NADPH Oxidase Nox5 Accelerates Renal Injury in Diabetic Nephropathy
Article Figures & Tables
Figures
- Figure 1
Expression of Nox5 in human kidney and mesangial cells and characterization of Nox5 transgenic mice (SM22+Nox5+) expressing Nox5β in VSMCs including mesangial cells in the glomeruli. A: Gene expression of Nox1, Nox2, Nox4, and Nox5 in response to NG (5 mmol/L), high glucose (25 mmol/L), and mannitol (25 mmol/L + 5 mmol/L NG) in human mesangial cells. *P < 0.05 vs. NG. B: Immunofluorescence for Nox5 (red staining) in kidney biopsies obtained from individuals without and with diabetes (n = 3/group). C and D: Colocalization of Nox5 (red staining) and the marker of smooth muscle cells, transgelin (SM22-α [green staining]), in kidney biopsies obtained from individuals with diabetes (C) as well as in frozen kidney sections of SM22+Nox5+ transgenic mice (D) (magnification ×20). E: Western blot for the expression of Nox5 in the glomerular fraction of the control (C) and diabetic (D) SM22+Nox5− and SM22+Nox5+ mice after 10 weeks of diabetes and its quantitation (F) (n = 3/group). *P < 0.05 vs. control SM22+Nox5+ mice. Data are shown as mean ± SEM. OD, optical density.
- Figure 2
Overexpression of Nox5 in mesangial cells enhances glomerular ROS production in diabetic mice. Immunostaining of glomerular nitrotyrosine (A) and its quantitation (B) (n = 6–8/group) (magnification ×40). ELISA for nitrotyrosine in glomerular fraction (C). Immunofluorescence staining for DHE (D) (scale bars = 50 μm) and NADPH-dependent lucigenin assay (E) (n = 5–6/group) for superoxide production in the glomeruli of control (C) and diabetic (D) SM22+Nox5− and SM22+Nox5+ transgenic mice after 10 weeks of diabetes. Data are shown as mean ± SEM. *P < 0.05 vs. control SM22+Nox5− mice; ^P < 0.05 vs. control SM22+Nox5+ mice; #P < 0.05 vs. diabetic SM22+Nox5− mice. RLU, relative light units.
- Figure 3
Overexpression of Nox5 in mesangial cells exacerbates glomerular injury in diabetic mice. PAS staining (A) (magnification ×40), mesangial area expansion (B), and glomerulosclerosis index (C) in control (C) and diabetic (D) SM22+Nox5− and SM22+Nox5+ transgenic mice (n = 6–9/group) after 10 weeks of diabetes. Data are shown as mean ± SEM. *P < 0.05 vs. control SM22+Nox5− mice; ^P < 0.05 vs. control SM22+Nox5+ mice; #P < 0.05 vs. diabetic SM22+Nox5− mice.
- Figure 4
Overexpression of Nox5 in mesangial cells exacerbates glomerular fibrosis in diabetic mice. Glomerular gene expression of fibronectin, collagen I, collagen IV, α-SMA, and cyclin-dependent kinase inhibitor 1A (p21) (A). Immunostaining of fibronectin (B) and its quantitation (C). Immunostaining of collagen IV (D) and its quantitation (E) in the glomeruli of control (C) and diabetic (D) SM22+Nox5− and SM22+Nox5+ transgenic mice (n = 5–8/group) after 10 weeks of diabetes (magnification ×40). Data are shown as mean ± SEM. *P < 0.05 vs. control SM22+Nox5− mice; ^P < 0.05 vs. control SM22+Nox5+ mice; $P < 0.05 vs. control SM22+Nox5− mice; #P < 0.05 vs. diabetic SM22+Nox5− mice.
- Figure 5
Overexpression of Nox5 in mesangial cells increases glomerular macrophage infiltration and PKC-α expression in diabetic mice. Immunostaining for macrophage infiltration marker F4/80 and its quantitation (A and B) (magnification ×40). Glomerular gene expression of MCP-1 (C) and Western blot for MCP-1 (25kDa) and its quantitation (D and E) (n = 2–3/group). Glomerular gene expression of PKC-α (F), immunostaining and its quantitation (G and H) as well as Western blot for PKC-α and its quantitation (I and J) in the glomeruli of control (C) and diabetic (D) SM22+Nox5− and SM22+Nox5+ transgenic mice (n = 5–8/group) after 10 weeks of diabetes (magnification ×40). Data are shown as mean ± SEM. *P < 0.05 vs. control SM22+Nox5− mice; ^P < 0.05 vs. control SM22+Nox5+ mice; $P < 0.05 vs. control SM22+Nox5− mice; †P = 0.05 vs. control SM22+Nox5− mice; #P < 0.05 vs. diabetic SM22+Nox5− mice. OD, optical density.
- Figure 6
Silencing of Nox5 using shRNA attenuates high glucose and TGF-β1–mediated ROS formation and gene expression of markers of fibrosis, inflammation, and putative pathways in human mesangial cells. Human mesangial cells infected with shRNA specific for Nox5 are grown in NG (5 mmol/L) or in high glucose (HG group) (25 mmol/L) or in high glucose plus TGF-β1 (5 ng/mL) or in mannitol (25 mmol/L + 5 mmol/L NG, as osmotic control) for 2 days for the gene expression analysis and 4 h for the ROS measurement. Gene expression of Nox5 (A), putative pathways PKC-α and PKC-β (B) and TRPC6 (C), markers of inflammation MCP-1 and markers of fibrosis α-SMA (C), and fibronectin, CTGF, and collagen type IV (D) in human mesangial cells transfected with shRNA specific for Nox5. Analysis of ROS production in human mesangial cells transfected with shRNA specific for Nox5 by DHE immunofluorescence (E) and by chemiluminescence (L-012) (F). Results were expressed relative to nontarget plus NG. Data are mean ± SEM (n = 4–6/group). *P < 0.05 vs. nontarget plus NG; ^P < 0.05 vs. nontarget plus NG; †P < 0.05 vs. nontarget plus NG; #P < 0.05 vs. nontarget plus HG or nontarget plus high glucose and TGF-β1. RLU, relative light units.
Tables
- Table 1
General and metabolic variables, albuminuria, creatinine clearance, plasma creatinine, and cystatin C in control and diabetic SM22+Nox5− and SM22+Nox5+ mice (n = 10–20/group)
SM22+Nox5− SM22+Nox5+ Control Diabetes Control Diabetes Body weight (g) 32 ± 0.9 28 ± 0.6* 32 ± 0.7 26 ± 0.6^ Kidney weight/body weight (%) 0.64 ± 0.02 0.99 ± 0.03* 0.68 ± 0.02 1.12 ± 0.03^# Plasma glucose (mmol/L) 11.9 ± 0.5 33.0 ± 2.4* 12.0 ± 0.8 34.2 ± 1.4^ Glycated hemoglobin (%) 4.3 ± 0.1 11.4 ± 0.5* 4.2 ± 0.1 11.2 ± 0.5^ Systolic BP (mmHg) 105 ± 0.9 109 ± 1.0 108 ± 0.9 110 ± 1.2 Food intake (g) 2.9 ± 0.2 5.8 ± 0.5* 3.1 ± 0.3 6.4 ± 0.3^ Water intake (mL) 2.85 ± 0.40 27.5 ± 2.0* 3.0 ± 0.4 26.9 ± 1.6^ Urine output (mL) 0.56 ± 0.08 22.7 ± 1.8* 0.57 ± 0.10 24.4 ± 1.61^ Creatinine clearance (mL/min/m2) 6.8 ± 0.5 19.8 ± 2.6* 6.9 ± 1.1 13.9 ± 2.7^ Albuminuria (µg/24 h) 38 ± 4.5 651 ± 80* 27 ± 2.6 864 ± 70^† ACR (µg/mg) 124 ± 21 1,130 ± 147* 97 ± 11 1,648 ± 203^†† Plasma creatinine (µmol/L) 22.7 ± 2.3 17.7 ± 2.5 29.6 ± 3.2 21.5 ± 4.7 Cystatin C (ng/mL) 346 ± 24 208 ± 32* 431 ± 28 134 ± 18* Data are shown as mean ± SEM. BP, blood pressure.
*P < 0.05 vs. control SM22+Nox5− mice;
^P < 0.05 vs. control SM22+Nox5+ mice;
†P = 0.059 and
††P = 0.057 vs. diabetic SM22+Nox5− mice;
#P < 0.05 vs. diabetic SM22+Nox5− mice.
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