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. 2009 May;58(5):1201-11.
doi: 10.2337/db08-1536. Epub 2009 Feb 10.

Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases

Assaad A Eid  1 , Yves Gorin, Bridget M Fagg, Rita Maalouf, Jeffrey L Barnes, Karen Block, Hanna E Abboud
Affiliations

Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases

Assaad A Eid et al. Diabetes. 2009 May.

Abstract

Objective: We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

Research design and methods: Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

Results: Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

Conclusions: Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.

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Figures

FIG. 1.
FIG. 1.
Temporal effect of high glucose on ROS generation in mouse podocytes. A: Mouse podocytes were exposed to either high glucose (HG; 25 mmol/l) or normal glucose (NG; 5 mmol/l) for the indicated time periods. ROS generation was measured by DCF with a multiwell fluorescence plate reader as described in research design and methods. Values are the means ± SE of three independent experiments (n = 3). *P < 0.05, high glucose vs. control. B: Mouse podocytes were exposed to either high glucose (25 mmol/l) or normal glucose (5 mmol/l) for the indicated time periods. NADPH-dependent superoxide generation was assessed by lucigenin-enhanced chemiluminescence. Superoxide anion was expressed as relative chemiluminescent light units (RLU) per milligram of protein per minute (n = 3). C: Expression of Nox1, Nox2, and Nox4 protein was determined by Western blotting analysis on homogenized podocytes. Actin was included as a control for loading and the specificity of change in protein expression. The Western blot is representative of three independent experiments (n = 3).
FIG. 2.
FIG. 2.
CYP4A-dependent 20-HETE production mediates high-glucose–induced ROS generation in mouse podocytes. Serum-deprived podocytes were preincubated with or without HET0016 (10 μmol/l) for 1 h and treated with high glucose (HG; 25 mmol/l) or with 1 μmol/l of pure recombinant 20-HETE for the indicated time periods. A: Expression of CYP4A protein was determined by Western blotting analysis on microsomes isolated from mouse podocytes. The Western blot is representative of three independent experiments (n = 3). B: Microsomes (0.25 mg of protein) prepared from podocytes were incubated with 10 μmol/l arachidonic acid in the presence of NADPH. 20-HETE was separated by reverse-phase HPLC. Values of three independent experiments (n = 3) are the means ± SE. *P < 0.05, high glucose vs. normal glucose. C: DCF fluorescence was measured as in Fig. 1. Values are the means ± SE of three independent experiments (n = 3). *P < 0.05, high glucose or 20-HETE vs. normal glucose; #P < 0.05, high glucose vs. high glucose + HET0016.
FIG. 3.
FIG. 3.
CYP4A-dependent 20-HETE production mediates high-glucose–induced podocyte apoptosis. Serum-deprived podocytes were preincubated with or without HET0016 (10 μmol/l) for 1 h and then treated with high glucose (HG; 25 mmol/l), normal glucose (NG; 5 mmol/l), or 1 μmol/l of recombinant 20-HETE for the indicated time periods. A: Representative photographs of annexin V and propidium iodide staining in different groups (n = 3 in each group). The number of apoptotic or necrotic cells was quantified by FACS analysis after staining with annexin V and propidium iodide. The cytograms show viable cells that did not bind annexin V or propidium iodide in the left lower quadrant. Cells at early stages of apoptosis that bound annexin V but that still had intact cell membranes and excluded propidium iodide are shown in the lower right quadrant. Cells with advanced stages of apoptosis or necrotic cells were both annexin V positive and propidium iodide positive and are shown in the upper right quadrant. B: Apoptotic nuclei were detected using Hoechst 33258. Chromatin condensation was examined by fluorescent microscopy. (A high-quality digital representation of this figure can be found in the online issue.)
FIG. 4.
FIG. 4.
CYP4A-dependent 20-HETE production mediates high-glucose–induced Nox1 and Nox4 protein expression and NADPH oxidase activation. Serum-deprived podocytes were preincubated with or without HET0016 (10 μmol/l) for 1 h and treated with high glucose (HG; 25 mmol/l), normal glucose (NG; 5 mmol/l), or 1 μmol/l of pure recombinant 20-HETE for the indicated time periods. Expression of Nox1 (A) and Nox4 (B) protein was determined by Western blotting analysis on homogenized podocytes. The Western blots are representative of three independent experiments (n = 3). Each histogram represents the ratio of the intensity of the Nox1 or Nox4 bands factored by the actin band. Values are the means ± SE. *P < 0.05, high glucose or 20-HETE vs. normal glucose; #P < 0.05, high glucose vs. high glucose + HET0016. C: Nox1 and Nox4 mRNA levels. The values represent the relative induction as measured by real-time RT-PCR relative to GAPDH mRNA levels (n = 3). The values are the means ± SE. *P < 0.05, high glucose or 20-HETE vs. normal glucose; #P < 0.05, high glucose vs. high glucose + HET0016. D: NADPH-dependent ROS generation in podocyte homogenates was measured by lucigenin-enhanced chemiluminescence. Superoxide anion is expressed as relative chemiluminescent light units (RLU) per milligram of protein per minute. Values of three independent experiments (n = 3) are the means ± SE. *P < 0.05, high glucose or 20-HETE vs. normal glucose; #P < 0.05, high glucose vs. high glucose + HET0016.
FIG. 5.
FIG. 5.
CYP4A-dependent 20-HETE production contributes to Nox1 and Nox4 protein expression and NADPH oxidase activity in glomeruli of type 1 diabetic mice. A: Glomeruli were isolated and pooled from five separate animals. CYP4A protein expression was determined in microsomes from isolated mouse glomeruli. B: Glomeruli were isolated from the kidney cortex of 6-month-old OVE 26 mice, OVE 26 mice treated for 3 weeks with HET0016 (2.5 mg · kg−1 · day−1 subcutaneously), and their littermate FVB mice. Nox1 and Nox4 protein expression was determined by Western blotting. Bottom panel: Each histogram represents the ratio of the intensity of Nox1 or Nox4 bands factored by the actin band. Values are the means ± SE. *P < 0.05, OVE26 mice vs. control FVB mice; #P < 0.05, decrease in Nox1 or Nox4 protein expression in HET0016 pretreated OVE26 mice vs. nontreated OVE 26 mice. C: NADPH-dependent ROS generation in glomerular homogenates isolated from the kidney cortex of FVB, OVE26, and OVE26 mice treated with HET0016 was measured by lucigenin-enhanced chemiluminescence. Superoxide anion is expressed as relative chemiluminescent light units (RLU) per milligram of protein per minute. Values are the means ± SE. *P < 0.05, OVE26 mice vs. control FVB mice; #P < 0.05, decrease in NADPH oxidase activity in HET0016 pretreated OVE26 mice vs. nontreated OVE 26 mice.
FIG. 6.
FIG. 6.
CYP4A contributes to podocyte apoptosis and foot process effacement in glomeruli of type 1 diabetic mice. A: Representative transmission electron micrographs of glomerular cross-section of FVB, OVE26, and OVE 26 mice treated with HET0016. The images show a condensed podocyte nucleus, foot process effacement, cytoplasmic rarefication, and basement membrane thickening of an OVE26 diabetic mouse. This effect was not seen in the OVE26 mice treated with HET0016. B: Semiquantitative analysis of foot process effacement of glomeruli from each group of animals (n = 5 per group). *P < 0.05, OVE26 mice vs. control FVB mice; #P < 0.05, decrease in the percentage of foot process effacement in OVE26 mice treated with HET0016 compared with OVE26 mice.
FIG. 7.
FIG. 7.
CYP4A contributes to podocyte apoptosis and reduction in synaptopodin protein expression in glomeruli of type 1 diabetic mice A: Representative immunofluorescence images of glomeruli stained with collagen IV (green), synaptopodin (red), and 4′,6-diamidino-2-phenylindole (blue). B: Podocyte number per glomerular section was lower in OVE26 mice, with a significant increase in podocyte number in the OVE26 mice treated with HET0016 (n = 5 per group). C: Synaptopodin protein expression in OVE 26 mice was decreased in the OVE26 mice and restored with the injection of HET0016. Values are the means ± SE. *P < 0.05, OVE26 mice vs. control FVB mice; #P < 0.05, decrease in albumin levels in HET0016-pretreated OVE26 mice vs. nontreated OVE 26 mice (n = 5 per group). (A high-quality digital representation of this figure can be found in the online issue.)
FIG. 8.
FIG. 8.
CYP4A contributes to albuminuria in type 1 diabetic mice. FVB, OVE26, and OVE 26 mice treated with HET0016 were placed in metabolic cages for 24 h. Urine was collected and albumin levels were measured and expressed as milligrams of albumin per 24 h. Values are the means ± SE. *P < 0.05, OVE26 mice vs. control FVB mice; #P < 0.05, decrease in albumin levels in HET0016-pretreated OVE26 mice vs. nontreated OVE 26 mice (n = 5 per group).
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