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NOX1 abet mesangial fibrogenesis via iNOS induction in diabetes
Ling Gao1, Weilu Huang, Jing Li
1Department of Endocrinology, Renmin Hospital of Wuhan University, Jiefang RD 238, Wuhan, 430060, China.
Abstract:
Both NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) are the main sources of reactive oxygen species in kidney. However, their interactions in oxidative stress and contributions to kidney fibrosis during diabetic nephropathy have not been studied. Human mesangial cells were treated with normal glucose (5.6 mmol/L), high glucose (30 mmol/L) in the presence or absence of AGE (200 mg/L). Protein expressions of NOX1, NOX2, NOX4, and iNOS were examined by immunoblotting. NOX was genetically silenced with specific RNAi to study the interactions between NOX and iNOS in diabetic milieu. Superoxide (O(·-)) and peroxynitrite (ONOO(·-)) productions were assessed by dihydroethidium and hydroxyphenyl fluorescein, respectively. Fibrotic factors were determined by biochemistry assay. Superoxide, peroxynitrite, TGF-β, and fibronectin productions as well as the protein expressions of NOX1, NOX2, NOX4, and iNOS were increased in the diabetic milieu (high glucose 30 mmol/L plus AGE 200 mg/L). However, abolishment of iNOS induction with 1400W or iNOS RNAi would restore peroxynitrite, TGF-β, and fibronectin productions completely to basal level and attenuate superoxide production. Moreover, NOX1 inhibition not only prevented iNOS induction but also abrogated changes consequent to iNOS induction such as mesangial fibrogenesis.
Insights
Diabetic nephropathy involves kidney fibrosis driven by reactive oxygen species from NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS). NOX1 inhibition prevents iNOS induction, mitigating fibrosis in kidney cells.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) are primary sources of reactive oxygen species in the kidney.
- The interplay between NOX and iNOS in kidney fibrosis during diabetic nephropathy remains understudied.
Purpose of the Study:
- To investigate the interactions between NOX and iNOS in oxidative stress.
- To determine their contributions to kidney fibrosis in a diabetic nephropathy model.
Main Methods:
- Human mesangial cells were cultured under normal and high glucose conditions with Advanced Glycation End-products (AGEs).
- Protein expression of NOX1, NOX2, NOX4, and iNOS was analyzed using immunoblotting.
- NOX was genetically silenced using RNA interference (RNAi).
- Superoxide and peroxynitrite production were measured using fluorescent probes.
- Fibrotic factors (TGF-β, fibronectin) were quantified via biochemical assays.
Main Results:
- High glucose plus AGEs increased superoxide, peroxynitrite, TGF-β, and fibronectin, alongside elevated NOX and iNOS expression.
- Inhibiting iNOS (using 1400W or iNOS RNAi) normalized peroxynitrite, TGF-β, and fibronectin levels and reduced superoxide.
- NOX1 inhibition prevented iNOS induction and subsequent fibrogenesis, including mesangial fibrogenesis.
Conclusions:
- NOX1 plays a crucial role in mediating iNOS induction and subsequent fibrotic processes in diabetic nephropathy.
- Targeting NOX1 may offer a therapeutic strategy to combat kidney fibrosis in diabetic nephropathy.
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