AMP-activated protein kinase (AMPK) negatively regulates Nox4-dependent activation of p53 and epithelial cell apoptosis in diabetes

Abstract

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.

FIGURE 1.

NADPH oxidase Nox4 mediates HG-induced podocyte apoptosis. Podocytes were infected with AdDN-Nox4 or AdGFP and treated with either HG (25 mmol/liter) or NG (5 mmol/liter) for 48 h. A, NADPH-dependent superoxide generation measured in podocytes infected with AdGFP and treated with NG or HG or in podocytes transfected with AdDN-Nox4 and treated with HG. RLU, relative light units. B, percent annexin V-positive cells 48 h after infection with AdGFP or AdDN-Nox4 in HG. C, ELISA for cellular DNA fragmentation. D, histogram representing caspase-3 activity. All values are the mean ± S.E. from four independent experiments. *, p < 0.05 versus the control; #, p < 0.05 versus HG treatment.

FIGURE 2.

AMPK regulates Nox4-dependent podocyte apoptosis. Podocytes were exposed either to HG (25 mmol/liter) with or without AICAR (1 mmol/liter) or to ARA (1 mmol/liter) in NG (5 mmol/liter) for 48 h. A, representative Western blot of phospho-Thr¹⁷² AMPKα and AMPKα. B, histogram representing AMPK activity measured in podocytes treated with HG and AICAR or treated with ARA in NG. C, relative mRNA levels of Nox4 in control and treated podocytes. D, representative Western blot of Nox4 and β-actin levels. E, NADPH-dependent superoxide generation measured in podocytes treated with HG and AICAR or treated with ARA in NG. In parallel experiments, podocytes were transfected with WT-AMPKα2 in HG or transfected with DN-AMPKα2 in NG. RLU, relative light units. F and G, WT-AMPKα2 in the presence of HG increases phospho-Thr¹⁷² AMPKα expression and AMPK activity, respectively. Shown is a representative Western blot of phospho-Thr¹⁷² AMPKα and AMPKα (F) and a histogram representing AMPK activity measured in podocytes transfected with WT-AMPKα2 and treated with HG or transfected with DN-AMPKα2 in NG (G). H, relative mRNA levels of Nox4. I, representative Western blot of Nox4 and β-actin levels. J, NADPH-dependent superoxide generation measured in podocytes transfected with WT-AMPKα2 and treated with HG or transfected with DN-AMPKα2 in NG. All values are the mean ± S.E. from four independent experiments. *, p < 0.05 versus the control; #, p < 0.05 versus HG treatment.

FIGURE 3.

AMPK regulates HG-induced podocyte apoptosis. Mouse podocytes were serum-deprived for 24 h and pretreated with the AMPK activator AICAR (1 mmol/liter) for 1 h before incubation with HG or were incubated with the AMPK inhibitor ARA (1 mmol/liter) in the presence of NG for 48 h. In parallel experiments, mouse podocytes were transfected with WT-AMPKα2 before treatment with HG or transfected with DN-AMPKα2 in NG. AICAR treatment inhibited HG-induced apoptosis, whereas ARA induced apoptosis as measured by annexin V binding (A), caspase-3 activity (B), and cellular DNA fragmentation (C). The effect of HG on podocyte apoptosis was also blocked by WT-AMPKα2 and reproduced by DN-AMPKα2 in NG medium as measured by annexin V binding (D), caspase-3 activity (E), and cellular DNA fragmentation (F). All values are the mean ± S.E. from four independent experiments. *, p < 0.05 versus the control; #, p < 0.05 versus HG.

FIGURE 4.

p53 mediates HG-induced podocyte apoptosis. Mouse podocytes were transfected with scrambled siRNA (nontargeting; Scr) or with siRNA for p53 (sip53) in NG or HG. A, relative mRNA amount of p53. B, representative Western blot of p53, phospho-Ser⁴⁶ p53, and β-actin. C, relative mRNA amount of PUMA in podocytes transfected with scrambled siRNA in NG or HG or transfected with p53 siRNA and treated with HG. D, percent annexin V-positive cells 48 h after transfection with scrambled or p53 siRNA in HG. E and F, histograms of caspase-3 activity and ELISA for cellular DNA fragmentation, respectively. All values are the mean ± S.E. from four independent experiments. *, p < 0.05 versus the control + Scr; #, p < 0.05 versus HG + Scr.

FIGURE 5.

AMPK/Nox4 axis regulates HG-induced p53 and PUMA. Mouse podocytes were serum-deprived for 24 h and pretreated with the AMPK activator AICAR (1 mmol/liter) for 1 h before incubation with HG, or mouse podocytes were incubated with the AMPK inhibitor ARA (1 mmol/liter) in the presence of NG for 48 h. A, representative Western blot of p53, phospho-Ser⁴⁶ p53, PUMA, and β-actin. B, relative mRNA levels of PUMA. In parallel experiments, mouse podocytes were transfected with WT-AMPKα2 before treatment with HG or transfected with DN-AMPKα2 in NG. C, representative Western blot of p53, phospho-Ser⁴⁶ p53, PUMA, and β-actin levels. D, relative mRNA levels of PUMA. In an another set of experiments, podocytes were infected with AdDN-Nox4 or AdGFP in NG or HG. E, representative Western blot of p53, phospho-Ser⁴⁶ p53, PUMA, and β-actin. F, relative mRNA levels of PUMA. All values are the mean ± S.E. from four independent experiments. *, p < 0.05 versus the control; #, p < 0.05 versus HG treatment.

FIGURE 6.

AMPK inactivation up-regulates Nox4 and enhances NADPH oxidase activity and p53 expression in type 1 diabetic mice. 17-week-old OVE26 mice were treated with AICAR (750 mg/kg/day, dissolved in saline, intraperitoneal) for 5 weeks. Mice in the control group received saline vehicle. Glomeruli were isolated from the kidneys of three groups of mice (n = 5): control FVB mice, OVE26 mice, and OVE26 mice treated with AICAR. A, representative Western blot of phospho-Thr¹⁷² AMPKα and total AMPKα of four of five mice from each group (FVB mice, lanes 1–4; OVE26 mice, lane 5–8; and OVE26 mice treated with AICAR, lanes 9–12). B, histogram of AMPK activity. C, representative Western blot of Nox4 and β-actin of four mice of five from each group (FVB mice, lanes 1–4; OVE26 mice, lane 5–8; and OVE26 mice treated with AICAR, lanes 9–12). D, NADPH-dependent superoxide generation. RLU, relative light units. E, representative Western blot of p53, PUMA, and β-actin of four mice of five from each group (FVB mice, lanes 1–4; OVE26 mice, lane 5–8; and OVE26 mice treated with AICAR, lanes 9–12). F, relative amount of PUMA mRNA. *, p < 0.05, OVE26 mice versus FVB mice; #, p < 0.05, OVE26 mice treated with AICAR compared with OVE26 mice treated with vehicle.

FIGURE 7.

AMPK regulates GBM thickening, foot process effacement, podocyte loss, and albuminuria in type 1 diabetic mice. 17-week-old OVE26 mice were treated with AICAR (750 mg/kg/day, dissolved in saline, intraperitoneal) for 5 weeks. Mice in the control group received saline vehicle. Glomeruli were isolated from the kidneys of three groups of mice (n = 5): control FVB mice, OVE26 mice, and OVE26 mice treated with AICAR. A, representative transmission electron micrographs of glomerular cross-sections of FVB, OVE26, and AICAR-treated OVE26 mice. The images show foot process effacement (panel b, red arrow), cytoplasmic rarefaction, and basement membrane thickening (panel b, blue arrow) of an OVE26 mouse. This effect was not seen in OVE26 mice treated with AICAR (panel c). B, histogram representing thickness of the GBM measured in nanometers. C, semiquantitative analysis of foot process effacement of glomeruli from each group of animals. D, representative immunofluorescent images of glomeruli stained with collagen IV (green), synaptopodin (red), and DAPI (blue). E, barogram representing podocyte number per glomerular section. *, p < 0.05, OVE26 mice versus FVB mice; #, p < 0.05, AICAR-treated OVE26 mice compared with OVE26 mice. F, FVB, OVE26, and AICAR-treated OVE26 mice were placed in metabolic cages for 24 h. Urine was collected, and albumin levels were measured and expressed as micrograms of albumin/24 h. Values are the mean ± S.E. *, p < 0.05, OVE26 mice versus control FVB mice; #, p < 0.05, decrease in albumin levels in AICAR-pretreated OVE26 mice versus untreated OVE26 mice (n = five per group).

FIGURE 8.

Proposed mechanism of HG/diabetes-induced glomerular epithelial cell (podocyte) apoptosis. See “Discussion” for details.