD.
Materials and methods: We investigated the expression of human NOX5 and NOX4, as well as reactive oxygen species (ROS) production, in human kidney biopsies, renal organoids, and cells under diabetic conditions. To evaluate their impact, we employed genetic silencing and pharmacological inhibition of NOX4, in human renal cells and organoids exposed to a diabetic milieu. In animal studies, we assessed the effects of human NOX5 overexpression in Nox5 transgenic mice, independent of NOX4, under diabetic and non-diabetic conditions. Additionally, we analysed urinary and plasma NOX5 levels in diabetic subjects at various stages of nephropathy.
Results: Compared to NOX4, silencing of NOX5 significantly reduced high glucose-induced expression of fibrotic markers (CTGF and collagen IV) and inflammatory markers (MCP-1 and TLR4), and also downregulated ROS-sensitive factors such as EGR-1 and PKC-α, along with the cell cycle regulator P21, in cellular and organoid models of DKD. Our findings also indicate that NOX5 functions upstream of NOX4, as silencing NOX5 resulted in the downregulation of NOX4, whereas the reverse was not observed.
In vivo studies demonstrated that independent overexpression of NOX5, not involving the NOX4 pathway, exacerbated renal pathology in STZ-diabetic mice, showing increased albuminuria, renal fibrosis, and inflammation. Furthermore, we observed a positive correlation between urinary NOX5 excretion and albuminuria levels in diabetic individuals with nephropathy.
Conclusion: These findings highlight the therapeutic promise of targeting NOX5 rather than NOX4 in human DKD. The study suggests that specifically targeting NOX5 alone may effectively attenuate DKD progression, advocating for the development of NOX5-specific inhibitors as a potential therapeutic strategy. Furthermore, NOX5 could serve as a biomarker for diabetic kidney disease.