Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) may play a critical role in the pathogenesis of diabetic nephropathy (DN). Amongst the seven Nox homologs, Nox4 is the most abundant isoform in kidney. Previous studies have shown that functional and structural manifestations in the kidney of diabetic mice using whole-body-Nox4-knockout mice yielded conflicting results [1] (full text). We used a novel specific model of Podocin-Nox4-knockout mice to elucidate the role of Nox4 in DN.
Wild-type (WT) and Podocin-Nox4-knockout (Pod-Nox4-KO) mice, 5 months age, were injected with Streptozocin (STZ) 150mg/kg intraperitoneally to induce experimental model of Type 1 Diabetes and followed up for 16 weeks. Blood glucose was determined weekly and 24h urine were collected for albumin measurements. Animals were sacrificed under anesthesia and both kidneys were removed. A slice of kidney cortex was frozen in liquid nitrogen or embedded in paraffin for microscopy and protein analyses, 1 kidney and half from each mouse were used for glomeruli isolation.
PAS-stained sections showed reduction in matrix expansion in STZ-Pod-Nox4-KO mice compared to the WT-STZ mice. Generation of hydrogen peroxide measured by an Amplex Red Kit was significantly decreased in STZ-Pod-Nox4-KO mice compared to WT-STZ mice and the detection of superoxide production in single isolated glomeruli using dihydroethidium (DHE) analysis confirmed the same trend. Again, immunochemistry and Western Blot analysis demonstrated an important cut down in proteins matrix deposition in STZ-Pod-Nox4-KO mice. All together these data suggests that Nox-4 is one of the major source of ROS production in kidney cortex, in particular in glomeruli, and it's a main actor in the development of DN (Fig. 1-5).
Our animal model of Podocin specific Nox4-KO mice clearly elucidate the role of Nox4 as one of the main mediator involved in the devolopment of DN in terms of renal hypertrophy, mesangial matrix accumulation and interstitial matrix protein expression.
[1] Jha JC, Gray SP, Barit D et al. Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy. Journal of the American Society of Nephrology : JASN 2014 Jun;25(6):1237-54 (full text)
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