The antioxidant silybin prevents high glucose-induced oxidative stress and podocyte injury in vitro and in vivo

Abstract

Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression, and 150% the number of apoptotic cells, effects that were completely blocked by 10 μM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out that was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.

Keywords: NADPH oxidase; albuminuria; apoptosis; diabetic nephropathy; phytochemicals.

Figures

Fig. 1.

Silybin inhibits high glucose (HG)-induced superoxide generation. Exposure of mouse podocyte cultures to normal glucose (NG; 5 mM glucose) or to HG (25 mM glucose) with or without 10 μM silybin. Assessment of superoxide generation with the dihydroethidium (DHE) stain using 2 different methods: A, left: representative fluorescence microscopic images of podocytes after incubation with 10 μM DHE for 15 min. A, right: bar graph of semiquantitative analysis of the DHE fluorescence (means ± SE of 3 different experiments). ***P < 0.0001 HG vs. NG; ###P < 0.0001 HG vs. HG + silybin. B, left: representative HPLC elution profiles of the dihydroxyethidium (2-OH-E+) peak, a DHE byproduct specific for superoxide generation, and the nonspecific ethidium (E+) peak in cells extracts from cultured podocytes. B, right: bar graph of quantitative analysis of 2-OH-E+ generation (means ± SE of 4 different experiments each experiment in triplicate). ***P < 0.001 HG vs. NG; ##P < = 0.01 HG vs. HG + 10 μM silybin. Results are expressed as nmol 2-OH-E+/μM consumed DHE. C: dose-dependent response of cultured podocytes to silybin. Cells exposed to HG in the presence of 0.1, 1, or 10 μM silybin or vehicle. 2-OH-E+ generation assessed by HPLC, as in B (***P < 0.001 HG vs. NG; #P < 0.05 HG vs. HG + 0.1 μM silybin; ##P < 0.01 HG vs. HG + 1 μM silybin; ##P < 0.01 HG vs. HG + 10 μM silybin).

Fig. 2.

Effect of silybin on NADPH oxidase activity and Nox4 expression. Exposure of mouse podocytes to NG or HG without and with 10 μM silybin. A, left: representation of a representative individual experiment of NADPH-dependent superoxide generation in cultured cells, analyzed by lucigenin-enhanced chemiluminescence. Superoxide anion generation was determined by photoemission every 30 s for 4–5 min and was expressed as relative light units (RLU)/mg protein. A, right: bar graph of quantitative analysis of photoemission intensity expressed as RLU·min−1·mg protein−1 (means ± SE). **P < 0.01 NG vs. HG; #P < 0.05 HG vs. HG + silybin. B, left: Western blotting analysis of Nox4 expression in homogenized podocytes. Actin was included as control for loading and for specificity of change in protein expression. B, right: bar graph of quantitative analysis of Nox4 densitometry corrected for actin band density (means ± SE; *P < 0.05 NG vs. HG; ##P < 0.01 HG vs. HG + silybin). C, left: representative Western blotting analysis of Nox1 expression in homogenized podocytes. Actin was included as control for loading and for specificity of change in protein expression. C, right: bar graph of quantitative analysis of Nox1 densitometry corrected for actin band density (means ± SE of 3 experiments).

Fig. 3.

Effect of silybin on HG-induced podocyte apoptosis. Exposure of serum-deprived mouse podocytes to NG or HG for 24 h without or with 10 μM silybin. A, left: detection of apoptotic nuclei using Hoechst 33258. Chromatin condensation (white arrows) examined by fluorescent microscopy. A, right: bar graph of apoptotic cells as percent count of total cells (3 different experiments). **P < 0.01 NG vs. HG; ##P < 0.01 HG vs. HG + silybin. B: ELISA for cellular DNA fragmentation. Bar graph expresses the fold increase (means ± SE of 3 experiments in triplicate). *P < 0.05 NG vs. HG; #P < 0.05 HG vs. HG + silybin.

Fig. 4.

Silybin inhibits superoxide generation in diabetic mice kidney cortex and decreases the Nox4 protein expression. Measurement of superoxide generation in kidney cortex of 16-wk-old FVB mice, OVE26 mice, and OVE26 mice treated with silybin 100 mg/kg ip for 6 wk. A: bar graph of the percent increase in 2-OH-E+ generation in kidney cortex (means ± SE; n = 4 per group). **P < 0.01 FVB vs. OVE26; #P < 0.05 OVE26 vs. OVE26 + silybin. B: representative immunoperoxidase staining images of Nox4 protein in kidney sections from FVB (nondiabetic control), OVE26 (diabetic control), and OVE26 + silybin mice (diabetic treated).

Fig. 5.

Silybin decrease podocyte injury in diabetic mice. A, left: representative immunofluorescence images of glomeruli stained for synaptopodin. A, right: bar graph of fluorescence intensity (means ± SE; n = 3 per group). ***P < 0.001 FVB vs. OVE26; ###P < 0.001 OVE26 vs. OVE26 + silybin. B, left: representative dual-label immunohistochemistry/fluorescence staining of glomeruli for podocyte count; basement membranes stained with collagen IV. B, right: bar graph of podocyte count per glomerulus (means ± SE; n = 3 per group). ***P < 0.001 FVB vs. OVE26; ###P < 0.001 OVE26 vs. OVE26 + silybin. C, left: representative immunoperoxidase staining of glomeruli for WT-1 protein expression. Right panel, bar graph of podocyte count per glomerulus (mean ± SE; n = 3 per group). ***P < 0.001 FVB vs. OVE26; ###P < 0.001 OVE26 vs. OVE26 + silybin. D: bar graph of albumin/creatinine ratio in 24-h urine samples from FVB, OVE26, and silybin-treated OVE26 mice. Urine collected over 24 h in metabolic cages after acclimatization. Albumin measured by ELISA and creatinine by colorimetry. Results expressed as μg protein/mg creatinine (means ± SE). ***P < 0.001 FVB vs. OVE26; P = 0.06 OVE26 vs. OVE26 + silybin.

Fig. 6.

Proposed mechanism of the protective effect of silybin on HG/diabetes-induced podocyte injury/apoptosis.