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. 2014 Jun;25(6):1237-54.
doi: 10.1681/ASN.2013070810. Epub 2014 Feb 7.

Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy

Jay C Jha  1 , Stephen P Gray  2 , David Barit  2 , Jun Okabe  3 , Assam El-Osta  3 , Tamehachi Namikoshi  4 , Vicki Thallas-Bonke  2 , Kirstin Wingler  5 , Cedric Szyndralewiez  6 , Freddy Heitz  6 , Rhian M Touyz  7 , Mark E Cooper  1 , Harald H H W Schmidt  5 , Karin A Jandeleit-Dahm  8
Affiliations

Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy

Jay C Jha et al. J Am Soc Nephrol. 2014 Jun.

Abstract

Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)-forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE(-/-) mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE(-/-) mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.

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Figures

Figure 1.
Figure 1.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate diabetes-induced increased albuminuria in diabetic ApoE−/− mice. Urinary albumin excretion (A–C) and ACR (D–F) in control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A and D), control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice (B and E), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 (C and F) for 10 and 20 weeks (n=10–15 per group). Data are the mean±SEM. *P<0.01 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A and D), control Nox1+/yApoE−/− and Nox1−/yApoE−/− mice (B and E), or control ApoE−/− and ApoE−/− plus GKT137831 mice (C and F); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A and D) or diabetic ApoE−/− mice (C and F). ACR, albumin/creatinine ratio; Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 2.
Figure 2.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate glomerular injury in diabetic ApoE−/− mice. Glomerulosclerosis index (A) and mesangial area expansion (B) in control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks (n=7–8 per group). Glomerulosclerosis index (C) and mesangial area expansion (D) in control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (n=7–8 per group). Glomerulosclerosis index (E) and mesangial area expansion (F) in control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (n=7–8 per group). Data are the mean±SEM. *P<0.01 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A and B), control Nox1+/yApoE−/− and Nox1-/yApoE−/− mice (C and D), or control ApoE−/− and ApoE−/− plus GKT137831 mice (E and F); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A and B) or diabetic ApoE−/− mice (E and F). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 3.
Figure 3.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate increased collagen IV accumulation in glomeruli of diabetic ApoE−/− mice. Immunostaining for collagen IV in glomeruli of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks (A), control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (B), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (C) (n=6–8 per group). Data are the mean±SEM. *P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A), control Nox1+/yApoE−/− and Nox1−/yApoE−/− mice (B), or control ApoE−/− and ApoE−/− plus GKT137831 mice (C); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A) or diabetic ApoE−/− mice (C). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 4.
Figure 4.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate increased fibronectin accumulation in glomeruli of diabetic ApoE−/− mice. Immunostaining for fibronectin in glomeruli of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks (A), control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (B), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (C) (n=6–8 per group). Data are the mean±SEM. *P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A), control Nox1+/yApoE−/− and Nox1-/yApoE−/− mice (B), or control ApoE−/− and ApoE−/− plus GKT137831 mice (C); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A) or diabetic ApoE−/− mice (C). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 5.
Figure 5.
Genetic deficiency of Nox4 and pharmacologic Nox inhibition attenuate increased VEGF expression in glomeruli of diabetic ApoE−/− mice. Immunostaining for VEGF in glomeruli of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks (A) and control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (B) (n=6–8 per group). Data are the mean±SEM. *P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A) or control ApoE−/− and ApoE−/− plus GKT137831 mice (B); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A); ‡P=0.08 versus diabetic ApoE−/− mice (B). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 6.
Figure 6.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate nitrotyrosine accumulation in glomeruli of diabetic ApoE−/− mice. Immunostaining for nitrotyrosine in glomeruli of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks (A), control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (B), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (C) (n=6–8 per group). Results are expressed relative to control Nox4+/+ApoE−/− mice (A), control Nox1+/yApoE−/− mice (B), or control (untreated) ApoE−/− mice (C), which were arbitrarily assigned a value of 1. Data are the mean±SEM. *P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A), control Nox1+/yApoE−/− and Nox1−/yApoE−/− mice (B), or control ApoE−/− and ApoE−/− plus GKT137831 mice (C); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A) or diabetic ApoE−/− mice (C). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 7.
Figure 7.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuate increased renal superoxide and ROS formation in diabetic ApoE−/− mice. Superoxide production in renal cortex (A, C, and E) and ROS production in cytosolic and mitochondrial fractions of the renal cortex (B, D, and F) in control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A and B), control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (C and D), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (E and F) (n=5–6 per group). Superoxide data (A, C, and E) are shown as the ratio of 2HE (nanomoles) to DHE (micromoles). With respect to ROS measurements, results were expressed relative to control Nox4+/+ApoE−/− mice (B), control Nox1+/yApoE−/− mice (D), or control (untreated) ApoE−/− mice (F), which was arbitrarily assigned a value of 100. Data are the mean±SEM. *P≤0.05 versus respective control Nox4+/+ApoE−/− or control Nox1+/yApoE−/− mice or control ApoE−/− mice; #P<0.05 versus diabetic Nox4+/+ApoE−/− or diabetic ApoE−/− mice. 2HE, 2 hydroethidium; Cont, control; Diab, diabetes; DHE, dihydroethidium; GKT, GKT137831; RLU, relative light unit.
Figure 8.
Figure 8.
Genetic deficiency of Nox4, but not of Nox1, and pharmacologic Nox inhibition attenuates macrophage infiltration in glomeruli of diabetic ApoE−/− mice. Immunostaining for F4/80 in glomeruli of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A) or in control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks (B), or control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks (C) (n=6–8 per group). Data are the mean±SEM. *P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (A), control Nox1+/yApoE−/− and Nox1-/yApoE−/− mice (B), or control ApoE−/− and ApoE−/− plus GKT137831 mice (C); #P<0.05 versus diabetic Nox4+/+ApoE−/− (A) or diabetic ApoE−/− mice (C). Cont, control; Diab, diabetes; GKT, GKT137831.
Figure 9.
Figure 9.
Silencing of Nox4 attenuates high glucose–as well as high glucose plus TGF-β1–mediated ROS formation and gene expression of profibrotic markers in human podocytes. (A) Nox4 and Nox5, but not Nox1, Nox2, or p47phox mRNA levels were upregulated in human podocytes by diabetic stimuli. Analysis of Nox isoform mRNA levels and the cytosolic regulator, p47phox, in cultured differentiated human podocytes in NG (5 mM) or HG (25 mM) and in the presence or absence of TGF-β1 (5 ng/ml, 2 days). Mannitol (20 mM/L+5 mM D-glucose) served as an osmotic control. Data are the mean±SEM. *P<0.05 versus NG; #P<0.05 versus HG. (B and C) Analysis of ROS production (B) and RT-PCR (C) for collagen IV, fibronectin, VEGF and α-SMA in differentiated human podocytes transfected with shRNA specific for Nox4 and then grown in NG (5 mM) or HG (25 mM) for 2 days (B and C). Results for ROS production are expressed relative to nontarget plus NG, which was arbitrarily assigned a value of 100 (B). Data are the mean±SEM (n=6/group). ∧P<0.05 versus nontarget plus NG; §P<0.05 versus nontarget plus HG. (D and E) Analysis of ROS production (D) and RT-PCR (E) for collagen IV, fibronectin, VEGF and α-SMA in differentiated human podocytes transfected with shRNA specific for Nox4 and then grown in the presence or absence of TGF-β1 (5 ng/ml) for 4 hours for ROS production (D) and 2days for RT-PCR (E). Data are the mean±SEM (n=6/group). †P<0.05 versus nontarget, ‡P<0.05 versus non-target plus TGF-β1. HG, high glucose; NG, normal glucose; RLU, relative light unit.
Figure 10.
Figure 10.
Nox inhibition ameliorates TGF-β1–induced ROS generation and gene expression of profibrotic markers in human podocytes. (A) Nox inhibition ameliorates TGF-β1–induced ROS generation in human podocytes. Analysis of ROS production in cultured differentiated human podocytes after pretreatment with and without GKT137831 (10 µM) for 2 hours in the presence and absence of TGF-β1 (5 ng/ml) for 4 hours. DMSO serves as the vehicle control. Results are expressed relative to control, which is arbitrarily assigned a value of 100. (B) Nox inhibition decreases the high glucose– and TGF-β1–induced increased gene expression of collagen IV, fibronectin, CTGF, VEGF, and α-SMA in human podocytes. The mRNA levels are quantified in cultured differentiated human podocytes after pretreatment with and without GKT137831 (10 µM) for 2 hours in the presence or absence of TGF-β1 (5 ng/ml) for 2 days. DMSO serves as the vehicle control. Data are the mean±SEM (n=6 per group). *P<0.05 versus control; #P<0.05 versus TGF-β1. COL4, collagen IV; CTGF, connective tissue growth factor; GKT, GKT137831; RLU, relative light unit.
Figure 11.
Figure 11.
Genetic targeting of Nox4 attenuates diabetes-induced increased expression of proinflammatory markers MCP-1 and NF-κB p65 in vitro and in vivo. (A and B) RT-PCR analysis of (A) MCP-1 and (B) NF-κB p65 in human podocytes transfected with shRNA specific for Nox4 and then grown in NG (5 mM) or HG (25 mM) for 2 days. Data are the mean±SEM (n=6/group). *P<0.05 versus nontarget plus NG; #P<0.01 versus nontarget plus HG. (C and D) RT-PCR analysis of (C) MCP-1 and (D) NF-κB p65 in renal cortex of control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice after 20 weeks. Data are the mean±SEM (n=6/group). ^P<0.05 versus control Nox4+/+ApoE−/−; §P<0.05 versus diabetic Nox4+/+ApoE−/− mice. (E and F) Measurement of MCP-1 by ELISA in protein extracts of renal cortex of (E) control and diabetic Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice or in (F) control and diabetic Nox1+/yApoE−/− and Nox1−/yApoE−/− mice after 20 weeks, (n=5–6/group) or in (G) control and diabetic ApoE−/− mice with and without treatment with GKT137831 for 20 weeks, (n=5–6/group). Data are the mean±SEM. †P<0.05 versus respective control Nox4+/+ApoE−/− and Nox4−/−ApoE−/− mice (E) or control Nox1+/yApoE−/− and Nox1−/yApoE−/− mice (F) or control ApoE−/− and ApoE−/− plus GKT137831 mice (G); ‡P<0.05 versus diabetic ApoE−/− mice (G). Cont, control; Diab, diabetes; GKT, GKT137831; HG, high glucose; NG, normal glucose.
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