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. 2015 Jun 1;308(11):F1276-87.
doi: 10.1152/ajprenal.00396.2014. Epub 2015 Feb 4.

Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes

Yves Gorin  1 , Rita C Cavaglieri  2 , Khaled Khazim  2 , Doug-Yoon Lee  2 , Francesca Bruno  2 , Sachin Thakur  2 , Paolo Fanti  3 , Cédric Szyndralewiez  4 , Jeffrey L Barnes  3 , Karen Block  5 , Hanna E Abboud  3
Affiliations

Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes

Yves Gorin et al. Am J Physiol Renal Physiol. .

Abstract

Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.

Keywords: NADPH oxidase; Nox1/Nox4 inhibitor; diabetic nephropathy.

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Figures

Fig. 1.
Fig. 1.
Treatment of OVE26 type 1 diabetic mice with Nox4/1 inhibitor GKT137831 reduces diabetes-induced reactive oxygen species (ROS) generation in the renal cortex without affecting Nox4 or Nox1 protein expression. A: NADPH oxidase activity in cortical homogenates. Superoxide anion generation was determined by photoemission every 30 s for 5–10 min. The initial rate of enzyme activity was calculated over the first 30–120 s of exposure to NADPH. NADPH-driven superoxide production was expressed as relative light units (RLU)·min−1·mg protein−1. Values are means ± SE. *P < 0.05 vs. FVB. #P < 0.05 vs. OVE26. B: hydrogen peroxide production was assessed using an Amplex Red Assay kit. Values are the mean ± SE. *P < 0.05 vs. FVB. #P < 0.05 vs. OVE26. C: in situ detection of intracellular ROS with dihydroethidium (DHE) staining and confocal microscopy in renal cortex sections from FVB, OVE26, OVE26+GKT137831 10 mg/kg, and OVE26+GKT137831 40 mg/kg groups. Dashed white lines indicate position of glomeruli. D: superoxide generation was evaluated in the renal cortex using DHE and HPLC as described in materials and methods. Superoxide production was expressed as μM 2-hydroxyethidium produced/mg renal cortex. Values are means ± SE from all the animals in each group. *P < 0.01 vs. FVB. #P < 0.05 vs. OVE26. E and F: Nox4 (E) and Nox1 (F) protein expression were determined by direct immunoblotting of cortical lysates. GAPDH was included as a control for loading and the specificity of change in protein expression. Representative results of Western blot analysis were obtained from 3 independent samples from each group. Each histogram at the bottom represents the ratio of the intensity of the Nox4 or Nox1 bands quantified by densitometry factored by the densitometric measurement of the actin band. Values are means ± SE from all the animals in each group. *P < 0.05 vs. FVB.
Fig. 2.
Fig. 2.
Treatment of OVE26 mice with GKT137831 reduces diabetes-induced extracellular matrix protein expression in the renal cortex including glomeruli. A: representative Western blot for fibronectin protein expression in the renal cortex. Representative results of Western blot analysis were obtained from 3 independent samples from each group. Each histogram at the bottom represents the ratio of the intensity of the fibronectin bands quantified by densitometry factored by the densitometric measurement of the actin band. Values are means ± SE from all the animals in each group. *P < 0.05 vs. FVB. #P < 0.05 vs. OVE26. B: representative Western blot for collagen IV protein expression in the renal cortex. Representative results of Western blot analysis were obtained from 2 independent samples from each group. The histogram on the right represents the ratio of the intensity of collagen IV bands quantified by densitometry factored by the densitometric measurement of GAPDH band. The data are expressed as percentage of control (FVB), where the ratio in the control was defined as 100%. Values are means ± SE from all the animals in each group. *P < 0.05 vs. FVB. #P < 0.05 vs. OVE26. C: fibronectin expression was detected by immunoperoxidase (top) and immunofluorescence (bottom) staining of kidney sections from FVB, OVE26, OVE26+GKT137831 10 mg/kg, and OVE26+GKT137831 40 mg/kg groups. D: quantitation of glomerular collagen fibronectin expression. The histograms represent means ± SE of glomeruli in sections from individual mice in each group. *P < 0.001 vs. FVB. #P < 0.001 vs. OVE26. E: collagen type IV was detected by immunoperoxidase (top) and immunofluorescence (bottom) staining of kidney sections from FVB, OVE26, OVE26+GKT137831 10 mg/kg, and OVE26+GKT137831 40 mg/kg groups. F: quantitation of glomerular collagen type IV expression. The histograms represent means ± SE of glomeruli in sections from individual mice in each group. *P < 0.001 vs. FVB. #P < 0.001 vs. OVE26.
Fig. 3.
Fig. 3.
Treatment of OVE26 mice with GKT137831 reduces diabetes-induced glomerular hypertrophy and mesangial expansion. A: representative photomicrographs of kidney sections stained with hematoxylin and eosin from FVB, OVE26, OVE26+GKT137831 10 mg/kg, and OVE26+GKT137831 40 mg/kg groups. Right: quantitation of glomerular size. Glomerular cross-sectional areas were measured from kidney sections stained with hematoxylin and eosin by using ImagePro Plus 4.5 software. The histograms represent means ± SE from 25 individual glomeruli in sections from 6 individual rats in each group. B: representative photomicrographs of kidney sections stained with periodic acid- Schiff from FVB, OVE26, OVE26+GKT137831 10 mg/kg, and OVE26+GKT137831 40 mg/kg groups. Right: evaluation of mesangial matrix expansion index. The histograms represent means ± SE from 25 individual glomeruli in the individual mice for each group. *P < 0.01 vs. FVB. #P < 0.05 vs. OVE26.
Fig. 4.
Fig. 4.
Treatment of OVE26 mice with GKT137831 ameliorates urine albumin excretion and reduces podocyte loss. A: urine albumin excretion was evaluated using a mouse albumin ELISA quantification kit and expressed as μg albumin/24 h. B: representative immunoperoxidase images of glomeruli stained with podocyte marker WT-1 (brown). Right: quantitation of WT-1-positive cells (WT-1+) in glomeruli using ImagePro Plus 4.5 software. The histograms represent means ± SE of 25 individual glomeruli in each section. Values are means ± SE. *P < 0.001 vs. FVB. #P < 0.05 vs. OVE26. &P < 0.01 vs. OVE26. C: representative immunofluorescence images of glomeruli stained with synaptopodin (red), collagen IV (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue), and quantification of podocytes per glomerulus. Values are means ± SE. *P < 0.001 vs. FVB.
Fig. 5.
Fig. 5.
Treatment of OVE26 mice with GKT137831 reduces diabetes-induced monocyte infiltration in the interstitial area and glomeruli. A: representative immunohistochemistry images of interstitial area stained with CD68 (pink). Right: quantitation of CD68-positive cells (CD68+) in interstitial area using ImagePro Plus 4.5 software. Values are means ± SE. *P < 0.01 vs. FVB. #P < 0.01 vs. OVE26. B: representative immunohistochemistry images of glomeruli stained with CD68. Right: quantitation of CD68-positive cells (CD68+) in glomeruli using ImagePro Plus 4.5 software. The histograms represent means ± SE of 25 individual glomeruli in each section. Values are means ± SE. *P < 0.01 vs. FVB. #P < 0.01 vs. OVE26.
Fig. 6.
Fig. 6.
Speculative schematic for the renoprotective effects of small-molecule Nox1/4 inhibitor GKT137831 in diabetic kidney disease.
See this image and copyright information in PMC

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