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Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy.

Zheng H, Whitman SA, Wu W, Wondrak GT, Wong PK, Fang D, Zhang DD - Diabetes (2011)

Bottom Line: Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed.Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs.In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona, USA.

ABSTRACT

Objective: To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.

Research design and methods: Diabetes was induced in Nrf2(+/+) and Nrf2(-/-) mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs).

Results: SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2(+/+) but not in Nrf2(-/-) mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2(+/+) mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.

Conclusions: We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.

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High glucose–mediated mesangial cell growth inhibition and hypertrophy can be reversed by activation of Nrf2. A: Cell growth of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media was monitored in real-time for 96 h (upper panel = average; lower panel = average with error). B: Cell death and proliferation were assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay (positive control is treated with cisplatin at 18 μmol/L for 24 h) or Ki67 immunolabeling. HG media induced cell growth inhibition, which was alleviated by coculture with an Nrf2 activator. C: Cell size of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 96 h was measured by forward light scatter/flow cytometry. Incubation in HG media induced cellular hypertrophy that was reduced with an Nrf2 activator. D: Cell area is reported from GFP-transfected HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h. Total cell area increased with HG conditions but was significantly reduced in the presence of an Nrf2 activator. Data in D are expressed as mean ± SD (n = 100). *P < 0.05 compared with NG group. #P < 0.05 Nrf2 activators compared with HG alone. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)
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Figure 6: High glucose–mediated mesangial cell growth inhibition and hypertrophy can be reversed by activation of Nrf2. A: Cell growth of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media was monitored in real-time for 96 h (upper panel = average; lower panel = average with error). B: Cell death and proliferation were assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay (positive control is treated with cisplatin at 18 μmol/L for 24 h) or Ki67 immunolabeling. HG media induced cell growth inhibition, which was alleviated by coculture with an Nrf2 activator. C: Cell size of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 96 h was measured by forward light scatter/flow cytometry. Incubation in HG media induced cellular hypertrophy that was reduced with an Nrf2 activator. D: Cell area is reported from GFP-transfected HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h. Total cell area increased with HG conditions but was significantly reduced in the presence of an Nrf2 activator. Data in D are expressed as mean ± SD (n = 100). *P < 0.05 compared with NG group. #P < 0.05 Nrf2 activators compared with HG alone. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)

Mentions: As a chronic disease, diabetic nephropathy is characterized by sequential pathological changes including cell growth inhibition and glomerular hypertrophy. First, we measured cell growth rate of HRMCs in differential glucose media using the xCELLigence system. As expected, the growth curve for HRMCs growing in HG media fell below those either growing in NG or in HG and supplemented with an Nrf2 activator (Fig. 6A). No cell death was observed under any condition and staining with Ki67 showed that hyperglycemia inhibited cell proliferation, which was counteracted by activation of Nrf2 (Fig. 6B). Next, the size of HRMCs under NG and HG conditions were measured by two independent methods. First, forward light scattering analysis by flow cytometry revealed a dramatic shift in size distribution (Fig. 6C), indicating an increase in the size of HRMCs growing in HG compared with NG media. Conversely, the light scattering curves for HRMCs growing in HG media in the presence of an Nrf2 activator were completely shifted back to the left where they overlapped with the curve of NG cells (Fig. 6C). Second, HRMCs were transfected with GFP to mark the entire area of cells. Incubation of HRMCs in HG media resulted in an increase in cell size as measured by the average area per GFP+ cell. The hypertrophy observed in HG media was suppressed by treatment with tBHQ, SF, or CA (Fig. 6D). Collectively, these results demonstrate that hyperglycemia induced cell growth inhibition, and hypertrophy can be attenuated by activation of the Nrf2 pathway.


Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy.

Zheng H, Whitman SA, Wu W, Wondrak GT, Wong PK, Fang D, Zhang DD - Diabetes (2011)

High glucose–mediated mesangial cell growth inhibition and hypertrophy can be reversed by activation of Nrf2. A: Cell growth of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media was monitored in real-time for 96 h (upper panel = average; lower panel = average with error). B: Cell death and proliferation were assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay (positive control is treated with cisplatin at 18 μmol/L for 24 h) or Ki67 immunolabeling. HG media induced cell growth inhibition, which was alleviated by coculture with an Nrf2 activator. C: Cell size of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 96 h was measured by forward light scatter/flow cytometry. Incubation in HG media induced cellular hypertrophy that was reduced with an Nrf2 activator. D: Cell area is reported from GFP-transfected HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h. Total cell area increased with HG conditions but was significantly reduced in the presence of an Nrf2 activator. Data in D are expressed as mean ± SD (n = 100). *P < 0.05 compared with NG group. #P < 0.05 Nrf2 activators compared with HG alone. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: High glucose–mediated mesangial cell growth inhibition and hypertrophy can be reversed by activation of Nrf2. A: Cell growth of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media was monitored in real-time for 96 h (upper panel = average; lower panel = average with error). B: Cell death and proliferation were assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay (positive control is treated with cisplatin at 18 μmol/L for 24 h) or Ki67 immunolabeling. HG media induced cell growth inhibition, which was alleviated by coculture with an Nrf2 activator. C: Cell size of HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 96 h was measured by forward light scatter/flow cytometry. Incubation in HG media induced cellular hypertrophy that was reduced with an Nrf2 activator. D: Cell area is reported from GFP-transfected HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h. Total cell area increased with HG conditions but was significantly reduced in the presence of an Nrf2 activator. Data in D are expressed as mean ± SD (n = 100). *P < 0.05 compared with NG group. #P < 0.05 Nrf2 activators compared with HG alone. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)
Mentions: As a chronic disease, diabetic nephropathy is characterized by sequential pathological changes including cell growth inhibition and glomerular hypertrophy. First, we measured cell growth rate of HRMCs in differential glucose media using the xCELLigence system. As expected, the growth curve for HRMCs growing in HG media fell below those either growing in NG or in HG and supplemented with an Nrf2 activator (Fig. 6A). No cell death was observed under any condition and staining with Ki67 showed that hyperglycemia inhibited cell proliferation, which was counteracted by activation of Nrf2 (Fig. 6B). Next, the size of HRMCs under NG and HG conditions were measured by two independent methods. First, forward light scattering analysis by flow cytometry revealed a dramatic shift in size distribution (Fig. 6C), indicating an increase in the size of HRMCs growing in HG compared with NG media. Conversely, the light scattering curves for HRMCs growing in HG media in the presence of an Nrf2 activator were completely shifted back to the left where they overlapped with the curve of NG cells (Fig. 6C). Second, HRMCs were transfected with GFP to mark the entire area of cells. Incubation of HRMCs in HG media resulted in an increase in cell size as measured by the average area per GFP+ cell. The hypertrophy observed in HG media was suppressed by treatment with tBHQ, SF, or CA (Fig. 6D). Collectively, these results demonstrate that hyperglycemia induced cell growth inhibition, and hypertrophy can be attenuated by activation of the Nrf2 pathway.

Bottom Line: Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed.Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs.In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona, USA.

ABSTRACT

Objective: To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.

Research design and methods: Diabetes was induced in Nrf2(+/+) and Nrf2(-/-) mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs).

Results: SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2(+/+) but not in Nrf2(-/-) mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2(+/+) mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.

Conclusions: We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.

Show MeSH
Related in: MedlinePlus