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. 2014 Nov 3;2(11):e12192.
doi: 10.14814/phy2.12192. Print 2014 Nov 1.

Nox-4 deletion reduces oxidative stress and injury by PKC-α-associated mechanisms in diabetic nephropathy

Vicki Thallas-Bonke  1 , Jay C Jha  2 , Stephen P Gray  3 , David Barit  3 , Hermann Haller  4 , Harald H H W Schmidt  5 , Melinda T Coughlan  6 , Mark E Cooper  2 , Josephine M Forbes  7 , Karin A M Jandeleit-Dahm  2
Affiliations

Nox-4 deletion reduces oxidative stress and injury by PKC-α-associated mechanisms in diabetic nephropathy

Vicki Thallas-Bonke et al. Physiol Rep. .

Abstract

Current treatments for diabetic nephropathy (DN) only result in slowing its progression, thus highlighting a need to identify novel targets. Increased production of reactive oxygen species (ROS) is considered a key downstream pathway of end-organ injury with increasing data implicating both mitochondrial and cytosolic sources of ROS. The enzyme, NADPH oxidase, generates ROS in the kidney and has been implicated in the activation of protein kinase C (PKC), in the pathogenesis of DN, but the link between PKC and Nox-derived ROS has not been evaluated in detail in vivo. In this study, global deletion of a NADPH-oxidase isoform, Nox4, was examined in mice with streptozotocin-induced diabetes (C57Bl6/J) in order to evaluate the effects of Nox4 deletion, not only on renal structure and function but also on the PKC pathway and downstream events. Nox4 deletion attenuated diabetes-associated increases in albuminuria, glomerulosclerosis, and extracellular matrix accumulation. Lack of Nox4 resulted in a decrease in diabetes-induced renal cortical ROS derived from the mitochondria and the cytosol, urinary isoprostanes, and PKC activity. Immunostaining of renal cortex revealed that major isoforms of PKC, PKC-α and PKC-β1, were increased with diabetes and normalized by Nox4 deletion. Downregulation of the PKC pathway was observed in tandem with reduced expression of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1 and restoration of the podocyte slit pore protein nephrin. This study suggests that deletion of Nox4 may alleviate renal injury via PKC-dependent mechanisms, further strengthening the view that Nox4 is a suitable target for renoprotection in diabetes.

Keywords: Diabetic nephropathy; NADPH oxidase; protein kinase C; reactive oxygen species.

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Figures

Figure 1.
Figure 1.
(A) Analysis of Nox4 mRNA levels in the renal cortex of control wild‐type and knockout mice. B) Genotyping by PCR of genomic DNA extracted from mouse tails of wild‐type and Nox4 knockout mice.
Figure 2.
Figure 2.
Renal functional data at week 20. A) Urinary albumin to creatinine ratio (ACR), B) Plasma cystatin C, *P <0.01 versus WT‐C; †P <0.05 versus WT‐D; ‡P <0.05 versus WT‐C; §P <0.05 versus KO‐C group, n =8–10/group.
Figure 3.
Figure 3.
Renal parameters at 20 weeks. A) Urinary VEGF excretion. B) Computer‐aided analysis of immunohistochemical staining of glomerular nephrin expression, C) Representative photomicrographs of nephrin immunohistochemical staining of renal cortex, (i) WT‐C; (ii) WT‐D, (iii) Nox4‐KO‐C; (iv) Nox4‐KO‐D, *P <0.01 versus WT‐C; †P <0.05 versus WT‐D; ‡P <0.001 versus WT‐C; §P <0.01 versus WT‐D, n =8–10/group; all scale bars 50 μm.
Figure 4.
Figure 4.
Renal structural data at week 20. A) Glomerulosclerotic index (GSI), B) Representative photomicrographs of PAS stained renal cortex of kidney (×400 magnification), (i) WT‐C; (ii) WT‐D, (iii) Nox4‐KO‐C; (iv) Nox4‐KO‐D, *P <0.001 versus WT‐C; †P <0.05 versus WT‐C and ‡P <0.05 versus WT‐D, n =8–10/group.
Figure 5.
Figure 5.
Various renal parameters at week 20, A) Biologically active TGF‐β1 excretion, B) Biologically active membranous TGF‐β1 expression, C) Computer‐aided image analysis of the extracellular matrix protein collagen IV by immunohistochemistry, expressed as percent area in glomeruli, D) Computer‐aided image analysis of the extracellular matrix protein fibronectin by immunohistochemistry, expressed as percent area in glomeruli, *P <0.001 versus WT‐C; †P <0.01 versus WT‐D; ‡P <0.05 versus WT‐C; §P <0.05 versus WT‐D; #P <0.001 versus WT‐D; ∂P <0.05 versus KO‐C; αP < 0.01 versus WT‐C, n =8–10/group.
Figure 6.
Figure 6.
Oxidative stress markers at 20 weeks from renal cortex, A) Ex vivo NADH‐dependent mitochondrial tissue superoxide production, B) Ex vivo NADPH‐dependent cytosolic tissue superoxide production, C) Superoxide production from isolated mitochondria, D) Urinary 8‐isoprostane excretion, E) Computer‐aided analysis of immunohistochemical staining of renal cortical nitrotyrosine expressed as percent area, *P <0.05 versus WT‐C; †P <0.05 versus WT‐D; ‡P <0.001 versus WT‐C; §P <0.05 versus KO‐C, #P <0.01 versus WT‐D, n =8–10/group.
Figure 7.
Figure 7.
PKC activity of subcellular renal cortical compartments from 20‐week‐old mice, A) Membranous, B) Mitochondrial, C) Nuclear, D) Cytosolic, *P <0.01 versus WT‐C; †P <0.05 versus WT‐D; ‡P <0.05 versus WT‐C; §P <0.01 versus WT‐D; n =8–10/group.
Figure 8.
Figure 8.
PKC‐α expression at 20 weeks A) Membranous fraction and B) Mitochondrial fraction of renal cortex by ELISA. C) Computer‐aided analysis of immunohistochemical staining of renal cortical PKC‐α expressed as percent area and D) Representative photomicrographs of PKC‐α immunostaining in renal cortex of kidney (×400 magnification), (i) WT‐C; (ii) WT‐D, (iii) Nox4‐KO‐C; (iv) Nox4‐KO‐D. *P <0.01 versus WT‐C; †P <0.01 versus WT‐D; ‡P <0.05 versus WT‐D, §P <0.001 versus WT‐D, n =8–10/group.
Figure 9.
Figure 9.
PKC‐β1 expression at 20 weeks, A) Computer‐aided analysis of immunohistochemical staining of renal cortical PKC‐β1 expressed as percent area (n =8–10/group), B) Representative photomicrographs of PKC‐β1 immunostaining in renal cortex of kidney (×400 magnification), (i) WT‐C; (ii) WT‐D, (iii) Nox4‐KO‐C; (iv) Nox4‐KO‐D. *P <0.001 versus WT‐C; †P <0.001 versus WT‐D.
Figure 10.
Figure 10.
Speculative schema for the involvement of Nox4 and PKC in diabetic nephropathy.
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